Principios de Citogenetica Clinica

Steven L.
Gersen
Martha B. Keagle
Editors
The Principles of
Clinical Cytogenetics
Third Edition
123
The Principles of Clinical Cytogenetics
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Steven L. Gersen
Martha B. Keagle
Editors
The Principles of Clinical

Cytogenetics
Third Edition
Editors
Steven L. Gersen Martha B. Keagle
AmeriPath Northeast Department of Allied Health Sciences
Shelton, CT College of Agriculture and Natural Resources
USA University of Connecticut
Storrs, CT
USA
ISBN 978-1-4419-1687-7 ISBN 978-1-4419-1688-4 (e-Book)

DOI 10.1007/978-1-4419-1688-4
Springer New York Heidelberg Dordrecht London
Library of Congress Control Number: 2012947405
© Springer Science+Business Media New York 2013

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Preface
In April 2011, the website The DNA Exchange ran a story about the origin of our convention
of referring to the short and long arms of chromosomes as “p” and “q.” Several possible expla-
nations for how this usage came into being were presented in a somewhat whimsical manner.
Did we really go with p from the French petite and q because it alphabetically follows p?
Was there really a “French vs. English” argument? Was it supposed to be p and g (from the
French grande) but changed due to a typesetting error? Was Hardy-Weinberg equilibrium
(p + q = 1) invoked?
This prompted a flurry of comments over the Listserv used by cytogeneticists. Ultimately,
several participants of the 1966 “Chicago Conference” weighed in, and Dr. Kurt Hirschhorn,
who chaired the session at that conference, confirmed that the decision to go with p and q
resulted from a combination of (sometimes spirited) debate, compromise (p really is for petite),
logic, and, yes, agreement that p + q = 1.
This is all great fun. But the story in The DNA Exchange also spawned other comments.
It opened with:
Karyotypes are sooooo 20th century. Time was when a ripe crop of G-banded chromosomes promised a
fruitful harvest of genetic secrets. But nowadays a Giemsa-stained karyotype seems like a quaint low
resolution black and white TV set – those cute little D & G groups even have rabbit-ear antennas – com-
pared with the bright, sexy colors of FISH, the fine oligonucleotide detail of microarrays, and the dense
volumes of data of generated by high throughput DNA sequencing.
Some cytogeneticists took offense at this.

People have been predicting the demise of cytogenetics for decades; this tended to happen
each time new technology, such as DNA analysis or fluorescence in situ hybridization, became
available. And yet we are still here.
Interestingly, this idea was significant as the previous edition of this book went to press in
2005 due to the increasingly important role of many FISH assays. In the preface to that edition,
we discussed that while some classically trained cytogeneticists were concerned that FISH was
going to put them out of work, Dorothy Warburton had predicted, years earlier, that FISH
would actually provide the cytogenetics lab with an even more important diagnostic and prog-
nostic role. She was of course correct.
Now we have microarrays. This edition of our book has a chapter dedicated to this technol-
ogy, and several authors also deal with it in their individual chapters. The term “cytogenomics”
(chromosome analysis using molecular techniques) is working its way into our lexicon.
Once again, there is talk, if not concern, that arrays could mean the unemployment line for
cytogeneticists and, if not arrays, then perhaps next-generation sequencing. And once again,
Dorothy put things into perspective:
The way I look at it is that cytogenetics is not about a technique, but a field of knowledge. We may
change the way we look at chromosomes, but the questions and problems remain the same. A technique
is only as good as our ability to interpret what we see in a way that helps families, and having molecular
training does not provide the experience necessary to do this. We would never have known about bal-
v
vi Preface
anced translocations without looking at chromosomes, but now we have a way to tell if they are really
balanced or not. I also believe that we will never be able to stop using chromosome preparations to
interpret what we see on arrays. We have many examples where confirming array data has revealed
unexpected kinds of rearrangements, as well as mosaicism. These are things that have much more
significance for counseling than a simple call of a dup or del. I don’t believe sequencing will change
this.
I was first advised to find another field in 1969 (right before banding). So far I still have a job, although
what I look at day to day has changed a great deal. “Classical” is pretty much a synonym for “in the
past,” so yes, classical cytogenetics may no longer be practiced. However, what is here is exciting and
challenging and requires every technique in our playbook.
This third edition of The Principles of Clinical Cytogenetics was prompted by significant
advances in the field since the last edition of this book was published. So while it is true that
the way we look at chromosomes will likely continue to evolve, we do not expect to stop look-
ing at them any time soon.
Shelton, CT, USA Steven L. Gersen, Ph.D.
Storrs, CT, USA Martha B. Keagle, M.Ed.
Contents
Part I Basic Concepts and Background

1 History of Clinical Cytogenetics ............................................................................. 3
Steven L. Gersen
2 DNA, Chromosomes, and Cell Division ................................................................. 9
Martha B. Keagle
3 Human Chromosome Nomenclature: An Overview
and Definition of Terms ........................................................................................... 23
Marilyn L. Slovak, Aaron Theisen, and Lisa G. Shaffer
Part II Examining and Analyzing Chromosomes
4 Basic Cytogenetics Laboratory Procedures ........................................................... 53
Martha B. Keagle and Steven L. Gersen
5 The Essentials of Light Microscopy ....................................................................... 67
Nathan S. Claxton and Stephen T. Ross
6 Quality Control and Quality Assurance ................................................................ 77
Martha B. Keagle
7 Instrumentation in the Cytogenetics Laboratory ................................................. 95
Steven L. Gersen
Part III Clinical Cytogenetics
8 Autosomal Aneuploidy............................................................................................. 113
Jin-Chen C. Wang
9 Structural Chromosome Rearrangements............................................................. 139
Kathleen Kaiser-Rogers and Kathleen W. Rao
10 Sex Chromosomes, Sex Chromosome Disorders, and Disorders
of Sex Development .................................................................................................. 175
Cynthia M. Powell
11 The Cytogenetics of Infertility ................................................................................ 213
Linda Marie Randolph
12 Prenatal Cytogenetics .............................................................................................. 229
13 The Cytogenetics of Spontaneous Abortion .......................................................... 275
Solveig M.V. Pflueger
vii
viii Contents
14 Chromosome Instability .......................................................................................... 293

Xiao-Xiang Zhang
Part IV Cancer Cytogenetics
15 The Cytogenetics of Hematologic Neoplasms ........................................................ 309
Aurelia Meloni-Ehrig
16 The Cytogenetics of Solid Tumors .......................................................................... 371
Linda D. Cooley and Kathleen S. Wilson
Part V Adjunct Technologies
17 Fluorescence In Situ Hybridization (FISH) ........................................................... 415
Daynna J. Wolff
18 Microarray-Based Cytogenetics ............................................................................. 441
Lisa G. Shaffer
Part VI Beyond Chromosomes
19 Fragile X—A Family of Disorders: Changing Phenotype
and Molecular Genetics ........................................................................................... 453
Elaine B. Spector
20 Genomic Imprinting and Uniparental Disomy ..................................................... 473
Jin-Chen C. Wang
21 Genetic Counseling .................................................................................................. 499
Sarah Hutchings Clark
Index .................................................................................................................................. 515
Contributors
Sarah Hutchings Clark, M.S., C.G.C. Department of Advanced Obstetrics and Gynecology,
Stamford Hospital, Stamford, CT, USA
Nathan S. Claxton, B.S. Product and Marketing Department, Nikon Instruments Inc.,
Melville, NY, USA
Linda D. Cooley, M.D., M.B.A. University of Missouri Kansas City School of Medicine,
Kansas City, MO, USA
Cytogenetics Laboratory, Children’s Mercy Hospital, Kansas City, MO, USA
Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas City,
MO, USA
Steven L. Gersen, Ph.D. Cytogenetics Laboratory, AmeriPath Northeast, Shelton, CT, USA
Kathleen Kaiser-Rogers, Ph.D. UNC Hospitals Cytogenetics Laboratory, Department of
Pathology and Laboratory Medicine, Pediatrics, and Genetics, University of North Carolina
Hospitals, Chapel Hill, NC, USA
Martha B. Keagle, M.Ed. Department of Allied Health, College of Agriculture and Natural
Resources, University of Connecticut, Storrs, CT, USA
Aurelia Meloni-Ehrig, Ph.D., D.Sc. Department of Cytogenetics, AmeriPath Central Florida,
Orlando, FL, USA
Solveig M.V. Pflueger, Ph.D., M.D. Department of Pathology, Baystate Medical Center,
Tufts University School of Medicine, Springfield, MA, USA
Cynthia M. Powell, M.D., M.S. Department of Pediatrics, The University of North Carolina
at Chapel Hill, Chapel Hill, NC, USA
Linda Marie Randolph, M.D., MA. Division of Medical Genetics, Keck School of Medicine,
University of Southern California, Los Angeles, CA, USA
Division of Medical Genetics, Childrens Hospital Los Angeles, Los Angeles, CA, USA
Kathleen W. Rao, Ph.D. Cytogenetics Laboratory, North Carolina Memorial Hospital,
Chapel Hill, NC, USA
Department of Pediatrics, Pathology and Laboratory Medicine, and Genetics, The University
of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Stephen T. Ross, Ph.D. Product and Marketing Department, Nikon Instruments, Inc.,
Melville, NY, USA
Lisa G. Shaffer, Ph.D. Signature Genomic Laboratories, Spokane, WA, USA
ix
x Contributors
Marilyn L. Slovak, Ph.D. Cytogenetics Laboratory, Palo Verde Laboratory/Sonora Quest

Laboratories, Tempe, AZ, USA
Elaine B. Spector, Ph.D. UCD DNA Diagnostic Laboratory, Department of Pediatrics,
University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, CO, USA
Aaron Theisen Theisen Consulting, Spokane, WA, USA
Jin-Chen C. Wang, M.D. Department of Cytogenetics, Genzyme Genetics, Monrovia,
CA, USA
Kathleen S. Wilson, M.D. McDermott Center for Human Growth and Development and the
Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX,
USA
Daynna J. Wolff, Ph.D. Department of Pathology and Laboratory Medicine, Medical
University of South Carolina, Charleston, SC, USA
Cytogenetics and Molecular Genetics, Medical University of South Carolina, Charleston,
SC, USA
Xiao-Xiang Zhang, M.D., Ph.D. US Labs, a Labcorp Company, Brentwood, TN, USA
Part I
Basic Concepts and Background
History of Clinical Cytogenetics
1
Steven L. Gersen
The beginning of human cytogenetics is generally attributed term karyotype to refer to the ordered arrangement of
to Walther Flemming, an Austrian cytologist and professor chromosomes [7].
of anatomy, who published the first illustrations of human Despite continued technical improvements, there was
chromosomes in 1882. Flemming also referred to the stain- clearly some difficulty in properly visualizing or discrimi-
able portion of the nucleus as chromatin and first used the nating between individual chromosomes. Even though
term mitosis [1]. In 1888, Waldeyer introduced the word Painter’s number of 48 human chromosomes was reported
chromosome, from the Greek words for “colored body,” and somewhat conservatively, it was increasingly treated as
several prominent scientists of the day began to formulate fact with the passage of time and was “confirmed” several
the idea that determinants of heredity were carried on chro- times over the next few decades. For example, in 1952,
mosomes [2]. After the “rediscovery” of Mendelian inheri- T. C. Hsu reported that, rather than depending upon histo-
tance in 1900, Sutton (and, independently at around the same logic sections, examination of chromosomes could be
time, Boveri) formally developed a “chromosome theory of facilitated if one studied cells grown with tissue culture
inheritance” [3, 4]. Sutton combined the disciplines of cytol- techniques published by Fisher [8]. Hsu then demonstrated
ogy and genetics when he referred to the study of chromo- the value of this method by using it to examine human
somes as cytogenetics. embryonic cell cultures, from which he produced both
Due in part to improvements in optical lenses, stains, and mitotic metaphase drawings and an idiogram of all 48
tissue manipulation techniques during the late nineteenth human chromosomes [9]!
and early twentieth centuries, the study of cytogenetics con- As with other significant discoveries, correcting this inac-
tinued, with an emphasis placed by some on determining the curacy required an unplanned event—a laboratory error. Its
correct number of chromosomes, as well as the sex chromo- origin can be found in the addendum that appears at the end
some configuration, in humans. Several reports appeared, of Hsu’s paper:
with differing estimates of these. For example, in 1912, von It was found after this article had been sent to press that the well-
Winiwarter concluded that men have 47 chromosomes and spread metaphases were the result of an accident. Instead of
women 48 [5]. Then, in 1923, T. S. Painter studied (meiotic) being washed in isotonic saline, the cultures had been washed in
chromosomes derived from the testicles of several men who hypotonic solution before fixation [9].
had been incarcerated, castrated, and ultimately hanged in The hypotonic solution caused water to enter the cells
the Texas State Insane Asylum. Based on this work, Painter via osmosis, which swelled the cell membranes and sepa-
definitively reported the human diploid chromosome number rated the chromosomes, making them easier to visualize.
to be 48 (double the 24 bivalents he saw), even though, This accident was the key that unlocked the future of
2 years earlier, he had preliminarily reported that some of his human cytogenetics. Within one year, Hsu, realizing the
better samples produced a diploid number of 46 [6]. At this potential of this fortuitous event, reported a “hypotonic
time, Painter also proposed the X and Y sex chromosome shock” procedure [10]. By 1955, Ford and Hamerton had
mechanism in man. One year later, Levitsky formulated the modified this technique and had also worked out a method
for pretreating cells grown in culture with colchicine so
as to destroy the mitotic spindle apparatus and thus accu-
mulate dividing cells in metaphase [11]. Joe Hin Tjio, an
S.L. Gersen, Ph.D. (*)
Cytogenetics Laboratory, AmeriPath Northeast,
American-born Indonesian, learned about these proce-
1 Greenwich Place, Shelton, CT 06484, USA dures and worked with Hamerton and Ford to further
e-mail: [email protected] improve upon them.
S.L. Gersen and M.B. Keagle (eds.), The Principles of Clinical Cytogenetics, Third Edition, 3
DOI 10.1007/978-1-4419-1688-4_1, © Springer Science+Business Media New York 2013
4 S.L. Gersen
In November of 1955, Tjio was invited to Lund, Sweden, The trisomy was reported to involve one of the smallest pairs
to work on human embryonic lung fibroblast cultures in the of chromosomes and would eventually be referred to as
laboratory of his colleague, Albert Levan, a Spaniard who trisomy 21. Lejeune had proved Waardenburg’s hypothesis
had learned the colchicine and hypotonic method in Hsu’s by reporting the first example of a chromosomal syndrome in
laboratory at the Sloan-Kettering Institute in New York. Tjio man, and in December of 1962, he received one of the first
and Levan optimized the colchicine/hypotonic method for Joseph Kennedy Jr. Foundation International Awards for his
these cells and in January of 1956 (after carefully reviewing work (Fig. 1.1).
images from decades of previously reported work) diplomat- Three more chromosomal syndromes, all believed to
ically reported that the human diploid chromosome number involve the sex chromosomes, were also described in 1959.
appeared to be 46, not 48 [12]. They referenced anecdotal Ford reported that females with Turner syndrome have 45
data from a colleague who had been studying liver mitoses chromosomes, apparently with a single X chromosome and
from aborted human embryos in the Spring of 1955 but tem- no Y, and Jacobs and Strong demonstrated that men with
porarily abandoned the research “because the workers were Klinefelter syndrome have 47 chromosomes, with the addi-
unable to find all the 48 human chromosomes in their mate- tional chromosome belonging to the group that contained the
rial; as a matter of fact, the number 46 was repeatedly counted X chromosome [16, 17]. A female with sexual dysfunction
in their slides.” Tjio and Levan concluded their paper: was also shown by Jacobs to have 47 chromosomes and was
…we do not wish to generalize our present findings into a state- believed to have an XXX sex chromosome complement [18].
ment that the chromosome number of man is 2n = 46, but it is The sex chromosome designation of these syndromes
hard to avoid the conclusion that this would be the most natural was supported by (and helped explain) a phenomenon that
explanation of our observations [12]. had been observed 10 years earlier. In 1949, Murray Barr
What was dogma for over 30 years had been overturned in was studying fatigue in repeatedly stimulated neural cells of
one now classic paper. Ford and Hamerton soon confirmed the cat [19]. Barr observed a small stained body on the
Tjio and Levan’s finding [13]. The era of clinical cytogenet- periphery of some interphase nuclei, and his records were
ics was at hand. It would take three more years to arrive, detailed enough for him to realize that this was present only
however, and it would begin with the identification of four in the nuclei of female cats. This object, referred to as sex
chromosomal syndromes. chromatin (now known as X chromatin or the Barr body), is
The concept that an abnormality involving the chromo- actually the inactivated X chromosome present in nucleated
somes could have a phenotypic effect was not original. In cells of all normal female mammals but absent in normal
1932, Waardenburg made the suggestion that Down syn- males. The observation that the Turner syndrome, Klinefelter
drome could perhaps be the result of a chromosomal aberra- syndrome, and putative XXX patients had zero, one, and
tion, but the science of the time could neither prove nor two Barr bodies, respectively, elucidated the mechanism of
disprove his idea; this would take almost three decades [14]. sex determination in humans, confirming for the first time
In 1958, Lejeune studied the chromosomes of fibroblast cul- that it is the presence or absence of the Y chromosome that
tures from patients with Down syndrome and in 1959, determines maleness, not merely the number of X chromo-
described an extra chromosome in each of these cells [15]. somes present, as in Drosophila. In 1961, the single active X
Fig. 1.1 Jérôme Lejeune

receives a Joseph
P. Kennedy Jr. Foundation
International Award for
demonstrating that Down
syndrome results from an extra
chromosome (Photo courtesy of
the John F. Kennedy Library,
Boston, MA)
1 History of Clinical Cytogenetics 5
chromosome mechanism of X-dosage compensation in “aggressive behavior, mental subnormality and the XYY
mammals was developed by Mary Lyon and has been since male,” and the chromosomal instabilities associated with
known as the Lyon hypothesis [20]. Bloom syndrome and Fanconi anemia were reported
It was not long after Lejeune’s report of the chromosomal [28–30].
basis of Down syndrome that other autosomal abnormalities Additional technical advancements had facilitated the
were discovered. In the April 9, 1960, edition of The Lancet, routine study of patient karyotypes. In 1960, Peter Nowell
Patau et al. described two similar infants with an extra observed that the kidney bean extract phytohemagglutinin,
“D-group” chromosome who had multiple anomalies quite used to separate red and white blood cells, stimulated lym-
different from those seen in Down syndrome [21]. In the same phocytes to divide. He introduced its use as a mitogen,
journal, Edwards et al. described “a new trisomic syndrome” permitting a peripheral blood sample to be used for chromo-
in an infant girl with yet another constellation of phenotypic some analysis [31]. This eliminated the need for bone mar-
abnormalities and a different autosomal trisomy [22]. The row aspiration, which had previously been the best way to
former became known as Patau syndrome or “D trisomy” and obtain a sufficient number of spontaneously dividing cells. It
the latter as Edwards syndrome or “E trisomy.” Patau paper was now feasible to produce mitotic cells suitable for chro-
incredibly contains a typographical error and announces that mosome analysis from virtually any patient.
the extra chromosome “belongs to the E group,” and Edwards Yet, within nine years of the discovery of the number of
reported that “the patient was … trisomic for the no. 17 chro- chromosomes in humans, only three autosomal trisomies,
mosome,” but we now know these syndromes to be trisomies four sex chromosome aneuploidies, a structural abnormality
13 and 18, respectively. (a deletion), an acquired chromosomal abnormality associ-
Also in 1960, Nowell and Hungerford reported the pres- ated with cancer, and two chromosome breakage disorders
ence of a small chromosome in patients with chronic myel- had been described as recognizable “chromosomal syn-
ogenous leukemia. Using the proposed nomenclature method dromes.” A new clinical laboratory discipline had been cre-
at the time, this was designated Philadelphia chromosome ated; was it destined to be restricted to the diagnosis of a few
1 (Ph1), and it demonstrated, for the first time, an association abnormalities?
between chromosomes and cancer [23–25] (Fig. 1.2). Still This seemed likely. Even though certain pairs were dis-
referred to as the “Philadelphia chromosome” for historical tinguishable by size and centromere position, individual
purposes, this phenomenon was eventually relegated to noth- chromosomes could not be identified, and as a result, patient-
ing more than a curiosity during the 1960s, as the concept of specific chromosome abnormalities could be observed but
a clinical association between chromosomes and cancer fell not defined. Furthermore, the existence of certain abnormali-
out of favor. ties, such as inversions involving a single chromosome arm
In 1963 and 1964, Lejeune et al. reported that three infants (so-called paracentric inversions) could be hypothesized, but
with the cri du chat (“cat cry”) syndrome of phenotypic not proven, because they could not be visualized. Indeed, it
anomalies, which includes severe mental retardation and a seemed that without a way to definitively identify each chro-
characteristic kitten-like mewing cry, had a deletion of the mosome (and more importantly, regions of each chromo-
short arm of a B-group chromosome, designated as chromosome), this new field of medicine would be limited in scope
some 5 [26, 27]. Within two years, Jacobs et al. described to the study of a few disorders.
Fig. 1.2 The first photograph of

a Q-banded cell published by
Caspersson in 1970. The figure
was originally labeled
“Quinacrine mustard treated
human metaphase chromosomes
(male) from leukocyte culture.
Fluorescence
microscope × 2,000” (Reprinted
with permission from Caspersson
et al. [33], Elsevier)
6 S.L. Gersen
For three more years, clinical cytogenetics was so and a room that could be darkened, and the fluorescence
relegated. Then, in 1968, Torbjörn Caspersson observed that tended to fade or “quench” after a few minutes, making real-
when plant chromosomes were stained with fluorescent qui- time microscopic analysis difficult.
nacrine compounds, they did not fluoresce uniformly but These difficulties were overcome a year later, when Drets
rather produced a series of bright and dull areas across the and Shaw described a method of producing similar chromo-
length of each chromosome. Furthermore, each pair somal banding patterns using an alkali and saline pretreat-
fluoresced with a different pattern, so that previously indis- ment followed by staining with Giemsa, a compound
tinguishable chromosomes could now be recognized [32]. developed for identification, in blood smears, of the proto-
Caspersson then turned his attention from plants to the zoan that causes malaria [35]. Even though some of the chro-
study of human chromosomes. He hypothesized that the qui- mosome designations proposed by Drets and Shaw have
nacrine derivative quinacrine mustard (QM) would preferen- been changed (essentially in favor of those advocated by
tially bind to guanine residues and that C-G rich regions of Caspersson), this method, and successive variations of it,
chromosomes should therefore produce brighter “striations,” facilitated widespread application of clinical cytogenetic
as he initially referred to them, while A-T rich regions would techniques. While the availability of individuals with the
be dull. Although it ultimately turned out that it is the A-T appropriate training and expertise limited the number and
rich regions that fluoresce brightly and that ordinary quina- capacity of laboratories that could perform these procedures
crine dihydrochloride works as well as QM, by 1971, (in some ways still true today), the technology itself was now
Caspersson had successfully produced and reported a unique within the grasp of any facility.
“banding” pattern for each human chromosome pair [33, 34]. What followed was a cascade of defined chromosomal
See Fig. 1.3. abnormalities and syndromes: aneuploidies, deletions, microde-
For the first time, each human chromosome could be posi- letions, translocations, inversions (including the paracentric
tively identified. The method, however, was cumbersome. variety), insertions, mosaicisms, and a seemingly infinite num-
It required a relatively expensive fluorescence microscope ber of patient- and family-specific rearrangements.
In 1973, Janet Rowley demonstrated that the “Philadelphia
chromosome” was actually the result of a translocation
involving chromosomes 9 and 22, and in that same year, she
also described an (8;21) translocation in AML [36, 37]. The
association between chromosomes and cancer could no lon-
ger be ignored. The decades that followed saw an ever-
increasing collection of rearrangements and other cytogenetic
anomalies associated with neoplasia. These were eventually
cataloged by Felix Mitelman in what has become an ongoing
project of incredible dedication; the first volume was pub-
lished in 1983, and the most recent version is an online data-
base with close to 60,000 entries [38, 39].
Thanks to the host of research applications made possible
by the precise identification of smaller and smaller regions
of the karyotype, genes began to be mapped to chromosomes
at a furious pace. The probes that resulted from such research
have given rise to the discipline of molecular cytogenetics,
Fig. 1.3 One of the first photomicrographs of a metaphase spread from which utilizes the techniques of fluorescence in situ hybrid-
a patient with chronic myelogenous leukemia, indicating the ization (FISH). In recent years, this exciting development
Philadelphia chromosome. Reported a decade before routine chromo-
some banding, the authors (correctly) interpreted the abnormal chromo-
and the many innovative procedures derived from it have cre-
some to represent the next-to-smallest human chromosome and reported ated even more interest in the human karyotype. A perfect
it as being a chromosome 21: “Note the Ph1 chromosome (arrow). To example involves the union of information gleaned from the
right are shown, from bottom to top, 21, Ph1, 22, 22, and Y. The Ph1 Human Genome Project with molecular techniques such as
chromosome is apparently a 21 which has lost approximately one half
of its long arm.” However, although chromosome banding demonstrated
comparative genomic hybridization (GCH) or single nucle-
that the chromosome involved in Down syndrome is actually the small- otide polymorphism (SNP) analysis. Combining these using
est human chromosome, the term “trisomy 21” was already too com- computer and droplet technologies has given rise to the chro-
mon to be changed, and so the numbering of the two smallest human mosome microarray, which is already becoming the next step
chromosomes was reversed. The Philadelphia chromosome is therefore
described as being derived from chromosome 22 (Figure courtesy of
in the evolution of clinical cytogenetics.
Alice Hungerford and reprinted with permission from Nowell and In the summer of 2006, geneticists from around the world
Hungerford [25]) met in Bethesda, Maryland, to celebrate “50 Years of 46
1 History of Clinical Cytogenetics 7
Fig. 1.4 In July 2006, geneticists from around the world met in Bethesda, Maryland, to celebrate “50 Years of 46 Human Chromosomes: Progress
in Cytogenetics”
Human Chromosomes: Progress in Cytogenetics” (Fig. 1.4), It was really not so long ago that humans had 48 chromo-
and in 2010, we gathered in Philadelphia for a “Philadelphia somes. One has to wonder whether any of the pioneers of this
Chromosome Symposium: Past, Present, and Future—The field could have predicted the modern widespread clinical
50th Anniversary of the Discovery of the Philadelphia use of chromosome analysis, in all its forms. But perhaps it
Chromosome.” This group had the honor of being addressed is even more exciting to wonder what lies ahead.
by Dr. Peter Nowell, Dr. Janet Rowley, Dr. Felix Mitelman,
and Mrs. Alice Hungerford, wife of the late Dr. David
Hungerford. References
More than one million cytogenetic and molecular cytoge-
1. Flemming W. Zellsubstanz, Kern und Zellteilung. Leipzig: Vogel;
netic analyses are now performed annually in more than 400
1882.
laboratories worldwide, and this testing is now often the 2. Waldeyer W. Über Karyokineze und ihre Beziehung zu den
standard of care [40, 41]. Pregnant women over the age of Befruchtungsvorgängen. Arch Mikr Anat. 1888;32:1.
35, or those with certain serum-screening results, are rou- 3. Sutton WS. The chromosomes in heredity. Biol Bull Wood’s Hole.
1903;4:231.
tinely offered prenatal cytogenetic analysis, and many also
4. Boveri T. Über mehrpolige Mitosen als Mittel zur Analyse des
have prenatal ploidy analysis via FISH. For children with Zellkerns. Verh Phys-med Ges Würzburg NF. 1902;35:67–90.
phenotypic and/or mental difficulties and for couples experi- 5. Winiwarter H. Études sur la spermatogenèse humaine. I. Cellule de
encing reproductive problems, cytogenetics has become a Sertoli. II. Hétérochromosome et mitoses de l’epitheleum seminal.
Arch Biol (Liege). 1912;27:91–189.
routine part of their clinical workup. FISH has permitted us
6. Painter TS. Studies in mammalian spermatogenesis. II. The sper-
to visualize changes that are too subtle to be detected with matogenesis of man. J Exp Zool. 1923;37:291–336.
standard chromosome analysis, and chromosome microar- 7. Levitsky GA. Materielle Grundlagen der Vererbung. Kiew:
rays provide even greater resolution. Cytogenetics and FISH Staatsverlag; 1924.
8. Fisher A. Biology of tissue cells. Cambridge: Cambridge University
also provide information vital to the diagnosis, prognosis,
Press; 1946.
therapy, and monitoring of treatment for a variety of cancers, 9. Hsu TC. Mammalian Chromosomes in vitro. I. The karyotype of
and cancer arrays are gaining utility as well. man. J Hered. 1952;43:167–72.
8 S.L. Gersen
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A method for spreading the chromosomes of cells in tissue partielle du bras court d’un chromosome 5. C R Acad Sci (Paris).
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sequence for mammalian chromosomes. Stain Technol. 1956;31: bras court du chromosome 5. Individualisation d’un nouvel état
247. morbide. Sem Hôp Paris. 1964;18:1069–79.
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1956;178:1020–3. 29. Schroeder TM, Anschütz F, Knopp F. Spontane chromosomenaber-
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DNA, Chromosomes, and Cell Division
2
Martha B. Keagle
Introduction guanine are purines with a double-ring structure, whereas

cytosine and thymine are smaller pyrimidine molecules with
The molecule deoxyribonucleic acid (DNA) is the raw material a single ring structure. Two nitrogenous bases positioned
of inheritance and ultimately influences all aspects of the struc- side by side on the inside of the double helix form one rung
ture and functioning of the human body. A single molecule of of the molecular ladder. The sugar and phosphate groups
DNA, along with associated proteins, comprises a chromo- form the backbone or outer structure of the helix. The fifth
some. Chromosomes are located in the nuclei of all human (5¢) carbon of one deoxyribose molecule and the third (3¢)
cells (with the exception of mature red blood cells), and each carbon of the next deoxyribose are joined by a covalent phos-
human cell contains 23 different pairs of chromosomes. phate linkage. This gives each strand of the helix a chemical
Genes are functional units of genetic information that orientation with the two strands running opposite or antipar-
reside on each of the 23 pairs of chromosomes. These units allel to one another.
are linear sequences of nitrogenous bases that code for pro- Biochemical analyses performed by Erwin Chargaff
tein molecules necessary for the proper functioning of the showed that the nitrogenous bases of DNA were not present
body. The genetic information contained within the chromo- in equal proportions and that the proportion of these bases
somes is copied and distributed to newly created cells during varied from one species to another [3]. Chargaff noted, how-
cell division. The structure of DNA provides the answer to ever, that concentrations of guanine and cytosine were always
how it is precisely copied with each cell division and to how equal, as were the concentrations of adenine and thymine.
proteins are synthesized. This finding became known as Chargaff’s rule. Watson and
Crick postulated that in order to fulfill Chargaff’s rule and to
maintain a uniform shape to the DNA molecule, there must be
DNA Structure a specific complementary pairing of the bases: adenine must
always pair with thymine, and guanine must always pair with
James Watson and Francis Crick elucidated the molecular cytosine. Each strand of DNA, therefore, contains a nucle-
structure of DNA in 1953 using X-ray diffraction data col- otide sequence that is complementary to its partner. The link-
lected by Rosalind Franklin and Maurice Wilkins, and model age of these complementary nitrogenous base pairs holds the
building techniques advocated by Linus Pauling [1, 2]. antiparallel strands of DNA together. Two hydrogen bonds
Watson and Crick proposed the double helix: a twisted, spi- link the adenine and thymine pairs, whereas three hydrogen
ral ladder structure consisting of two long chains wound bonds link the guanine and cytosine pairs (Fig. 2.1). The
around each other and held together by hydrogen bonds. complementarity of DNA strands is what allows the molecule
DNA is composed of repeating units—the nucleotides. Each to replicate faithfully. The sequence of bases is critical for
nucleotide consists of a deoxyribose sugar, a phosphate DNA function because genetic information is determined by
group, and one of four nitrogen-containing bases: adenine the order of the bases along the DNA molecule.
(A), guanine (G), cytosine (C), or thymine (T). Adenine and
DNA Synthesis
M.B. Keagle, M.Ed. (*)
Department of Allied Health, College of Agriculture and Natural
The synthesis of a new molecule of DNA is called replica-
Resources, University of Connecticut,
358 Mansfield Road, Unit 2101, Storrs, CT 06269, USA tion. This process requires many enzymes and cofactors. The
e-mail: [email protected] first step of the process involves breakage of the hydrogen
10 M.B. Keagle
enzyme DNA primase uses ribonucleotides to form a ribo-

nucleic acid primer.
The structure of ribonucleic acid (RNA) is similar to that
of DNA, except that each nucleotide in RNA has a ribose
sugar instead of deoxyribose and the pyrimidine thymine is
replaced by another pyrimidine, uracil (U). RNA also dif-
fers from DNA in that it is a single-stranded molecule. This
RNA primer is at the beginning of each Okazaki segment to
be copied, provides a 3¢-hydroxyl group, and is important
for the efficiency of the replication process. The ribonucleic
acid primer then attracts DNA polymerase I. DNA poly-
merase I brings in the nucleotides and also removes the
RNA primer and any mismatches that occur during the pro-
cess. Okazaki fragments are later joined by the enzyme
DNA ligase. The process of replication is semiconservative
because the net result is creation of two identical DNA mol-
ecules, each consisting of a parent DNA strand and a newly
synthesized DNA strand. The new DNA molecule grows as
hydrogen bonds form between the complementary bases
(Fig. 2.2).
Protein Synthesis
Fig. 2.1 DNA structure. Schematic representation of a DNA double The genetic information of DNA is stored as a code; a linear
helix unwound to show the complementarity of bases and the antiparal- sequence of nitrogenous bases in triplets. These triplets code
lel structure of the phosphate (P) and sugar (S) backbone strands for specific amino acids that are subsequently linked together
to form protein molecules. The process of protein synthesis
involves several types of ribonucleic acid.
bonds that hold the DNA strands together. DNA helicases The first step in protein synthesis is transcription. During
and single-strand binding proteins work to separate the this process, DNA is copied into a complementary piece of
strands and keep the DNA exposed at many points along the messenger RNA (mRNA). Transcription is controlled by the
length of the helix during replication. The area of DNA at the enzyme RNA polymerase, which functions to link ribonucle-
active region of separation is a Y-shaped structure referred to otides together in a sequence complementary to the DNA
as a replication fork. These replication forks originate at template strand. The attachment of RNA polymerase to a
structures called replication bubbles, which, in turn, are at promoter region, a specific sequence of bases that varies
DNA sequences called replication origins. The molecular from gene to gene, starts transcription. RNA polymerase
sequence of the replication origins has not been completely moves off the template strand at a termination sequence to
characterized. Replication takes place on both strands, but complete the synthesis of an mRNA molecule (Fig. 2.3).
nucleotides can only be added to the 3¢ end of an existing Messenger RNA is modified at this point by the removal
strand. The separated strands of DNA serve as templates for of introns—segments of DNA that do not code for an mRNA
production of complementary strands of DNA following product. In addition, some nucleotides are removed from the
Chargaff’s rules of base pairing. 3¢ end of the molecule, and a string of adenine nucleotides
The process of DNA synthesis differs for the two strands are added. This poly(A) tail helps in the transport of mRNA
of DNA because of its antiparallel structure. Replication is molecules to the cytoplasm. Another modification is the
straightforward on the leading strand. The enzyme DNA addition of a cap to the 5¢ end of the mRNA, which serves to
polymerase I facilitates the addition of complementary aid in attachment of the mRNA to the ribosome during trans-
nucleotides to the 3¢ end of a newly forming strand of DNA. lation. These alterations to mRNA are referred to as mRNA
In order to add further nucleotides, DNA polymerase I processing (Fig. 2.4). At this point, mRNA, carrying the
requires the 3¢-hydroxyl end of a base-paired strand. information necessary to synthesize a specific protein, is
DNA synthesis on the lagging strand is accomplished by transferred from the nucleus into the cytoplasm of the cell,
the formation of small segments of nucleotides called where it then associates with ribosomes. Ribosomes, com-
Okazaki fragments [4]. After separation of the strands, the posed of ribosomal RNA (rRNA) and protein, are the site of
2 DNA, Chromosomes, and Cell Division 11
Leading Strand
3’
Direction of Replication
5’ Okazaki DNA and RNA

Fragments Polymerases
3’
5’
Lagging
Strand
3’
5’ DNA and RNA

DNA Polymerases
3’ Ligase
5’
Fig. 2.2 Semiconservative replication. Complementary nucleotides are added directly to the 3¢ end of the leading strand, whereas the lagging
strand is copied by the formation of Okazaki fragments
protein synthesis. Ribosomes consist of two subunits that Ribosomes read mRNA one codon at a time. Transfer
come together with mRNA to read the coded instructions on RNA molecules transfer the specific amino acids to the syn-
the mRNA molecule. thesizing protein chain (Fig. 2.5). The amino acids are joined
The next step in protein synthesis is translation. A chain to this chain by peptide bonds. This process is continued
of amino acids is synthesized during translation by using the until a stop codon is reached. The new protein molecule is
newly transcribed mRNA molecule as a template, with the then released into the cell milieu and the ribosomes split
help of a third ribonucleic acid, transfer RNA (tRNA). Leder apart (Fig. 2.6).
and Nirenberg and Khorana determined that three nitrogen
bases on an mRNA molecule constitute a codon [5, 6]. With
four nitrogenous bases, there are 64 possible three-base DNA Organization
codons. Sixty-one of these code for specific amino acids, and
the other three are “stop” codons that signal the termination Human chromatin consists of a single continuous molecule of
of protein synthesis. There are only 20 amino acids, but 61 DNA complexed with histone and nonhistone proteins. The
codons. Therefore, most amino acids are coded for by more DNA in a single human diploid cell, if stretched out, would be
than one mRNA codon. This redundancy in the genetic code approximately 2 m in length and therefore must be condensed
is referred to as degeneracy. considerably to fit within the cell nucleus [7]. There are sev-
Transfer RNA molecules contain “anticodons”—nucle- eral levels of DNA organization that allow for this.
otide triplets that are complementary to the codons on The DNA helix itself is the first level of condensation.
mRNA. Each tRNA molecule has attached to it the specific Next, two molecules of each of the histones H2A, H2B, H3,
amino acid for which it codes. and H4 form a protein core: the octamer. The DNA double
12 M.B. Keagle
Fig. 2.3 Transcription. A DNA

molecule is copied into mRNA
with the help of RNA polymerase
helix winds twice around the octamer to form a 10-nm Chromosome Structure
nucleosome, the basic structural unit of chromatin. Adjacent
nucleosomes are pulled together by a linker segment of the A chromosome consists of two sister chromatids, each of
histone H1. Repeated, this gives the chromatin the appear- which is comprised of a contracted and compacted double
ance of “beads on a string.” Nucleosomes are further coiled helix of DNA. The centromere, telomere, and nucleolar orga-
into a 30-nm solenoid, with each turn of the solenoid con- nizer regions are functionally differentiated areas of the
taining about six nucleosomes. The solenoids are packed chromosomes (Fig. 2.8).
into DNA looped domains attached to a nonhistone protein
matrix. Attachment points of each loop are fixed along the
DNA. The looped domains coil further to give rise to highly The Centromere
compacted units, the chromosomes, which are visible with
the light microscope only during cell division. Chromosomes The centromere is a constriction visible on metaphase chro-
reach their greatest extent of condensation during mitotic mosomes where the two sister chromatids are joined together.
metaphase (Fig. 2.7). The centromere is essential to the survival of a chromosome
rRNA. In humans, there are theoretically ten nucleolar

organizer regions, although all may not be active during any
given cell cycle.
The Telomeres
The telomeres are the physical ends of chromosomes.

Telomeres act as protective caps to chromosome ends, pre-
venting end-to-end fusion of chromosomes and DNA degra-
dation resulting after chromosome breakage. Nonhistone
proteins complex with telomeric DNA to protect the ends of
chromosomes from nucleases located within the cell [9]. The
telomeric region also plays a role in synapsis during meiosis.
Chromosome pairing appears to be initiated in the subtelo-
meric regions [10].
Telomeres contain tandem repeats of the nitrogenous
base sequence TTAGGG over 3–20 kb at the chromosome
ends [11]. At the very tip of the chromosome, the two strands
do not end at the same point, resulting in a short G-rich tail
that is single stranded. Because of this, DNA synthesis
breaks down at the telomeres and telomeres replicate differ-
ently than other types of linear DNA. The enzyme telom-
erase synthesizes new copies of the telomere TTAGGG
repeat using an RNA template that is a component of the
telomerase enzyme. Telomerase also counteracts the pro-
gressive shortening of chromosomes that results from many
Fig. 2.4 Messenger RNA processing. The transcribed strand of DNA cycles of normal DNA replication. Telomere length gradu-
is modified to produce a mature mRNA transcript ally decreases with the aging process and with increased
numbers of cell divisions in culture. The progressive short-
during cell division. Interaction with the mitotic spindle dur- ening of human telomeres appears to be a tumor-suppressor
ing cell division occurs at the centromeric region. Mitotic mechanism [12]. The maintenance of telomeric DNA per-
spindle fibers are the functional elements that separate the mits the binding of telomeric proteins that form the protec-
sister chromatids during cell division. tive cap at chromosome ends and regulate telomere length
Human chromosomes are classified based on the position [12]. Cells that have defective or unstable telomerase will
of the centromere on the chromosome. The centromere is exhibit shortening of chromosomes, leading to chromosome
located near the middle in metacentric chromosomes, near instability and cell death.
one end in acrocentric chromosomes, and between the middle
and end in submetacentric chromosomes. The kinetochore
apparatus is a complex structure consisting of proteins that Types of DNA
function at the molecular level to attach the chromosomes to
the spindle fibers during cell division. Although the kineto- DNA is classified into three general categories: unique
chore is located in the region of the centromere, it should not sequence, highly repetitive sequence DNA (>105 copies),
be confused with the centromere. The latter is the DNA at the and middle repetitive sequence DNA (102–104 copies).
site of the spindle-fiber attachment. Unique sequence or single-copy DNA is the most common
class of DNA, comprising about 75% of the human genome
[13]. This DNA consists of nucleotide sequences that are
The Nucleolar Organizer Regions represented only once in a haploid set. Genes that code for
proteins are single-copy DNA. Repetitive or repeated
The satellite stalks of human acrocentric chromosomes consequence DNA makes up the remaining 25% of the genome
tain the nucleolar organizer regions (NORs), so-called and is classified according to the number of repeats and
because this is where nucleoli form in interphase cells. NORs whether the repeats are tandem or interspersed among unique
are also the site of ribosomal RNA genes and production of sequence DNA [13].
14 M.B. Keagle
Fig. 2.5 Translation. Transfer RNA molecules bring in specific amino acids according to the triplet codon instructions of mRNA that are read at
the ribosomes
Repetitive, tandemly arranged DNA was first discovered The genes for 18S and 28S ribosomal RNAs are middle
with a cesium chloride density gradient. Repetitive, tandem repetitive sequences. Several hundred copies of these genes
sequences were visualized as separate bands in the gradient. are tandemly arranged on the short arms of the acrocentric
This DNA was termed satellite DNA [14]. Satellite DNA is chromosomes.
categorized, based on the length of sequences that make up Dispersed repetitive DNA is classified as either short or
the tandem array and the total length of the array, as a long. The terms SINEs (short interspersed elements) and
(alpha)-satellite, minisatellite, and microsatellite DNA. LINEs (long interspersed elements) were introduced by
Alpha-satellite DNA is a repeat of a 171-base pair Singer [17]. SINEs range in size from 90 to 500 base pairs.
sequence organized in a tandem array of up to a million base One class of SINEs is the Alu sequence. Many Alu sequences
pairs or more in total length. Alpha-satellite DNA is gener- are transcribed and are present in nuclear pre-mRNA and in
ally not transcribed and is located in the heterochromatin some noncoding regions of mRNA. Alu sequences have high
associated with the centromeres of chromosomes (see later). G-C content and are found predominantly in the Giemsa-
The size and number of repeats of satellite DNA is chromo- light bands of chromosomes [18]. LINEs can be as large as
some specific [15]. Although a-satellite DNA is associated 7,000 bases. The predominant member of the LINE family is
with centromeres, its role in centromere function has not a sequence called L1. L1 sequences have high A-T content
been determined. A centromeric protein, CENP-B, has been and are predominantly found in the Giemsa-dark bands of
shown to bind to a 17-base pair portion of some a-satellite chromosomes [17]. See Chaps. 3 and 4.
DNA, but the functional significance of this has not been
determined [16].
Minisatellites have repeats that are 20–70 base pairs in Chromatin
length, with a total length of a few thousand base pairs.
Microsatellites have repeat units of two, three, or four base There are two fundamental types of chromatin in eukaryotic
pairs, and the total length is usually less than a few hundred cells: euchromatin and heterochromatin. Euchromatin is
base pairs. Minisatellites and microsatellites vary in length loosely organized, extended, and uncoiled. This chromatin
among individuals and, as such, are useful markers for gene contains active, early replicating genes, and stains lightly
mapping and identity testing. with GTG-banding techniques (see Chap. 4).
Fig. 2.6 Overview of protein

synthesis. DNA is transcribed to
mRNA, which is modified to
mature transcript and then
transferred to the cytoplasm of
the cell. The codons are read at
the ribosomes and translated with
the help of tRNA. The chain of
amino acids produced during
translation is joined by peptide
bonds to form a protein molecule
There are two special types of heterochromatin that war- The heterochromatic regions of these chromosomes stain
rant special mention: facultative heterochromatin and consti- differentially with various special staining techniques, revealing
tutive heterochromatin. Both are genetically inactive, late that the DNA structure of these regions is not the same as the
replicating during the synthesis (S) phase of mitosis, and are structure of the euchromatic regions on the same chromosomes.
highly contracted. The only established function of constitutive heterochromatin
is the regulation of crossing-over—the exchange of genes from
one sister chromatid to the other during cell division [19].
Constitutive Heterochromatin
Constitutive heterochromatin consists of simple repeats of Facultative Heterochromatin

nitrogenous bases that are generally located around the cen-
tromeres of all chromosomes and at the distal end of the Y One X chromosome of every female cell is randomly inacti-
chromosome. There are no transcribed genes located in consti- vated. The inactivated X is condensed during interphase and
tutive heterochromatin, which explains the fact that variations replicates late during the synthesis stage of the cell cycle. It
in constitutive heterochromatic chromosome regions appar- is termed facultative heterochromatin. Because these regions
ently have no effect on the phenotype. Chromosomes 1, 9, 16, are inactivated, it has been proposed that facultative hetero-
and Y have variably sized constitutive heterochromatic regions. chromatin regulates gene function [20].
16 M.B. Keagle
Fig. 2.7 The levels of DNA organization (Reprinted with permission from Jorde et al. [8])
The Cell Cycle

Cell Division
The average mammalian cell cycle lasts about 17–18 h and is
An understanding of cell division is basic to an understanding of the transition of a cell from one interphase through cell divi-
cytogenetics. Dividing cells are needed in order to study chro- sion and back to interphase [21]. The cell cycle is divided into
mosomes using traditional cytogenetic techniques, and many four major stages. The first three stages, gap 1 (G1), synthesis
cytogenetic abnormalities result from errors in cell division. (S), and gap 2 (G2), comprise interphase. The fourth and final
There are two types of cell division: mitosis and meiosis. stage of the cell cycle is mitosis (M) (Fig. 2.9).
Mitosis is the division of somatic cells, whereas meiosis is a The first stage, G1, is the longest and typically lasts about
special type of division that occurs only in gametic cells. 9 h [21]. Chromosomes exist as single chromatids during this
Fig. 2.8 The functional and Metacentric Submetacentric Acrocentric

structural components of
metaphase chromosomes Telomere Satellites
Short arm (p) Stalks
Centromere
Long arm (q)
Chromatids
The final step in the cell cycle is mitosis. This stage lasts
only 1–2 h in most mammalian cells. Mitosis is the process
by which cells reproduce themselves, creating two daughter
cells that are genetically identical to one another and to the
original parent cell. Mitosis is itself divided into stages
(Fig. 2.10).
Mitosis
Prophase
Chromosomes are at their greatest elongation and are not

visible as discrete structures under the light microscope
during interphase. During prophase, chromosomes begin to
Fig. 2.9 The cell cycle: gap 1, synthesis, gap 2, and mitosis coil, become more condensed, and begin to become visible
as discrete structures. Nucleoli are visible early in prophase
but disappear as the stage progresses.
stage. Cells are metabolically active during G1, and this is
when protein synthesis takes place. A cell might be per-
manently arrested at this stage if it does not undergo further Prometaphase
division. This arrested phase is referred to as gap zero (G0).
Gap 1 is followed by the synthesis phase, which lasts Prometaphase is a short period between prophase and
about 5 h in mammalian cells [21]. This is when DNA synthesis metaphase during which the nuclear membrane disappears
occurs. The DNA replicates itself, and the chromosomes and the spindle fibers begin to appear. Chromosomes attach
then consist of two identical sister chromatids. to the spindle fibers at their kinetochores.
Some DNA replicates early in S phase, and some repli-
cates later. Early replicating DNA contains a higher portion
of active genes than late-replicating DNA. By standard Metaphase
G-banding techniques, the light-staining bands usually replicate
early, whereas the dark-staining bands and the inactive X During metaphase, the mitotic spindle is completed, the
chromosome in females replicate late in the S phase. centrioles divide and move to opposite poles, and the chromo-
Gap 2 lasts about 3 h [21]. During this phase, the cell somes line up on the equatorial plate. Chromosomes reach their
prepares to undergo cell division. The completion of G2 maximum state of contraction during this phase. It is metaphase
represents the end of interphase. chromosomes that are traditionally studied in cytogenetics.
18 M.B. Keagle
Fig. 2.10 Mitosis. Schematic

representation of two pairs of
chromosomes undergoing cell
division: (a) interphase,
(b) prophase, (c) metaphase,
(d) anaphase, (e) telophase,
(f) cytokinesis, and (g) interphase
of the next cell cycle
Anaphase of mitosis are two genetically identical daughter cells, each

of which contains the complete set of genetic material that
Centromeres divide longitudinally and the chromatids sepa- was present in the parent cell. The two daughter cells enter
rate during this stage. Sister chromatids migrate to opposite interphase, and the cycle is repeated.
poles as anaphase progresses.
Meiosis
Telophase
Meiosis takes place only in the ovaries and testes. A process
The final stage of mitosis is telophase. The chromosomes involving one duplication of the DNA and two cell divisions
uncoil and become indistinguishable again, the nucleoli (meiosis I and meiosis II) reduces the number of chromosomes
reform, and the nuclear membrane is reconstructed. Telophase from the diploid number (2n = 46) to the haploid number (n = 23).
is usually followed by cytokinesis, or cytoplasmic division. Each gamete produced contains only one copy of each chromo-
Barring errors in DNA synthesis or cell division, the products some. Fertilization restores the diploid number in the zygote.
Meiosis I
Meiosis I is comprised of several substages: prophase I,

metaphase I, anaphase I, and telophase I (Fig. 2.11).
Prophase I
Prophase I is a complex stage that is further subdivided as
follows.
Leptotene
In leptotene, there are 46 chromosomes, each comprised of
two chromatids. The chromosomes begin to condense but are
not yet visible by light microscopy. Once leptotene takes
place, the cell is committed to meiosis.
Zygotene
Zygotene follows leptotene. Homologous chromosomes,
which in zygotene appear as long thread-like structures, pair
locus for locus. This pairing is called synapsis. A tripartite
structure, the synaptonemal complex, can be seen with elec-
tron microscopy. The synaptonemal complex is necessary
for the phenomenon of crossing-over that will take place
later in prophase I.
Synapsis of the X and Y chromosomes in males occurs
only at the pseudoautosomal regions. These regions are
located at the distal short arms and are the only segments of
the X and Y chromosomes containing homologous loci. The
nonhomologous portions of these chromosomes condense to Fig. 2.11 Schematic representation of two chromosome pairs
form the sex vesicle. undergoing meiosis I: (a) prophase I, (b) metaphase I, (c) anaphase I,
(d) telophase I, and (e) products of meiosis I
Pachytene
Synapsis is complete during pachytene. Chromosomes con-
tinue to condense and now appear as thicker threads. The Diakinesis
paired homologs form structures called bivalents, sometimes Chromosomes reach their greatest contraction during this
referred to as tetrads because they are composed of four last stage of prophase.
chromatids.
The phenomenon of crossing over takes place during Metaphase I
pachytene. Homologous or like segments of DNA are Metaphase I is characterized by disappearance of the nuclear
exchanged between nonsister chromatids of the bivalents. membrane and formation of the meiotic spindle. The biva-
The result of crossing over is a reshuffling or recombination lents line up on the equatorial plate with their centromeres
of genetic material between homologs, creating new combi- randomly oriented toward opposite poles.
nations of genes in the daughter cells.
Anaphase I
Diplotene During anaphase I, the centromeres of each bivalent separate
In diplotene, chromosomes continue to shorten and thicken, and migrate to opposite poles.
and the homologous chromosomes begin to repel each other.
This repulsion continues until the homologous chromosomes Telophase I
are held together only at points where crossing-over took In telophase, the two haploid sets of chromosomes reach
place. These points are referred to as chiasmata. In males, the opposite poles, and the cytoplasm divides. The result is two
sex vesicle disappears, and the X and Y chromosomes asso- cells containing 23 chromosomes, each comprised of two
ciate end to end. chromatids.
20 M.B. Keagle
Meiosis II
The cells move directly from telophase I to metaphase II

with no intervening interphase or prophase. Meiosis II pro-
ceeds much like mitotic cell division except that each cell
contains only 23 chromosomes (Fig. 2.12).
The 23 chromosomes line up on the equatorial plate in
metaphase II, the chromatids separate and move to opposite
poles in anaphase II, and cytokinesis occurs in telophase II.
The net result is four cells, each of which contains 23 chro-
mosomes, each consisting of a single chromatid. Owing to
the effects crossing-over and random assortment of homologs,
each of the new cells differs genetically from one another
and from the original cell.
Spermatogenesis and Oögenesis
The steps of spermatogenesis and oögenesis are the same in

human males and females; however, the timing is very differ-
ent (Fig. 2.13).
Spermatogenesis
Spermatogenesis takes place in the seminiferous tubules of
the male testes. The process is continuous and each meiotic
cycle of a primary spermatocyte results in the formation of
four nonidentical spermatozoa. Spermatogenesis begins with
sexual maturity and occurs throughout the postpubertal life
of a man.
The spermatogonia contain 46 chromosomes. Through
mitotic cell division, they give rise to primary spermatocytes.
The primary spermatocytes enter meiosis I and give rise to
the secondary spermatocytes, which contain 23 chromo-
somes, each consisting of two chromatids. The secondary
spermatocytes undergo meiosis II and give rise to sperma- Fig. 2.12 Schematic representation of two chromosome pairs under-
tids. Spermatids contain 23 chromosomes, each consisting of going meiosis II: (a) products of meiosis I, (b) metaphase II, (c) ana-
phase II, (d) telophase II, and (e) products of meiosis
a single chromatid. The spermatids differentiate to become
spermatozoa, or mature sperm.
Oögenesis which has been ovulated, begins meiosis II. Meiosis II con-
Oögenesis in human females begins in prenatal life. Ova tinues only if fertilization takes place. The completion of
develop from oögonia within the follicles in the ovarian cor- meiosis II results in a haploid ovum and a second polar body.
tex. At about the third month of fetal development, the oögo- The first polar body might undergo meiosis II, or it might
nia, through mitotic cell division, begin to develop into degenerate. Only one of the potential four gametes produced
diploid primary oöcytes. Meiosis I continues to diplotene, each menstrual cycle is theoretically viable.
where it is arrested until sometime in the postpubertal repro-
ductive life of a woman. This suspended diplotene is referred Fertilization
to as dictyotene. The chromosomes of the egg and sperm produced in mei-
Subsequent to puberty, several follicles begin to mature osis II are each surrounded by a nuclear membrane within
with each menstrual cycle. Meiosis I rapidly proceeds with the cytoplasm of the ovum and are referred to as pronu-
an uneven distribution of the cytoplasm in cytokinesis of clei. The male and female pronuclei fuse to form the dip-
meiosis I, resulting in a secondary oöcyte containing most of loid nucleus of the zygote, and the first mitotic division
the cytoplasm, and a first polar body. The secondary oöcyte, begins.
Fig. 2.13 Spermatogenesis and Spermatogenesis Oögenesis

oögenesis. The events of
spermatogenesis and oögenesis 2n 2n
are the same, but the timing and
net results are different. Oögenesis
begins prenatally and is arrested Spermatogonia mitosis Oögonia
in meiosis I until the postpubertal
life of a woman; spermatogenesis
begins with the sexual maturity of Primary Primary
the male and is continuous. Each Spermatocyte 2n 2n Oöcyte
meiosis I
cycle of spermatogenesis results arrest
in four functional gametes, while
each cycle of oögenesis results in Secondary Secondary
a single egg Spermatocytes n Oöcyte &
n n meiosis II n 1st Polar Body
Spermatids Ovum (egg) &

2nd Polar Body
n n n n n n
differentiation
Spermatozoa
(sperm)
n n n n
11. Moyzis RK, Buckingham JM, Cram LS, Dani M, Deaven LL, Jones
References MD, Meyne J, Ratcliffe RL, Wu J. A highly conserved repetitive
DNA sequence, (TTAGGG)n present at the telomeres of human
1. Watson JD, Crick FHC. A structure for deoxyribose nucleic acid. chromosomes. Proc Natl Acad Sci USA. 1988;85:6622–6.
Nature. 1953;171:737–8. 12. Smith S, De Lange T. TRF1, a mammalian telomeric protein.
2. Watson JD, Crick FHC. The structure of DNA. Cold Spring Harb Trends Genet. 1997;13:21–6.
Symp Quant Biol. 1953;18:123–31. 13. Spradling A, Penman S, Campo MS, Bishop JO. Repetitious and
3. Chargaff E. Structure and function of nucleic acids as cell constitu- unique sequences in the heterogeneous nuclear and cytoplasmic mes-
ents. Fed Proc. 1951;10:654–9. senger RNA of mammalian and insect cells. Cell. 1974;3:23–30.
4. Okazaki R, Okazaki T, Sakabe K, Sugimoto K, Sugino A. 14. Hsu TC. Human and mammalian cytogenetics: an historical per-
Mechanism of DNA chain growth, I. Possible discontinuity and spective. New York: Springer; 1979.
unusual secondary structure of newly synthesized chains. Proc Natl 15. Willard HF, Waye JS. Hierarchal order in chromosome-specific
Acad Sci USA. 1968;59:598–605. human alpha satellite DNA. Trends Genet. 1987;3:192–8.
5. Leder P, Nirenberg M. RNA codewords and protein synthesis. 16. Willard HF. Centromeres of mammalian chromosomes. Trends
Science. 1964;145:1399–407. Genet. 1990;6:410–6.
6. Khorana HG. Synthesis in the study of nucleic acids. Biochem J. 17. Singer MF. SINEs and LINEs: highly repeated short and long inter-
1968;109:709–25. spersed sequences in mammalian genomes. Cell. 1982;28:433–4.
7. Sharma T, editor. Trends in chromosome research. New Delhi: 18. Korenberg JR, Rykowski MC. Human genome organization: Alu,
Narosa Publishing House; 1990. LINEs and the molecular structure of metaphase chromosome
8. Jorde LB, Carey JC, Bamshad MJ, White RL. Chapter 2. Basic cell bands. Cell. 1988;53:391–400.
biology: structure and function of genes and chromosomes. In: 19. Miklos G, John B. Heterochromatin and satellite DNA in man:
Medical genetics. 3rd ed. St. Louis: Mosby/Elsevier; 2006. p. 9. properties and prospects. Am J Hum Genet. 1979;31:264–80.
9. Zakian VA. Structure and function of telomeres. Annu Rev Genet. 20. Therman E, Susman M. Human chromosomes: structure, behavior,
1989;23:579–604. and effects. New York: Springer; 1993.
10. Lese CM, Ledbetter DH. The means to an end: exploring human 21. Barch MJ, Knutsen T, Spurbeck JL, editors. The AGT cytogenetic
telomeres. J Assoc Genet Technol. 1998;24(5):165–70. laboratory manual. Philadelphia: Raven-Lippincott; 1997.
Human Chromosome Nomenclature:
An Overview and Definition of Terms 3
Marilyn L. Slovak, Aaron Theisen, and Lisa G. Shaffer
ISCN [2] is one of several “dog-eared” books within an arm’s

Introduction reach of every practicing clinical cytogeneticist.
For those unfamiliar with the language of cytogenetics,
Science is about generating, interpreting, and communicat-
the deceptively simple act of communication can be a barrier
ing information. The need to establish a common language
to understanding or becoming involved in the field. The goal
or communication tool to describe human chromosomes and
of this chapter is to present the general concepts of the ISCN
chromosomal aberrations associated with human disease
and offer guidance on writing abnormal cytogenetic results
became apparent in the mid-1950s soon after Tjio and Levan
by providing nomenclature examples. It is not intended to
reported that the correct chromosome number in humans was
replace the latest edition of the ISCN. To get started, an easy-
46 [1]. Variations in chromosome number and structure were
reference glossary of the most commonly used ISCN terms
quickly associated with multiple congenital anomalies, intel-
may be found at the end of this chapter. After mastering the
lectual disabilities, and cancer. To effectively describe chro-
lingua franca of cytogeneticists offered in this primer, ISCN
mosomal changes in a systematic manner, a group of 17
[2] should be consulted to sharpen human nomenclature
forward-thinking investigators who had previously published
skills and extend proficiency to include chromosome break-
human karyotypes teamed up in Denver, Colorado, in 1960
age and meiotic chromosome nomenclature (Chaps. 10 and
to create the foundation of the celebrated communication
12, respectively, in ISCN [2]).
tool known today as An International System for Human
Cytogenetic Nomenclature or ISCN. ISCN is an abbreviated
symbolic writing method used to describe genetic changes
Visualizing Human Chromosomes
by copy number (dosage) and position (locus). This interna-
tional language allows cytogeneticists to describe the results
Human cells have 23 pairs of chromosomes (22 pairs of
of cytogenetic-based assays, communicate across cultures
autosomes and one pair of sex chromosomes) for a total of
and languages, create databases, publish scientific activities,
46 chromosomes per cell (Fig. 3.1). The autosomes are
and foster collaborations worldwide. To keep the nomencla-
assigned a number (1–22) based on size (with one exception;
ture current with the latest technological advances without
chromosome 22 is slightly larger than chromosome 21). The
losing the foundation on which it was built, the ISCN recom-
sex chromosomes are noted by the letters X and Y. The
mendations are updated periodically by an elected standing
female sex chromosome complement is XX and the male
committee with global representation; ten conferences have
complement is XY. Chromosomes are divided into long and
been held since its first printing in 1960. The latest version of
short arms, separated by a centromere, or primary constric-
tion. A chromosome may be metacentric, with its centrom-
ere in the middle; submetacentric, with the centromere closer
M.L. Slovak, Ph.D. (*) to one end of the chromosome; or acrocentric, in which the
Cytogenetics Laboratory, Palo Verde Laboratory/Sonora Quest
centromere is near one end of the chromosome and the short
Laboratories, 1255 W. Washington St., Tempe, AZ 85281, USA
e-mail: [email protected] arm is essentially comprised of repetitive DNA that consti-
tutes the satellites and nucleolar organizing regions. Chromo-
A. Theisen
Theisen Consulting, Spokane, WA, USA somes 1 and 3 are examples of metacentric chromosomes,
chromosomes 4 and 5 are large submetacentric chromosomes,
L.G. Shaffer, Ph.D.
Signature Genomic Laboratories, and chromosomes 13–15 are considered medium sized acro-
2820 North Astor, Spokane, WA 99207, USA centric chromosomes.
24 M.L. Slovak et al.
Fig. 3.1 G-banded normal male karyotype illustrating the characteristic size, centromere position, and G-banding banding pattern for each human
chromosome pair
Chromosome Banding and Identification heritable features (Table 3.1; see also Chap. 4). To identify
each chromosome in the human karyotype, it is important to
Launched in the early 1970s, banding methods allow for the be familiar with the characteristic morphological features or
identification of chromosomes not only by length and cen- landmarks of each chromosome, such as the telomeres that
tromere position, but also by their unique banding properties. cap the ends of the chromosomes, the centromere or “pri-
Figure 3.1 illustrates the characteristic size, centromere posi- mary constriction” that divides a chromosome into two
tion, and G-banding banding pattern for each human chro- arms, and certain defined bands (Fig. 3.2). The symbols p
mosome pair. The commonly used G-, Q-, and R-banding and q are used to designate the short and long arms, respec-
techniques show bands distributed along the entire chromo- tively. Convention places the short arm or “p” (from the
some, whereas the C-, T-, or NOR-banding techniques are French petite) arm at the top in diagrammatic representa-
used to identify specific chromosome structures that are tions and the long or “q” arm at the bottom. Characteristic
3 Human Chromosome Nomenclature: An Overview and Definition of Terms 25
Table 3.1 Commonly used banding and staining techniques in human cytogenetics
Commonly used banding and staining techniques in human cytogenetics
Q Q-banding—a fluorescent stain (quinacrine dihydrochloride) produces specific banding patterns for each pair of homologous
chromosomes similar to G-banding, excellent for identifying centromeric regions of chromosomes 3, 4, and 13, some
acrocentric chromosomes and the Y chromosome. AT-rich (gene poor) regions fluoresce brightly with Q-banding
QF Q-bands by fluorescence
QFQ Q-bands by fluorescence using quinacrine
QFH Q-bands by fluorescence using Hoechst 33258 dye
G G-banding—Giemsa stain produces specific banding patterns for each pair of homologous chromosomes similar to Q-banding.
The chromosomes are treated with trypsin to partially digest the chromosome prior to being stained. AT-rich (gene poor)
regions stain darkly with G-banding
GT G-bands by trypsin
GTG G-bands by trypsin using Giemsa
GTL G-bands by trypsin using Leishman stain
GTW G-bands by trypsin using Wright stain
C C-banding—after barium hydroxide treatment, Giemsa stain is used to stain constitutive heterochromatin close to the centrom-
eres and on the long arm of the Y chromosome. C-banding is used to identify dicentric chromosomes and variations of
constitutive heterochromatin
CB C-bands by barium hydroxide
CBG C-bands by barium hydroxide using Giemsa
R R-banding—a staining method in which chromosomes are heated in a phosphate buffer and then stained to produce a banding
pattern that is the reverse of that produced with G-banding
RF R-bands by fluorescence
RFA R-bands by fluorescence using acridine orange
RH R-bands by heating
RHG R-bands by heating using Giemsa
RB R-bands by 5-bromo-2-deoxyuridine (BrdU)
RBG R-bands by BrdU using Giemsa
RBA R-bands by BrdU using acridine orange
DAPI 4¢,6-diamidino-2-phenylindole (DAPI) staining—permits characterization of AT-rich (DAPI+) or AT-poor (DAPI-) heterochro-
matic regions, especially when counterstained with chromomycin A3, which preferentially binds to GC-rich DNA
DA-DAPI DAPI-bands by Distamycin A and DAPI
NOR Nucleolus organizing region staining—a staining method utilizing silver nitrate, which preferentially accumulates in the NORs
located on the stalks of the acrocentric chromosomes that contain active ribosomal RNA genes
T T-banding—a Giemsa staining technique that stains the telomeres (ends) and the centromeres of chromosomes
regions and bands within a given chromosome are observed The gene content of chromosome bands is also variable and,
when banding techniques are used. Chromosome regions in general, reflects functionality.
refer to those areas lying between two distinct landmarks and Giemsa or G-banding is the most common banding method
are divided into bands. For example, the long arm of chro- employed in North American cytogenetics laboratories.
mosome 7 has three regions: 7q1, 7q2, and 7q3 (Fig. 3.2). G-dark (positive) bands are AT rich, gene poor, and late rep-
These regions are further subdivided into bands. A band is licating. The early replicating G-light (negative) bands are
defined as a part of the chromosome that is clearly distin- GC rich, gene rich, and late replicating. Reverse or R-banding
guishable from its adjacent segments based on its staining shows this banding pattern in reverse (i.e., reversal of light
properties. and dark G-bands). However, the numbering of the bands is
As a general rule, a chromosome band contains ~5–10 identical with both banding methods. Additional banding/
megabases (Mb) of DNA. “High-resolution” cytogenetic tech- staining methods are used to detect specific chromosome
niques (see Chap. 4) produce elongated chromosomes that regions or abnormalities. For example, centromeric and peri-
allow further refinement of karyotypic aberrations by sub- centromeric DNA are comprised of alpha-satellite and
dividing bands into smaller sub-bands. Banding resolu- various other families of repetitive satellite DNA, which
tion and patterns may vary depending on the banding are easily visualized using constitutive heterochromatin
method employed (Table 3.1), so it is important to state the (C-banding) methods. C-banding is particularly useful when
level of banding resolution and banding method employed identifying the morphologically variable heterochromatin
on the final report when describing a cytogenetic result. regions of the Y chromosome and chromosomes 1, 9, and 16.
Fig. 3.2 Characteristic morphological features of a human chromo- The centromere divides the chromosomes into the short or “p” and long
some. Chromosome 7 is used in this example. Chromosomes have or “q” arms. Each arm is divided into 1–4 regions. Each band within a
major landmarks, including the centromere or primary constriction, region is numbered centromere to telomere. Bands may be subdivided
certain bands, and the telomeres that cap both ends of the chromosomes. into sub-bands
The short arms of the acrocentric chromosomes house the Karyotype Descriptions
ribosomal RNA gene clusters in the nucleolar organizing
regions (NORs), which form the nucleolus of the cell. Karyotype descriptions convey the total number of chromo-
NORs are detected by silver-based NOR staining. Finally, somes, the sex chromosome complement, and a description
telomeres are comprised of (TTAGGG)n mini-satellite of any chromosome abnormalities present. The correct use
repeats that stain darkly with T-banding. Technical details of punctuation in the nomenclature string brings structure
of the various chromosome banding methods may be found and meaning to the description. Table 3.2 provides a quick
in Chap. 4. reference guide of the conventional cytogenetic ISCN punc-
Figure 3.3 shows the idiograms or diagrammatic repre- tuation symbols and their meaning.
sentations of the G-banding patterns for normal human chro- The description of any human karyotype begins with two
mosomes 1 and 13 at five successive levels of resolution. The basic components separated by a comma; the total number of
centromere itself is designated as “10,” with the part adjacent chromosomes is listed first, followed by the sex chromosome
to the short arm as “p10” and the part adjacent to the long complement. Thus, a normal male karyotype is written as
arm as “q10.” The bands and regions are numbered outward 46,XY, and a normal female karyotype is designated as 46,XX.
from the centromere to the telomeres. Four distinct chromo- There are a few additional basic rules to describing chro-
some units—the chromosome number, the chromosome arm, mosome aberrations:
the region number, and the band number within a region—
are needed to describe a precise location within a specific 1. As with normal karyotypes, chromosome number (or
chromosome. For example, 7q34 refers to chromosome 7, chromosome range, see later in chapter) is listed first,
long arm, region 3, band 4 (Fig. 3.2). This is referred to as followed by the sex chromosome complement and any
“seven q three four,” NOT “seven q thirty-four.” If “high- aberrations. Commas separate chromosome number, the
resolution” banding is used, the band may be further subdi- sex chromosome complement, and each abnormality from
vided using a decimal point after the band designation. one another within the nomenclature string. An exception
Having a copy of the human chromosome idiograms and a exists when a triplet abbreviation is needed before the
reference set of well-banded karyotypes representing the chromosome number (e.g., mos for mosaic, see later). In
banding methods and banding level of resolution routinely these circumstances, a space must be used after any abbre-
employed by the laboratory is helpful. viation and before the chromosome number (Table 3.3).
Fig. 3.3 Idiograms or diagrammatic representations of the G-banding dark bands represent the G-positive and bright Q-bands, with the excep-
patterns for normal human chromosomes (a) 1 and (b) 13 at five succes- tion of the variable regions. R-bands will have the reverse banding pat-
sive levels of resolution. From left to right, the chromosomes represent tern, but the numbering of the bands remains unchanged (Reproduced
a haploid karyotype of 300-, 400-, 550-, 700-, and 850-band level. The from ISCN [2] with permission from Nicole L. Chia)
Table 3.2 ISCN punctuation and significance

Description Symbol Significance
Approximate sign ~ Denotes intervals and expresses uncertainty about breakpoints, number of chromosomes, fragments, or
markers; denotes chromosome range when the exact number cannot be determined
Arrow → Denotes from → to in the detailed system
Bracket, angle <> Denotes ploidy level
Bracket, square [] Denotes number of cells in a cell line/clone
Colon, single : Break in the detailed system
Colon, double :: Break and reunion in the detailed system
Comma , Separates chromosome, numbers, sex chromosomes, and chromosome aberrations
Decimal point . Denotes sub-bands
Equal sign = Number of chiasmata
Letter “c” c Indicates a constitutional abnormality in a cancer karyotype. Always placed immediately after the
constitutional abnormality
Minus sign − Loss
Multiplication sign ´ Multiple copies of rearranged chromosomes or number of copies of a chromosomal region
Parentheses () Surround structurally altered chromosomes and breakpoints
Period . Separates various banding/staining techniques
Plus sign + Gain
Question mark ? Questionable identification of a chromosome or chromosome structure
Semicolon ; Separates altered chromosomes and breakpoints in structural rearrangements involving more than one
chromosome
Slant line, single / Separates cell lines/clones
Slant line double // Separates chimeric cell lines/clones
Underlining, single __ Used to distinguish homologous chromosomes
Table 3.3 Reporting mosaicism, chimerism, and chimerism secondary to bone marrow stem cell transplantation
Example Interpretation or rule used
mos 47,XY,+18/46,XY When present, a normal cell line is listed last
mos 45,X[20]/47,XXX[10]/46,XX[20] When several cell lines are present, size matters: the largest is presented first, then the second
largest, etc.; normal cell lines are listed last, when present
chi 46,XY[25]/46,XX[10] Largest clone is presented first in chimeras
45,X[20]/46,X,i(X)(p10)[20] In the event of equivalent clone size, numerical abnormalities are reported before structural ones
47,XX,+8[15]/47,XX,+21[15] In the event of equivalent clone size with numerical abnormalities, the cell lines are listed from
lowest to highest autosome number
47,XXX[15]/47,XX,+21[15] Clones with sex abnormalities are always reported first
46,XX[4]//46,XY[16] Four cells from the female recipient were detected along with 16 cells from the male donor
46,XX,t(8;21)(q22;q22)[5]//46,XY[15] Five female recipient cells showed an (8;21) translocation, along with 15 normal male donor cells
//46,XY[20] All 20 cells analyzed were derived from the male donor
46,XX[20]// All 20 cells analyzed were derived from the female recipient (host)
2. Sex chromosome abnormalities are listed before any 5. Letters or triplets are used to specify structurally altered
autosomal aberrations, and X chromosome abnormalities chromosomes (see Table 3.4).
are presented before those involving the Y chromosome. 6. Parentheses are used to identify chromosomes involved
3. Autosomal abnormalities follow any sex chromosome in a specific aberration. The first set of parentheses
aberration and are listed in numerical order irrespective identifies which chromosome or chromosomes are
of aberration type. Multiple structural changes of homol- involved. The second set of parentheses denotes the
ogous chromosomes are listed in alphabetical order exact chromosome band of the aberration for each of
according to their abbreviated term (e.g., a deletion the chromosomes listed in the first set of parentheses.
would be written before an insertion). In both sets of parentheses, semicolons are used to sepa-
4. If a chromosome has both numerical and structural aber- rate multiple chromosomes or bands.
rations, numerical aberrations are listed first followed by 7. If the aberration involves a sex chromosome, it is always
structural aberrations; for example, trisomy 8 is listed listed first; otherwise, the autosome with the lowest num-
before a translocation involving chromosomes 8 and 14. ber is specified first. However, if an aberration involves a
Table 3.4 Short and detailed ISCN for common cytogenetic aberrations
Aberration type Description
add Additional material of unknown origin attached to a chromosome region or band
Short add(1)(q21)
Detailed add(1)(pter→q21::?)
Description Material of unknown original attached to the long arm of chromosome 1 at band 1q21. Chromosome 1 material distal to
band q21 is lost
del Deletion or loss of chromosome material. May be either terminal or interstitial
Interstitial
Short del(7)(q22q31)
Detailed del(7)(pter→q22::q31→qter)
Description Interstitial deletion with breakage and reunion (::) of bands 7q22 and 7q31. The segment lying between those two bands
is deleted
Terminal
Short del(7)(q22)
Detailed del(7)(pter→q22:)
Description Terminal deletion with a break in band 7q22. Segment distal to 7q22 is deleted
der Derivative or structurally rearranged chromosome with an intact centromere
Short der(5)inv(5)(p13q13)del(5)(q31q33)
Detailed der(5)(pter→p13::q13→cen→p13::q13→q31::q33→qter)
Description Derivative chromosome 5 with a pericentric inversion (breakage and reunion of bands 5p13 and 5q13 and a 180° rotation
of the segment) with an interstitial deletion with breakage and reunion (::) of band 5q31 and 5q33. The segment lying
between the latter two bands is deleted
dic Dicentric chromosome has two centromeres but counted as one chromosome. There is no need to indicate that one normal
chromosome is missing.
Short 45,XY,dic(13;14)(q14;q24)
Detailed 45,XY,dic(13;14)(13pter→13q14::14q24→14pter)
Description Dicentric chromosome with breaks and reunion at bands 13q14 and 14q24. The missing chromosomes13 and 14 are not
indicated because they are replaced by the dicentric. The karyotype has one normal chromosome13, one normal
chromosome14, and the dic(13;14). The resulting net imbalance is loss of the segments distal to 13q14 and 14q24.
dup Duplication of genetic material is present. Band order indicates whether this is direct or inverted
Short dup(1)(q21q32)
Detailed dup(1)(pter→q32::q21→qter)
Description Direct duplication of the segment between bands 1q21 and 1q32
Short dup(1)(q32q21) or dup(1)(q32q21)
Detailed dup(pter→q32::q32→q21::q32→qter)
Description Inverted duplication of the segment 1q21 to 1q32. The detailed system clarifies the location of the duplicated segment
hsr Intrachromosomal homogeneously staining region indicating gene amplification
Short hsr(8)(q24.1)
Detailed hsr(8)(pter→q24.1::hsr::q24.1→qter)
Description Homogeneously staining region in band 8q24.1
ins Insertion of material from one site into another site. Band order indicates whether this is direct or inverted. May involve
one or more chromosomes.
Short ins(5)(p13q31q15)
Detailed ins(5)(pter→p13::q15→q31::p13→q15::q31→qter)
Description Inverted insertion of the long arm 5q15 to 5q31 segment into the chromosome 5 short arm at 5q13. Band orientation
within the segment is reversed with respect to the centromere, that is, 5q15 is more distal to the centromere than 5q31
inv Inversion of a chromosome segment: breakpoints may be on either side of the centromere (pericentric) or within the same
chromosome arm (paracentric)
Short inv(9)(p13q21)
Detailed inv(9)(pter→p13::q21→p13::q21→qter)
Description Pericentric inversion with breakage and reunion at bands 9p13 and 9q21
i Isochromosome, a mirror image of chromosome from its centromere
Short i(17)(q10)
Detailed i(17)(qter→q10::q10→qter)
Description Isochromosome for the entire long arm of one chromosome 17. The centromeric band q10 indicates an isochromosome of
the long arm. If band p10 was listed, the isochromosome would be comprised of the short arm. The shorter designation of
i(17q) may be used in the text but never in the nomenclature string
(continued)
Table 3.4 (continued)

Aberration type Description
r Ring chromosome
Short r(6)(p23q25)
Detailed r(6)(::p23→q25::)
Description Ring chromosome with breakage and reunion at bands 6p23 and 6q25. The segments distal to the two breakpoints are
deleted
rec Recombinant chromosome due to meiotic crossing-over. This term is only used when the parental karyotype is known
(include “mat” or “pat”); otherwise, use “der”
Short rec(6)dup(6p)inv(6)(p22.2q25.2)pat
Detailed rec(6)(pter→q25.2::p22.2→pter)pat
Unknown rec der(6)(pter→q25.2::p22.2→pter)
t Balanced translocation involving two or more chromosomes. Also use “t” to describe balanced whole-arm translocations.
See text for reporting Robertsonian translocations.
Short t(8;9;22)(p21;q34.1;q11.2)
Detailed t(8;9;22)(22qter→22q11.2::8p21→8qter;9pter→9q34.1::8p21→8pter;22pter→22q11.2::9q34.1→9qter)
Description 3-way balanced translocation where the segment distal to 8p21 is translocated to chromosome 9 at band 9q34.1, the
segment distal to 9q34.1 is translocated to chromosome 22 at band 22q11.2, and the segment distal to 22q11.2 is
translocated to chromosome 8 at band 8p21
trc Tricentric chromosome is counted as one chromosome (note chromosome count). The chromosome with the lowest
number is specified first followed by the order of appearance within this chromosome
Short 44,XY,trc(5;11;8)(q31;q13p15;q22)
Detailed 44,XY,trc(5;11;8)(5pter→q31::11q13→11p15::8q22→8pter)
Description A tricentric chromosome where band 5q31 is fused with 11q13 and 11p15 is fused with 8q22
trp Triplication of chromosome material. Orientation of the triplicated segment is only obvious using the detailed system
Short 46,XX,trp(1)(q31q21)
Detailed 46,XX,trp(1)(pter→q31::q31→q21::q21→qter)
Description Inverted triplication of the 1q21 and 1q31 segments
three-break rearrangement, such as observed in “Insertions” This is a female with a balanced paracentric inversion
(see later), the receptor chromosome is specified before (an inversion involving a single chromosome arm) of the
the donor chromosome (also see “Translocations” later). long arm of chromosome 3. One break occurred at band
8. A semicolon is used between chromosomes and break- 3q21 and the other at 3q26.2. The chromosome segment
points within sets of parentheses if two or more chromo- between those breakpoints is present but inverted 180°.
somes have been altered in a rearrangement. No There are no spaces after any commas in the description, and
semicolon is used in the second set of parentheses for there is no semicolon in the second set of parentheses because
any rearrangement that involves a single chromosome. the aberration affects a single chromosome. When normal
9. A break suspected at the interface of two bands should chromosomes are replaced by structurally altered chromo-
be assigned the higher band number or the number more somes, there is no need to record the normal chromosome as
distal to (farther from) the centromere. missing. In this case, the nomenclature implies that one
10. Different clones or cell lines are separated by a single morphologically normal chromosome 3 and one inv(3q) are
slant line (/). present in this XX karyotype with 46 chromosomes. See also
11. Square brackets [ ] are placed after the karyotype string “Inversions” later.
to designate the number of cells of each cell line or
clone. In constitutional studies, the size of the cell lines 46,Y,t(X;9)(p22.3;q22)
determines the order of presentation in the karyotype. In
This is a male karyotype showing a balanced transloca-
cancer studies, the use of square brackets is critical
tion between the X chromosome and chromosome 9. The
because multiple clones indicating clonal evolution of
breakpoints for this translocation are Xp22.3 and 9q22,
disease may be observed at varying levels, and various
respectively. The chromosomal segments distal to these
therapies may eliminate or lessen one subclone but give
breakpoints have been exchanged. Note that the normal sex
another subclone a growth advantage.
chromosome in this example is written before the X chromo-
The following are examples using these basic guidelines; refer some aberration. Semicolons are present in both sets of
also to each specific section below for additional information: parentheses because two chromosomes are involved.
46,XX,inv(3)(q21q26.2) 46,XX,ins(5;2)(q13;q22q32)
Fig. 3.4 Diagrammatic dissection of translocation nomenclature. Autosome abnormalities are listed in numerical order. The second set
A translocation is indicated by the letter “t” followed by two sets of of parentheses denotes the exact chromosome band of the aberration
parentheses. The first set of parentheses will describe the chromo- for each of the chromosomes listed in the first set of parentheses.
somes involved in the translocation. In this example, chromosomes 9 The breakpoints involved in this translocation involve bands 9q34.1
and 22 are involved. If a sex chromosome was involved, it would and 22q11.2. Semicolons are used to identify the different chromo-
be listed before any autosomal aberration with X chromosome somes and their corresponding breakpoints.
abnormalities presented before those involving the Y chromosome.
This is a female karyotype in which material from the long the chromosome number and the (optional) use of square
arm of chromosome 2 between bands q22 and q32 (donor chro- brackets to indicate the number of cells in each cell line. See
mosome) is inserted into the long arm of chromosome 5 at band also “Mosaicism and Chimerism” later.
q13 (receptor chromosome). This is a direct insertion because
the original orientation of the inserted segment has been main- 47,XX,+8,t(8;14)(q24.1;q32)
tained in its new position; that is, band 2q22 remains more prox-
This is a female with two different aberrations involving
imal, or closer, to the centromere than band 2q31. If the insertion
chromosome 8. The numerical aberration is listed before the
were inverted in the receptor chromosome 5, the ISCN would
structural aberration.
be written as ins(5;2)(q13;q32q22), indicating that band 2q22 is
now more distal to the centromere than band 2q32.
46,XY,t(9;22)(q34.1;q11.2)[16]/47,XY,+8[4]
46,X,t(X;18)(q22;p11.2) - or - 46,Y,t(X;18)(q22;p11.2) This male patient has two clones; these are separated by a
slant line (/). The first clone of 16 cells shows a translocation
These denote the same aberration, a translocation involv- between the long arms of chromosomes 9 and 22, with breaks
ing an X chromosome and chromosome 18, in either a female at bands 9q34.1 and 22q11.2, respectively. The segments
or male, respectively. The normal sex chromosome is listed distal to the breakpoints have been exchanged (Fig. 3.4). The
before the rearranged one. second clone of four cells shows gain of chromosome 8 as
the sole clonal aberration. The abnormalities observed in the
46,t(X;18)(q22;p11.2),t(Y;13)(q11.2;q12) first clone are not seen in the second clone and vice versa.
If both the X and Y chromosome are involved in aberra- This situation is seen in neoplasia; see “Describing Cancer
tions, the abnormality involving the X chromosome is listed Karyotypes” later.
before that of the Y chromosome. Constitutional and acquired (neoplastic) karyotypes may
show a tremendous range of structural abnormalities. ISCN
mos 45,X[25]/46,XX[15] allows for both an abbreviated or short system as well as a
detailed system of nomenclature. Whenever possible, use of
This describes a mosaic karyotype with one cell line the short system is strongly encouraged; all examples to this
showing a single X chromosome in 25 cells and a second cell point are written in this way. Using the short system, the chro-
line with a normal female (XX) sex chromosome comple- mosome number, sex chromosome complement, type of rear-
ment in 15 cells. Note the use of a space between mos and rangement, the chromosome(s) involved, and the breakpoints
are indicated. However, complex rearrangements, especially ploidy levels most commonly used in human karyotyping,
structural aberrations with multiple gains and losses or involv- most often in acquired diseases, are:
ing multiple chromosomes, may necessitate the use of a • Near-haploid (1n), which describes chromosome counts
detailed system that describes the involved chromosomes up to 34 chromosomes; numerical abnormalities are
from end to end. The detailed system is particularly useful expressed in relation to 23 chromosomes.
when describing complex acquired aberrations in malignant • Near-diploid (2n), which describes counts with 35–57
disorders. chromosomes; numerical abnormalities being expressed
The rules used in the short system are retained in the in relation to 46 chromosomes.
detailed system with one exception. Instead of writing • Near-triploid (3n), which describes karyotypes with
the breakpoints within the last parentheses, an abbrevi- 58–80 chromosomes; numerical aberrations are expressed
ated description of the band composition of the rear- in relation to 69 chromosomes.
ranged chromosome(s) starting from the end of the short • Near-tetraploid (4n), which describes karyotypes with
arm (pter) and proceeding to the end of the long arm 81–103 chromosomes; numerical changes are expressed
(qter) is specified, that is, the bands are identi fied in the in relation to 92 chromosomes.
order in which they occur in the derivative chromosome.
In the detailed system, a single colon denotes a break, a 25,X,+4,+10
double colon denotes breakage and reunion, and an arrow
This represents a near-haploid karyotype with two copies
indicates “from→to.” If there are doubts as to whether to
of chromosomes 4 and 10 and single copies of all other
use the short or detailed system, fi rst write the short sys-
chromosomes.
tem and determine if the aberration(s) can be accurately
drawn as described in the nomenclature string. If the 70,XXY,+13
abnormalities cannot be correctly visualized using the
short system, the detailed system should be used. The This describes a near-triploid karyotype with four copies of
detailed system was devised to be fl exible; therefore, if chromosome 13 and three copies of all other chromosomes.
only one of several chromosome aberrations requires the
94,XXYY,−2,−5,+8,+8,+21,+21
use of the detailed system, it is acceptable to combine
the short and detailed systems to describe the karyotype. This represents a near-tetraploid karyotype with three
Table 3.4 lists the most common structural aberrations copies of chromosomes 2 and 5, five copies of chromosomes
found in human karyotypes with interpretation of the 8 and 21, and four copies of all other chromosomes.
findings and examples of how to write them in both the For more complex ploidy changes, please refer to ISCN [2].
short and detailed systems. At times, the biology of the study or the chromosome
number will vary between two ploidy levels. Because precise
communication of the karyotypic data is key, these cases
Numerical Abnormalities and Ploidy may be written with the ploidy level in angle brackets “< >”
immediately after the chromosome number and before the
Gains and losses of whole chromosomes in the karyotype sex chromosome complement. For example, high hyperdip-
string are usually denoted by the use of either a plus (+) or loidy, a favorable finding in pediatric acute lymphoblastic
minus (−) sign before the aberrant chromosome; for exam- leukemia (ALL), may be written relative to 2n ploidy even
ple, 47,XY,+21. The exception is the sex chromosomes in though it represents a near-triploid clone; for example,
constitutional studies, where sex chromosome gains and 59<2n>,XX,+X,+4,+5,+6,+10,+10,+14,+14,+17,+17,+18,+18,+21.
losses are indicated by listing the chromosome(s) present Endoreduplication (end) is a special form of duplication
(e.g., 45,X or 47,XXY) without use of plus or minus signs. of the genome without mitosis, giving rise to four-stranded
Acquired sex chromosome aberrations are written with chromosomes at prophase and metaphase. Endoreduplication
plus and minus signs (see “Describing Cancer Karyotypes” should be written as end 46,XY. Note the space after the
later). triplet and before the chromosome number.
Ploidy refers to the number of sets of chromosomes pres-
ent. Thus, diploid refers to the normal situation of two sets of
each chromosome (e.g., 46,XX or 46,XY). A haploid, trip- Structural Chromosome Abnormalities
loid, or tetraploid karyotype is evident from the chromosome
number; for example, 23,X, 69,XXY, or 92,XXYY, respec- Abbreviations are used to specify structural abnormalities
tively. If additional chromosome changes are evident, these (see Table 3.4) and precede the chromosome(s) involved in
are expressed in relation to the appropriate ploidy level. The the aberration in the nomenclature string.
Deletions (del) This indicates that two distinctly different clonally occurring
rings and a marker chromosome are present.
Deletions result in loss of a chromosome segment. Terminal If multiple rings are present but it is not known if any of
deletions are caused by a single break with loss of the seg- the rings are identical, the rings are denoted by a plus sign
ment distal to the break. Interstitial deletions result from two and the number of rings identified; for example, the presence
breaks in a chromosome, loss of the intervening segment, of three rings is described as +3r.
and reunion of the breakpoints.
del(5)(p15.3) Inversions (inv)

This describes a terminal deletion of the short arm of
In an inversion, a chromosomal segment breaks, reorients
chromosome 5. All chromosomal material distal to band
180°, and reinserts itself. If an inversion involves the cen-
p15.3 is missing.
tromere, with one break in each chromosome arm, it is
said to be pericentric. A paracentric inversion is isolated
del(20)(q11.2q13.3)
to one chromosome arm and does not involve the
This represents an interstitial deletion of the long arm of centromere.
chromosome 20. The material between bands q11.2 and
q13.3 is deleted. Note that no semicolon separates the break- 46,XX,inv(16)(p13.1q22)
points, as this abnormality involves a single chromosome.
This is a pericentric inversion of chromosome 16. A break
Additional examples are presented in Table 3.4. “High-
has occurred in the short arm at band 16p13.1 and the long
resolution” banding (see Chap. 4) allows for the detection of
arm at band 16q22. The chromosome segment between these
deletions within a single chromosome band. Such deletions
bands is present but inverted. This aberration is commonly
should be written denoting that two breaks have occurred in
observed in acute myelomonocytic leukemia with eosino-
a single band, for example, del(4)(q12q12).
philia (see also Chap. 15).
46,XY,inv(3)(q21q26.2)
Ring Chromosomes (r)
This is a paracentric inversion involving bands q21 and
Ring chromosomes, or rings, are donut-shaped structures q26.2 in the long arm of chromosome 3. This rearrange-
that may involve one or more chromosomes. When a single ment is also seen in acute myeloid leukemia (see also
chromosome is involved, a semicolon is not used between Chap. 15).
the band designations (see additional example in Table 3.4): For additional examples, see Table 3.4.
46,XX,r(7)(p22q36)
This describes a ring derived from chromosome 7. Breaks Duplications (dup)
have occurred at bands p22 and q36, and the ends of the segment
between the breakpoints have rejoined. The acentric (without The orientation of duplications is either direct or inverted
a centromere) segments distal to the breakpoints have been lost. and is indicated by the order of the bands with respect to the
When two chromosomes are involved and a monocentric centromere in the karyotype designation. The band closest to
(one centromere) ring chromosome and an acentric segment the centromere is written first in the short system; only the
results, “der” should be used (see “Derivative (der) and detailed system can pinpoint the exact location of the dupli-
Recombinant (rec) Chromosomes” later). cated segment.
46,XY,der(18;?)(p11.2q22;?) 46,XY,dup(1)(q21q42)
This indicates a ring derived from the segment between This is a direct duplication of the segment between bands
the breakpoints p11.2 and q22 of chromosome 18 and an 1q21 and 1q42 in the long arm of chromosome 1.
acentric fragment of unknown origin.
If the origin of a ring chromosome is not known, it is 46,XX,dup(13)(q34q21)
listed after all known aberrations but before other markers: This is an inverted duplication of the segment between
bands 13q21 and 13q21 in the long arm of chromosome 13.
51,XX,+8,+13,+r1,+r2,+mar For additional examples, see Table 3.4.
Insertions (ins) The material distal to 9q34.1 is translocated to chromosome

22 at band 22q11.2, the material distal to 22q11.2 is translo-
As the name implies, an insertion involves the movement of cated to chromosome 11 at band 11q13, and the material dis-
a segment of intrachromosomal material from one chromo- tal to 11q13 has been translocation to chromosome 9 at band
somal location into another. The recipient can be another 9q34.1. This 3-way translocation appears to be balanced at
chromosome or a different part of the chromosome of origin. the cytogenetic level.
The orientation of the inserted segment may be direct, For additional examples, see Table 3.4
retained in its original orientation, or inverted. In inverted
insertions, the “normal” orientation of the bands will be
reversed with respect to the centromere. Derivative (der) and Recombinant (rec)
Chromosomes
46,XY,ins(2)(q13p11.2p14)
Derivative chromosomes are structurally abnormal chromo-
This is an example of an insertion within a chromosome,
somes that can be generated in three ways: more than one
a direct insertion of the short arm segment between bands
rearrangement within a single chromosome, one rearrange-
2p11.2 and 2p14 into the long arm at band 2q31.
ment involving two or more chromosomes, including rear-
rangements between chromosome homologues, or more than
46,XX,ins(12;?)(q13;?)
one rearrangement involving two or more chromosomes. All
This is an example of an insertion between two chromo- three possible scenarios may result in an unbalanced karyo-
somes. Material has been inserted into chromosome 12 at type. The term “der” refers to a chromosome that has an
band 12q13; however, the origin of the inserted material is intact centromere.
not known.
Derivative Chromosomes Generated by More than
46,XY,ins(19;11)(p13.1;q23q13) One Rearrangement Within a Single Chromosome
This is an example of an inverted insertion between two 46,XY,der(7)del(7)(p11.2)del(7)(q11.2)
chromosomes. The long arm segment between bands 11q13
and 11q23 is present but is inverted and inserted into the
short arm of chromosome 19 at band 19p13.1 Note that the This describes a male karyotype with a chromosome 7
11q13 band of the inserted segment is more distal to the centromere showing deletions in both the short and long
centromere, indicating that the inverted segment is inverted arms, namely, deletion of the material distal to bands 7p11.2
compared to its normal orientation. and 7q11.2. This karyotype may appear as monosomy 7 with
For additional examples, see Table 3.4. a small centric fragment. Fluorescence in situ hybridization
(FISH) studies may have identified the centric fragment as
a chromosome 7 centromere using a FISH probe (see
Translocations (t) Chap. 17).
A translocation is an abnormality resulting from an exchange Derivative Chromosome Generated by One

of genetic material between two chromosomes. Translocations Rearrangement Involving Two or More
may be balanced or unbalanced (the latter resulting in deriva- Chromosomes
tive chromosomes and loss or gain or material). See “Derivative Whole-Arm Translocations
(der) and Recombinant (rec) Chromosomes” later.
45,XX,der(1;7)(q10;p10)
46,XY,t(12;14)(q13;q32)
This is a translocation involving two chromosomes. This describes a female karyotype with an unbalanced whole-
Breaks have occurred at bands 12q13 and 14q32. The seg- arm translocation (see also previous section “Translocations
ments distal to the two breakpoints are present but exchanged (t)”). The derivative chromosome is comprised of the long
with no apparent loss of genetic material. arm of chromosome 1 and the short arm of chromosome 7.
The derivative chromosome has replaced one normal chro-
46,XX,t(9;22;11)(q34.1;q11.2;q13) mosome 1 and one normal chromosome 7, resulting in a
chromosome count of 45. The two missing normal chromo-
This is a 3-way translocation. Breaks have occurred in
somes are not specified. The resulting net imbalance is
three chromosomes at bands 9q34.1, 22q11.2, and 11q13.
monosomy 1p and monosomy7q.
46,XX,+1,der(1;7)(q10;p10) The arrows in the detailed system describe the derivative

chromosome 1 from pter to qter.
This describes a female karyotype with two normal chro- In some instances, the centromere of the derivative chro-
mosomes 1 and a derivative chromosome comprised of the mosome is not known, but other parts of the chromosome are
long arm of chromosome 1 and the short arm of chromosome 7. clearly recognizable. These abnormal chromosomes should
The derivative chromosome has replaced one normal chro- be designated as der(?) and placed after all identified aberra-
mosome 1 and one normal chromosome 7, but +1 in this tions. Other unidentified ring chromosomes, markers, and
karyotype changes the net imbalance to trisomy 1q and double minutes (see Chap. 15) are listed behind the der(?), in
monosomy 7q. that order.
52,XY,….+der(?)t(?;5)(?;q15),+r,+mar,6~15dmin
Other Derivative Chromosomes Generated
by One Rearrangement Involving Two This represents a hyperdiploid clone with multiple aberra-
or More Chromosomes tions (not listed in this example) including a derivative chro-
mosome resulting from an unbalanced translocation between
46,XX,der(1)t(1;2)(p34.1;q31) chromosome 5 and a chromosome of unknown origin; a
ring chromosome, the chromosomal origin of which is
unknown; one marker, and six to 15 double minutes.
This describes a female karyotype with a derivative chro-
mosome 1. The unbalanced translocation results in loss of
1p34.1→pter and gain of 2q31→qter. Recombinant Chromosomes
Recombinant chromosomes are structurally rearranged chro-
47,XY,t(9;22)(q34.1;q11.2),+der(22)t(9;22) mosomes with a new segmental composition resulting from
meiotic crossing-over (see Chap. 9); thus, this term should
This describes a male karyotype with the standard (9;22) never be used to describe acquired (cancer) aberrations.
“Philadelphia chromosome” translocation and duplication Recombinants usually originate from heterozygotes carrying
of the Philadelphia (Ph) chromosome—the der(22). Note inversions or insertions, and the term always refers to the chro-
that once the breakpoints are written in the description, there mosome that has an intact centromere. The triplet “rec” should
is no need to repeat the breakpoints again for each addi- be used when the parental karyotypes are known and a parental
tional copy of the derivative chromosome. Also, the Ph inversion is identified (see Table 3.4). If parental karyotypes are
chromosome abbreviation may be used in the text of the unknown in a suspected recombinant, the abnormal chromo-
report, but only der(22)t(9;22) should be used in the ISCN some should be designed as a derivative chromosome (der).
description.
Derivative Chromosome Generated by Isochromosomes (i)

More than One Rearrangement Involving Two
or More Chromosomes An isochromosome is an abnormal chromosome with two iden-
The derivative chromosome is specified in parentheses, fol- tical arms due to duplication of one arm and loss of the other
lowed by all aberrations involved in its generation. These arm (mirror image of a chromosome from its centromere).
aberrations are listed according to the breakpoints of the
derivative chromosome from pter to qter and should not be 46,XY,i(6)(p10)
separated by a comma.
An isochromosome for the short arm of chromosome 6
Short system: 46,XX,der(1)t(1;2)(p34.1;q31)dup(1)(q21q32) has replaced one copy of chromosome 6.
or detailed system: 46,X,i(X)(q10)

46,XX,der(1)(2qter→2q31::1p34.1→1q32::1q21→1qter)
This is a female with one normal X chromosome and one
Both the short and detailed systems describe a female isochromosome for the long arm of the X chromosome. This
karyotype with a derivative chromosome 1 resulting from an karyotype is a frequent finding in patients with Turner syn-
unbalanced (1;2) translocation with a 1p24.1 breakpoint drome (see also Chap. 10).
and a duplication of the segment between 1q21 and 1q32. For additional examples, see Table 3.4.
Dicentric (dic), Isodicentric (idic), (p10 or q10) depending on which arm is present in the
and Pseudodicentric (psu dic) Chromosomes isoderivative chromosome.
These are structurally altered chromosomes with two 46,XY,ider(22)(q10)t(9;22)(q34.1;q11.2)

centromeres. The term “der” may be used instead of “dic”
but the combination of “der dic” is not appropriate. In the This is an isoderivative chromosome comprised of the
karyotype description, both dicentric and isodicentric chro- long arm of the “Philadelphia chromosome.” It is one of the
mosomes are counted as one chromosome without the need most common isoderivative chromosomes seen in cancer
to indicate the missing normal chromosome(s). cytogenetics (see “Describing Cancer Karyotypes” later).
45,XY,dic(14;14)(q11.2;q32)
Robertsonian Translocations (rob)
This represents a dicentric chromosome formed by
breakage and reunion at bands 14q11.2 and 14q32 on the Robertsonian translocations are a special type of translo-
two homologous chromosomes 14. However, if a dicentric cation in humans involving the acrocentric chromosomes
chromosome is proven to originate through breakage and (chromosomes 13, 14, 15, 21, and 22). Typically, the partici-
reunion of sister chromatids, it may be designated as dic(14) pating chromosomes break in their short arms and give the
(q11.2q32). To avoid ambiguity, it is always good practice appearance that the long arms fuse to form a single chro-
to describe complex dicentric chromosomes as derivative mosome with a single centromere. If the location of the
chromosomes. breakpoints is unproven, “rob” may be used. Because the
short arms of acrocentric chromosomes contain repetitive
47,XY,+idic(14)(q13) ribosomal gene clusters, loss of these arms due to this
47,XY,+idic(14)(pter®q13::q13®pter) type of translocation has no phenotypic consequences.
A karyotype with a single Robertsonian translocation by
This karyotype exhibits two normal chromosomes 14 and definition will have a 45 chromosome count. If the “rob” is
an isodicentric chromosome 14 consisting of two copies of proven to be a dicentric chromosome with breakpoints of
the short arm, centromere, and proximal long arm. The p11.2 or q11.2, the abbreviation “dic” should be used.
detailed nomenclature clearly describes the abnormal Either “rob” or “der” adequately describes these whole-arm
chromosome. translocations in a constitutional karyotype; however, for
At times, it is clear that only one centromere of the dicen- acquired Robertsonian translocations in cancer, “rob”
tric is active and the other is inactive (visualized by a decon- should not be used.
densed region of the inactive centromere in the majority of
mitotic cells analyzed). These structurally altered chromo- 45,XY,der(14;21)(q10;q10)
somes are commonly referred to as pseudodicentric chromo- - or -
somes (psu dic), and the presumed active centromere should 45,XY,rob(14;21)(q10;q10)
be written first in the karyotype string.
This describes a male karyotype with one normal chro-
45,XX,psu dic(15;14)(q11.2;q11.2) mosome 14, one normal chromosome 21, and a der(14;21) or
whole-arm translocation involving the long arms of chromo-
This is an abnormal karyotype with a pseudodicentric somes 14 and 21. The resulting net imbalance is loss of
chromosome comprised of the short arms, centromeres, and the short arms of chromosomes 14 and 21, which is of no
proximal long arms of chromosomes 14 and 15 replacing clinical consequence.
one normal copy each of chromosomes 14 and 15. The
nomenclature implies that the centromere of chromosome 15 46,XY,der(14;21)(q10;q10),+21
is active. - or -
46,XY,rob(14;21)(q10;q10),+21
Isoderivative Chromosomes (ider) These are similar to the previous example but with gain of
chromosome 21. This male karyotype has one normal chro-
An isoderivative chromosome designates an isochromo- mosome 14, two normal chromosomes 21, and the der(14;21).
some formation for one of the arms of a derivative chromo- The resulting net imbalance is inconsequential loss of the
some. The breakpoints are assigned to the centromeric bands short arms of chromosomes 14 and 21 and trisomy for the
long arm of chromosome 21, resulting in Down syndrome in Neocentromeres (neo)

a live-born individual.
Neocentromeres are fully functional centromeres that are
46,XY,+21,der(21;21)(q10;q10)
present in non-centromeric regions. They lack a-satellite
- or - DNA. A derivative chromosome with a neocentromere may
46,XY,+21,rob(21;21)(q10;q10) be described using either the triplet “neo” or “der,” both of
which are equally acceptable:
These denote a male karyotype with a derivative chromo-
some comprised of both copies of the long arms of chromo-
47,XY,+der(18)(qter→q21.1:)
some 21. One additional normal chromosome 21 is also
present, denoted with a plus sign. The net imbalance is - or -
referred to as translocation trisomy 21. 47,XY,+neo(18)(qter→q21.1:)
45,XY,dic(14;21)(p11.2;p11.2) For example, a derivative chromosome containing a neo-

- or - centromere within the segment 18q21 through 18qter, which
45,XY,dic(14;21)(14qter→14p11.2::21p11.2→21qter) normally lacks a centromere, is written with the short sys-
tem. However, to adequately describe the location of a neo-
These describe a translocation proven to be a dicentric, centromere, the detailed system is required:
with breakage and reunion at 14p11.2 and 21p11.2. In this
case, the triplet “dic” should be used in both the short or
detailed version, and the dicentric chromosome is counted as 47,XY,+der(18)(qter→q21.1→neo→q21.1:)
single chromosome. The resulting net clinically inconse-
quential “imbalance” of this karyotype is loss of the seg-
ments distal to 14p11.2 and 21p11.2. Additional Material of Unknown Origin (add)
The triplet “add” is used to describe material of unknown

Balanced Rearrangements Involving origin attached to a chromosome region or band. The material
Three or More Chromosomes may have come from the same chromosome or another chro-
mosome, and no known mechanism is implied (see addi-
As with any science, the general principles create a strong
tional example in Table 3.4). Despite the implication that
foundation, but karyotype complexity sometimes requires
such material is always additional, it often actually replaces
modifications or exceptions to the guidelines. As noted for
part of a chromosome arm.
balanced translocation nomenclature involving two chromo-
somes, a sex chromosome or the autosome with the lowest
46,XX,add(5)(q13)
number is always specified first, with subsequent abnormali-
ties listed in numerical order regardless of type. However,
when three or more chromosomes are involved in a balanced Material of indeterminate origin is present on chromo-
rearrangement, the next chromosome listed is the one that some 5 at band q13, replacing the material distal to this band.
received a segment from the first chromosome, and the chro- Note that the nomenclature does not describe the size of this
mosome specified last is the one that donates material to the piece of chromosomal material.
first chromosome listed.
46,Y,t(X;22;2)(q22.1;q22.2;p21) Marker Chromosomes (mar)

This represents an XY karyotype with the segment of
Marker chromosomes are structurally abnormal chromo-
the X chromosome distal to Xq22.1 translocated to chro-
somes in which no part can be unambiguously identified by
mosome 22 at band 22q22.2, the segment distal to chromo-
conventional banding techniques. A plus sign is always used
some 22q22.2 translocated to chromosome 2 at band 2p21,
when describing the presence of a marker in the karyotype,
and the segment distal to chromosome 2p21 translocated
and marker chromosomes are usually listed last in the
to the X chromosome at band Xq22.1. Note that the
nomenclature string. If multiple markers can be distinguished
segment from the last chromosome listed in the first set of
as distinct from one another, they should be written as +mar1,
parentheses has been translocated to the first chromosome
+mar2; otherwise, +2mar should be used. If any part of
listed.
the abnormal chromosomal marker can be recognized, even Incomplete Karyotypes (inc)
if the origin of the centromere is unknown, the correct
designation of the abnormal chromosome is “der” and not Every attempt should be made to describe all aberrations in
“mar,”—e.g., +der(?)t(?;7)(?;q11.2). an abnormal cell or clone. However, when this is not possible
(such as when chromosome morphology is poor), the triplet
49,XY,+8,+2mar “inc” is placed at the end of the nomenclature string, after the
description of the identifiable abnormalities:
An additional copy of chromosome 8 is present, as are
two marker chromosomes.
46,XY,del(7)(q22),inc[10]
Ten cells were examined; for cancer karyotypes, this is

Multiple Copies of Rearranged Chromosomes indicated using square brackets (see “Describing Cancer
Karyotypes” later). The number of chromosomes is deter-
When multiple copies of a rearranged chromosome are pres- mined, as is the presence of a deletion involving the long arm
ent, the multiplication sign (¥) is placed directly after the of chromosome 7. The triplet “inc” indicates that other
aberration. Note that a multiplication sign should never be abnormalities are also present but cannot be described.
used to describe multiple copies (gains) of a normal
chromosome.
Parental Inheritance
46,XX,del(5)(q13q31)×2
When parental inheritance is known, the triplet “mat” for
This indicates that two copies of a deletion involving 5q
maternally inherited or “pat” for paternally inherited should
were found.
be used, immediately following the designation of the abnor-
52,XY..........+mar1×3 mality. If multiple different aberrations are inherited from
the parents, the parental origin should be designated for each
This indicates that three copies of marker 1 was a clonal individual aberration even if both aberrations came from the
finding. same parent. If the parental chromosomes are normal with
respect to the abnormality, the abnormality may be designed
as “dn” for de novo.
Chromosome Breakage
46,XX,t(8;9)(q13;p13)mat,inv(13)(q14q32)mat
Cultured cells from patients with chromosomal breakage
syndromes, such as ataxia telangiectasia, Bloom syndrome, Both aberrations were inherited from the mother.
Fanconi anemia, and xeroderma pigmentosum may exhibit
spontaneous or elevated levels of chromosomal breakage 46,XX,t(8;9)(q13;p13)mat,inv(13)(q14q32)dn
following clastogenic exposure. Examples of abbreviations
used here are chrg (chromosome gap), chrb (chromosome The translocation was inherited from the mother and the
break), and chte (chromatid exchange). inversion arose de novo.
Fragile Sites (fra) Additional Symbols
Chromosomal fragile sites are inherited in a codominant Additional punctuation signs and symbols within the nomencla-
Mendelian fashion and are commonly considered to be nor- ture string clarify gains, losses, and variable heteromorphisms
mal variants with no phenotypic consequences. However, (chromosome morphology) or uncertain chromosome features.
they may result in chromosome abnormalities such as dele-
tions, multi-radial figures, or acentric fragments. Fragile Plus (+) and Minus (−) Signs
sites have been known to be associated with a specific dis- Plus and minus signs are used in conjunction with other sym-
ease or phenotype, such as the fragile X syndrome (see bols such as “h” and “s,” to distinguish normal variation in
Chap. 19). Regardless of their biological consequences, length from additions or deletions of other origins; however,
fragile sites are denoted by the triplet “fra,” for example, they should not be used after the chromosome arm in the
fra(X)(q27.3). nomenclature string. For example, 16qh+ describes an
increase in length of the heterochromatin on the long arm of variety of combinations within the uncertain region is
chromosome 16, but the use of 16q+ in an ISCN nomencla- possible, use “~.”
ture string is inappropriate. This should be written as add(16)
(q?), indicating that material of unknown origin has been
added to the long arm of chromosome 16, but the exact loca- Uniparental Disomy (UPD)
tion of the added material is unknown. Wyandt and Tonk
have compiled an excellent resource for the range of varia- Uniparental disomy (UPD) is a condition in which both
tion representing human chromosomal heteromorphisms [3], homologous chromosomes are derived from the one parent.
and a comprehensive guide for describing these variations In most cases, UPD is detected by polymorphic markers such
may be found in ISCN [2]. as microsatellite polymorphisms, but in some instances, UPD
may be identified cytogenetically or through single nucleotide
Questionable Karyotypic Results polymorphism (SNP) microarray analysis (see Chap. 20).
Uncertainty within a chromosome is denoted by using a
question mark (?), the approximate sign (~), or the term “or” 46,XX,upd(15) pat
in the nomenclature string.
A question mark (?) can be for uncertainty in a chromo- This is a female karyotype indicating UPD for a pater-
some band or a chromosome structure; the symbol must nally derived chromosome 15.
be placed immediately in front of the uncertain band or
structure that is questioned. For example, if a translocation Mosaicism and Chimerism
is suspected in a karyotype but additional testing is required
for confirmation, the question mark should go in front of the Mosaicism is the presence of two or more populations of
“t” and not behind it, for example, 46,XX,?t(15;17)(q22;q12). cells with different genotypes originating from the same
If additional testing proves that the translocation is present, zygote. To differentiate mosaicism from a chimerism, which
the report should be revised (i.e., issue an addendum stating has two or more different populations of genetically distinct
that new information allowed for ISCN refinement) to include cells or cell lines originating from different zygotes, the
the new confirmed data with revision of the nomenclature triplet “mos” or “chi” may be used, for example, mos
string. If the banding quality is particularly poor, a question 45,X/47,XXX/46,XX versus chi 46,XX/46,XY. When writ-
mark may replace a chromosome, region, or band designa- ing these karyotype descriptions, the triplet is only needed
tion; for example, 46,XY,del(1)(q?) indicates a long arm for the initial karyotype description within the report, and a
deletion of chromosome 1 in which neither the region nor space must be used after the triplet abbreviation and before
band can be identified. the chromosome number. A normal diploid cell line, when
The approximate sign (~) is most useful when describing present, is always listed last.
uncertain breakpoints or boundaries of a chromosome seg- Constitutional chimerism in humans is rare, but chime-
ment such as t(2;5)(p21~23;q33~35), but it may also be used rism secondary to bone marrow stem cell transplantation
to describe a range of chromosomes when the specific chro- (SCT) is a common finding in patients who have had such a
mosome modal number is not known (38~48,XX…) or when transplant. In these cases, the recipient or host cell lines
the number of markers within a karyotype appears to be vari- (clones) are listed first, followed by the donor cell line(s). A
able as in 48~53,XY,+2~7mar[12]. double slant line (//) is used to clearly separate the chimeric
The word “or” with a space on either side is used in human recipient//donor cell populations. Table 3.3 provides exam-
chromosome nomenclature to indicate an alternative inter- ples on how to write and report karyotypes with mosaicism/
pretation of an aberration, based on the banding level. For chimerism with respect to clone size and type of aberration.
example, 46,XY,add(19)(p13.1 or q13.1) denotes a karyo-
type in which additional material of unknown origin is 46,XX,t(9;22)(q34;q11.2)[15]//46,XY[5]
attached to chromosome 19, but the banding is too poor to
show with certainty whether the material is attached to the Fifteen of the twenty metaphase cells examined exhibit a
short or long arm. (9;22) translocation and represent the female recipient. The
The term “or” is also used to describe two possible break- remaining five cells represent her male bone marrow donor.
point interpretations of a translocation. In the example of
t(7;10)(q22;q24) or t(7;10)(q32;q22), the two alternative //46,XX[20]
interpretations give rise to identical-looking derivative chro-
mosomes. If the breakpoints could be either one band or All 20 metaphase cells examined from this male patient
another, use “or,” but if the breakpoints are less certain and a are of (female) donor origin.
Examples of using cancer nomenclature follow:

Describing Cancer Karyotypes
46,XY,t(8;14)(q24.1;q32)[20]/
Acquired abnormalities associated with neoplastic disorders
47,XY,t(8;14)(q24.1;q32),+der(14)t(8;14)[6]
imply the presence of both normal (constitutional) and
abnormal (neoplastic) cells in the same patient and frequently 46,XY,t(8;14)(q24.1;q32)[20]/
in a single specimen. Terms used exclusively in neoplasia 47,idem,der(14)t(8;14)[6]
nomenclature include clone, mainline, stemline, sideline,
composite karyotype, and unrelated clones. 46,XY,t(8;14)(q24.1;q32)[20]/
47,sl,der(14)t(8;14)[6]
Clone
These three nomenclature strings describe the same kary-
A clone is a cell population derived from a single progenitor otype: a clone (stemline) with 46 chromosomes and an (8;14)
cell. As in the constitutional setting where cell line is the translocation in 20 cells, with a subclone of six cells with 47
preferred term, a clone constitutes at least two mitotic cells chromosomes showing the t(8;14) and gain of a derivative
with the same chromosome gain or structural aberration, or chromosome 14 resulting from the t(8;14). The subclone
at least three cells with the same chromosome loss. The denotes clonal evolution of the stemline. The terms “idem”
requirement of three cells for the identification of clonal and “sl” are alternative ways to describe the same result—in
chromosome loss is due to the fact that chromosome loss this case the t(8;14) in the subclone. The terms “sl” and
may occur during the slide-making process. “idem” should never be intermixed when describing a single
The modal number (mn) is the most common chromo- tumor sample. Note that the stemline is the mainline in this
some number in a tumor cell population and may be expressed example.
as a range.
46,XY,t(8;14)(q24.1;q32)[12]/45,sl,-X[18]/46,sdl1,+8[5]/
47,sdl2,+der(14)t(8;14)[6]
Stemline, Sideline, and Mainline
46,XY,t(8;14)(q24.1;q32)[12]/45,idem,-X[18]/
The stemline (sl) is the most basic clone of a tumor cell popu- 46,idem,-X,+8[5]/47,idem,-X,+8,der(14)t(8;14)[6]
lation and is always written first in the nomenclature string.
Based on the well-founded assumption that clones with
These two examples show a male karyotype with t(8;14)
more abnormalities tend to have evolved from those with
as the sole abnormality in the stemline. Three related sub-
fewer abnormalities, clone order in oncology samples reflects
clones or sidelines are present. Note that the sidelines are
order of increasing complexity to describe the presence of
listed in order of increasing complexity. In this example, the
clonal evolution of disease or genetic instability. Square
mainline (18 cells) is sideline 1 with 45 chromosomes and
brackets are used to enumerate clone size.
two clonal aberrations: t(8;14) and loss of one chromosome
Additional related clonal aberrations (sublcones) are
X. Sideline 2 (5 cells) shows t(8;14), -X, and gain of chromo-
referred to as sidelines (sdl). Sidelines follow the stemline
some 8. Sideline 3 (6 cells) shows t(8;14), -X, +8, and gain
in the nomenclature string in order of increasing complex-
of the derivative chromosome 14. Note that the use of “sdl”
ity (note the difference from constitutional cell line order).
in the karyotype string indicates that all clonal aberrations
If multiple sidelines are present in a tumor karyotype,
found in that subclone are present in the new subclone in
they may be numbered sdl1, sdl2, sdl3, etc., and used within
addition to the new aberrations. The term “idem” refers back
the nomenclature string to refer back to that part of the stem-
to the stemline only. If using “idem,” the additional clonal
line or previous sideline that is also present in the new sub-
aberrations found in each subclone relative to the stemline
clone. The term “idem” (Latin for “the same”) may also be used
must be restated.
to represent a subclone. However, when used in a nomencla-
ture string, “idem” refers back only to the stemline clonal
aberrations. The purpose for these terms is to reduce redun- 46,XY,t(8;14)(q24.1;q32)[14]/92,sl×2[4]/93,sdl1,+8[6]
dancy when multiple clones are present in a tumor karyotype. 46,XY,t(8;14)(q24.1;q32)[14]/92,idem×2[4]/93,idem×2,+8[6]
The mainline (ml) is the largest clone in a tumor, but this
abbreviation is rarely used in the nomenclature string because
it is implied by the number of cells in brackets. The mainline In these two examples, the clone with t(8;14) is the stem-
may or may not be the stemline. line. Two additional subclones were detected: one subclone
(sideline 1) is a doubling product (×2) of the stemline. The When describing cancer karyotypes, several ISCN
second subclone (sideline 2) is a near-tetraploid subclone recommendations are critical.
of sd1 with gain of chromosome 8 (sideline 2). Note that
complexity, not size, determines clone order.
Acquired sex chromosome numerical
abnormalities
Composite Karyotype (cp)
Acquired sex chromosome numerical abnormalities are
A composite karyotype may be created in samples showing expressed with plus and minus signs. A tumor karyotype
vast tumor heterogeneity, a common finding in solid tumors, with loss of one X chromosome is written as 45,X,-X.
acute leukemias, and aggressive lymphomas. A composite Similarly, a tumor with loss of the Y chromosome is written
karyotype lists all clonally occurring abnormalities. In such as 45,X,-Y. Gains of sex chromosomes in cancer karyotypes
cases, the nomenclature string usually begins with a chromo- are written with plus sign:
some range, the sex chromosome component, and clonal
aberrations per ISCN standard guidelines, and is closed with 48,XY,+X,+Y
the total number of cells in which the clonal aberrations
This denotes a male tumor karyotype with one additional
were observed in square brackets:
X and one additional Y chromosome.
45~50,XX,+X,add(1)(p34.1),+8,i(9)(q10),add(13)(q32),
t(14;18)(q32;q21.3),+2~10mar[cp24] Presence of a constitutional chromosome
aberration
This female composite karyotype has from 45–50 chro-
mosomes in 24 cells denoted by “cp” in the square brackets If a known constitutional chromosome aberration is present in a
and the chromosome number written as a range. A com- cancer karyotype, the letter “c” should be used in the
posite karyotype contains all clonally occurring abnormali- karyotype.
ties observed in a tumor. Each of the abnormalities listed in
this example has been seen in at least two cells, but there 46,XXYc,−X
may be no cells with all abnormalities. The chromosome
range present in cancers, particularly solid tumors, may This is a male with Klinefelter syndrome (47,XXY, see
reflect incompletely analyzed or over/under-spread meta- Chap. 10) who has an acquired loss of one X chromosome in
phase cells, or too few analyzable mitotic cells. When his tumor karyotype.
reporting neoplastic cases, every effort should be made
to describe subclones so that clonal evolution is made 48,XXYc,+X
evident. Nevertheless, it is often practical, if not necessary, This indicates gain of one X chromosome in the previ-
to describe observed abnormalities as a single com- ous patient.
posite clone.
49,XY,+8,+21c,+21[20]
Unrelated Clones This indicates a tumor from a patient with constitu-

tional trisomy 21 (Down syndrome) and acquired gain of
Unrelated clones are clones found within a single tumor chromosomes 8 and 21.
that fail to show “relatedness” by conventional cytogenetics.
This term does not rule out the possibility of a common
molecular mutation. If a normal diploid clone is also
Clone size
present, it is listed last.
The number of cells that constitutes a clone is given in square
brackets [ ] after the description of that clone. Moreover, to
47,XX,+8[7]/46,XX,t(9;22)(q34.1;q11.2)[4]/46,XX[9]
provide an estimate of tumor load or effectiveness of treat-
This is an example of a female tumor karyotype with unre- ment, the number of cells that constitute a clone must be given
lated clones. Gain of chromosome 8 was observed in seven in square brackets after the karyotype, even if the karyotype
cells. The (9;22) translocation was observed as an unrelated appears to be a normal. Normal karyotypes are always written
clone in four cells. Nine cells showed a normal female karyo- last. Cancer karyotyping standards require a total of 20
type. Note that the number of cells that constitute each clone mitotic cells that are analyzed band for band (see Chap. 4).
is given in square braciets, and the normal diploid clone is
listed last. 46,XX[20]
This indicates the presence of a normal karyotype, and the and a plus (+) sign should never be placed in front of the
cell count in brackets indicates that the study was performed symbol “dmin.” It is also common to report a range of
to rule out an acquired aberration. double minutes since they do not segregate equally with
each division:
46,XY,t(8;14)(q24.1;q32)[20]
This describes a male karyotype with t(8;14) detected in 46,XY,t(8;14)(q24.1;q32),10~28dmin
all twenty cells examined.
In addition to an (8;14) translocation, cells with as few as
ten and as many as 28 double minutes were observed.
Non-clonal aberrations
Non-clonal or single cell aberrations should not be reported

Molecular Cytogenetic Techniques
in the nomenclature string, with two exceptions:
Molecular cytogenetic assays such as fluorescence in situ
If a single abnormal metaphase cell showed an abnor-
hybridization (FISH), comparative genomic hybridization
mality found to be clonal by a second genetic testing method
(CGH), and array-based comparative genomic hybridization
(e.g., FISH), the karyotype should be reported:
(aCGH) are valuable adjuncts to conventional chromosomal
banding techniques (see Chaps. 17 and 21). These hybridiza-
46,XY,del(13)(q12q12)[1]/46,XY[19].nuc ish
tion-based assays use labeled complementary DNA probes
(D13S319×1,LAMP1×2)[25/200]
to localize a specific DNA sequence (or demonstrate the lack
A single cell with an interstitial deletion of chromosome thereof) in a patient or tissue sample. Table 3.5 has a partial
13 was observed but is reported since 25 cells with such a list of abbreviations and symbols pertaining to molecular
deletion were also detected with FISH using a D13S319 cytogenetic techniques. For a complete list, please refer to
probe, which maps to 13q13.3 (see “Molecular Cytogenetic ISCN [2].
Techniques” later).
If at follow-up (post treatment), a patient shows the same
clonal aberration in a single cell detected at disease onset, the Fluorescence In Situ Hybridization (FISH)
cell should be reported in the karyotype:
The fluorescence in situ hybridization assay is a targeted
46,XY,t(9;22)(q34.1;q11.2)[1]/46,XY[19] approach to determine whether a specific DNA sequence, as
visualized with a probe, is present within chromosomes on
A single cell with a (9;22) translocation was observed in a microscope slides. The DNA, which may be in the nucleus
patient in whom this rearrangement was observed previously. (nuc ish) in nondividing or interphase cells or in metaphase
chromosomes (ish), is fixed on a slide and denatured in place
(in situ) to expose the two strands of DNA and allow a dena-
Gene amplification tured-labeled probe to hybridize to the chromosomal DNA.
The location of the hybridization signal is visualized by
Homogeneously staining regions (hsr) and double minutes
fluorescence microscopy and pinpoints the location of
(dmin) are two different cytological forms of amplified gene
the DNA segment to which the probe is hybridized (see
sequences. The term “hsr” refers to uniformly staining
Chap. 17).
regions within a chromosome arm, even though the staining
is not always homogeneous. These hsrs can range in size,
and the nomenclature does not indicate how large an hsr is. Metaphase FISH
To describe abnormal metaphase FISH results, the follow-
46,XX,hsr(8)(q24.1) ing sequence is used: the triplet “ish” followed by a space,
the triplet denoting the abnormality, then, in separate
A homogeneously staining region is present in band q24.1 parentheses, the chromosome, the band designation for the
of chromosome 8. breakpoint(s), and the locus or loci from which clones
Double minutes are extrachromosomal amplified gene were used. Whenever possible, the clone name should be
sequences that appear as two small cojoined chromosomal used. If the clone name is not available, the locus designa-
fragments in the nuclear matrix. A single version of a double tion or D-number should be used [4]. If the locus designa-
minute is known as a minute or min. When writing the tion is not available, then the gene name, using HUGO
nomenclature string, double minutes are not included in the nomenclature [5], may be substituted. Locus designations
chromosome count because they do not possess centromeres, are immediately followed by “×2” to indicate a normal
Table 3.5 Common symbols and abbreviations used in molecular cytogenetics

Symbol/Abbreviation Interpretation
minus sign (−) Loss of a probe signal; single minus sign represents one copy loss, double minus sign indicates two copy or
homozygous deletion
plus sign (+) Signal is present on a specific chromosome; two plus signs indicate gain of signal
multiplication sign (×) Precedes the number of signals observed
period (.) Separates cytogenetic observations from results of in situ hybridization
semicolon (;) Separate probes on different derivative chromosomes
aCGH Array-based comparative genomic hybridization
arr Microarray or results from microarray testing
amp Amplified gene sequences
cgh Comparative genomic hybridization
con Signals are connected
htz Heterozygous
hmz Homozygous
ish Refers to in situ hybridization result. ish usually refers to metaphase FISH
nuc ish Refers to an interphase in situ hybridization result. nuc indicates nuclear or non-mitotic cells
sep Signals that are separated. Used to denote separated signals using break-apart probe set (implies translocation)
subtel Subtelomeric
hybridization pattern to an autosome or pseudoautosomal findings, with a period separating the two results. The triplet
region and “×1” for normal hybridization to X or Y probes “ish” indicates the beginning of the FISH results:
in a male.
A minus sign is used to indicate that one copy (-) or both 46,XY.ish del(22)(q11.2q11.2)(HIRA−)
copies (--) of a probe are absent. When multiple probes are
used, those that are present are indicated by a plus sign. This describes a male with a normal karyotype by cytoge-
Additional plus signs indicate duplication of a locus. Locus netics with a microdeletion within the DiGeorge syndrome
designations should be separated by commas. critical region on chromosome 22 identified by metaphase
Normal in situ hybridization results are designated with FISH using a probe for HIRA.
the symbol “ish” followed by the chromosome, region, band,
or sub-band designation of the locus or loci tested, followed 46,XY.ish del(22)(q11.2q11.2)(HIRA−,N25−,D22Z1+)
in parentheses by the locus or loci tested, multiplication or This describes a male with a normal karyotype who has a
plus/minus signs, and the number of signals detected. When deletion of the DiGeorge syndrome critical region identified
conventional cytogenetics is not performed, the nomencla- via ish using probes for the HIRA, N25, and D22Z1 loci.
ture string begins with “ish”: HIRA and N25 are deleted, whereas D22Z1 is retained.
ish 22q11.2(HIRA×2) Subtelomere FISH (subtel)

Use of FISH probes for the 41 unique human subtelomeric
This indicates a normal hybridization result for the
regions is a diagnostic tool used in many cytogenetics labo-
DiGeorge syndrome locus HIRA.
ratories, although this is becoming less common due to the
increasing utilization of microarrays (see later and see also
ish.del(7)(q11.23q11.23)(ELN−)
Chap. 18).
This indicates a microdeletion associated with Williams When describing normal results for subtelomeric FISH,
syndrome identified by ish with a probe for the elastin gene. the short form is preferred:
ish.del(2)(q13q13)(NPHP1− −) ish subtel(41×2)
This indicates a homozygous deletion for NPHP1, the This indicates a normal subtelomere FISH result using
gene associated with nephronophthisis. probes to the 41 unique subtelomeric regions.
If conventional cytogenetics was also performed, the Abnormal results following subtelomeric FISH are
karyotype results are written prior to the in situ hybridization written the same way as standard in situ hybridization
(see previous); however, instead of using distal bands and nuc ish(RB1×3),(D18Z1,DSCR)×2
probe names, the presence or absence of a signal may be
designated by a plus or minus sign, following the symbol for Three copies of RB1 and two copies each of D18Z1 and
the appropriate short or long arm (pter or qter), respectively: DSCR were detected.
Many cytogenetics laboratories use one or multiple probe
ish del(8)(qter−) FISH strategies for acquired chromosomal abnormalities
(single fusion, single-fusion with extra signals, dual-fusion,
This indicates a terminal deletion of 8qter identified by and break-apart) to interrogate a specific or targeted regions
subtelomere FISH. of interest for a disease subtype (see Chap. 17). Each type of
probe can be reported with ISCN.
ish der(8)t(8;10)(qter−;pter+)
Dual-Fusion Probes
This indicates an unbalanced translocation between the
distal long arm of chromosome 8 and the distal short arm
nuc ish(ABL1,BCR)×2[200]
of chromosome 10, resulting in loss of the 8q subtelo-
meric region and gain of the 10p subtelomeric region.
This is a negative result using the dual-fusion BCR-ABL1
Alternately, distal band designations and probe names
fusion probe set. The result is from single hybridization, and
may be used:
each probe produced two signals in 200 interphase cells.
ish der(8)t(8;10)(q24.3−,p15.3+)
nuc ish(ABL1,BCR)×3(ABL1 con BCR×2)[100/200]
(RP11−6515−,RP11−581022+)
This is an abnormal (positive) result using the dual-fusion
Interphase FISH (Nuclear In Situ Hybridization, BCR-ABL1 fusion probe set. The result is from a single
nuc ish) hybridization. One hundred cells show three signals for each
When writing a karyotype description using results obtained probe because one probe for each locus is split to form two
from non-mitotic or interphase cells (nuc ish), the number of BCR-ABL1 fusion signals (connected or “con”) on both the
signals and their relative positions are indicated. Because der(9) and der(22), as indicated in the second set of parenthe-
chromosomal bands cannot be visualized in the nucleus of ses (ABL1 con BCR×2). There are also two signals localized
interphase cells, the band location is not necessary in the to the normal chromosomes 9 and 22, for a total of three
nomenclature string. signals for each probe. The abnormal or BCR-ABL1 positive
When single probes are used, the probe name, followed cells were found in 100 of the 200 interphase cells scored.
by a multiplication sign and the number of signals seen, is Normal results do not need to be reported because it is under-
given within parentheses. If multiple probes are used in the stood that the remaining 100 cells scored in this study pro-
same hybridization experiment, they are placed within paren- duced a normal FISH signal pattern.
theses and separated by a comma. The number of signals
seen is placed outside the parentheses if equal for both Break-Apart FISH Probes
probes; otherwise, the number of signals follows each probe
within the parentheses. The description of multiple probes Short system : nuc ish(MLL×2)[200]
follows the same general principles as basic karyotype
description; multiple probes on the same chromosome are Detailed system :
listed pter to qter, and multiple probes on different chromo- nuc ish(5′MLL,3′MLL)×2(5′MLL con 3′MLL×2)[200]
somes are listed in the same order as with cytogenetic
Break-apart probes are made up of two probes that are in
nomenclature—probes for the sex chromosomes are listed
close proximity to one another. The two examples above
first, followed by probes for autosomes in ascending numeri-
depict negative results for MLL. The detailed form indicates
cal order:
that the two probes associated with the 5¢ and the 3¢ part of
nuc ish(DSCR×2) the MLL gene, respectively, are connected (con) and there-
fore represent a normal signal pattern. The short form does
Two copies of DSCR were detected. not convey the normal location of probes, that is, 5¢ versus 3¢
to the breakpoint site, but a negative result is clearly
nuc ish(RB1,D18Z1,DSCR)×2 evident.
Two copies each of RB1, D18Z1, and DSCR were

detected. nuc ish(MLL×2)(5′MLL sep 3′MLL×1)[150/200]
This indicates the 5¢ and 3¢ MLL signals are separated This indicates a normal (diploid) result indicating no
(sep), presumably as the result of a translocation in 150 of evidence of ERBB2 amplification in 60 cells.
the 200 interphase cells scored. If conventional cytogenetics
studies were not performed previously or are negative, meta- nuc ish amp(MYC)[100]
phase FISH studies would be needed to identify the translo-
cation partner. This indicates that 100 cells showed amplification of
MYC, but the number of amplified copies cannot reliably be
Oncology FISH Probe Panels determined.
The following are examples of probe panels for chronic lym-
phocytic leukemia (CLL) where the ATM and TP53 probes Chimerism Studies in Sex-Mismatched Bone
are hybridized in one experiment and probes for chromo- Marrow Stem Cell Transplants
somes 12 and 13 are hybridized in a second experiment: Interphase FISH may be used to quantify the number of
donor and recipient cells after bone marrow stem cell trans-
nuc ish(ATM×1,TP53×2)[75/200], plantation. As used in conventional cytogenetics, the double
(D12Z3×2,D13S319×2,LAMP×2)[75/200] slash (//) denotes a chimeric state.
This abnormal result indicates that an ATM deletion and nuc ish(DXZ1×2)[150]//(DXZ1,DYZ3)×1[200]
gain of chromosome 12 were detected by FISH in 75 of the
200 interphase cells scored. Note that the probes co-hybrid- This FISH result describes 150 recipient (host) female
ized together are reported together. cells and 200 male donor cells seen using probes specific for
the X and Y chromosomes. Note that the recipient (host) cell
nuc ish(ATM,TP53×2)[200], line is listed before the double slant line, followed by the
(D12Z3×2,D13S319×0,LAMP×2)[120/200] donor cell line.
This FISH study is positive for a biallelic 13q deletion. In

this case, the ATM and TP53 probes are hybridized together, Comparative Genomic Hybridization (cgh)
and they are normal, so the ×2 can be placed outside the
parentheses. Compare this example to the previous one. Comparative genomic hybridization (CGH) is a technique in
which genomic DNA from test and reference (control) sam-
nuc ish(MYB,ATM,D12Z3,D13S319,LAMP1,TP53)×2[200] ples are differentially labeled and competitively hybridized
to metaphase chromosomes. CGH detects relative DNA
This describes a normal hybridization pattern in 200 inter- copy-number gains or losses. Alterations detected by CGH
phase cells for all probes scored. When the results are normal may be written as follows:
and the number of cells counted are identical for each hybrid-
ization, the ×2 (normal diploid result) may be written outside ish cgh del(12)(q24.3qter)
the parentheses. However, since not all probes are hybridized
together and more than 200 cells must be examined in total, A deletion of the long arm of chromosome 12 is identified
this can also be described as with CGH.
If FISH is used to confirm the CGH results, the nomencla-
nuc ish(MYB×2)[200],(ATM,TP53)×2[200], ture may be rewritten to incorporate the new findings if they
(D12Z3,D13S319,LAMP)×2[200] clarify the CGH results.
Gene Amplification Detected by FISH

Microarray Analysis (arr)
nuc ish(D17Z1×2,ERBB2×15~20)[52/60]
Array-based comparative genomic hybridization (aCGH)
This indicates that 15–20 copies of ERBB2 (HER2) were has become an important technique for interrogating the
detected in 52 of 60 interphase cell scored. Only two copies entire genome for unbalanced chromosomal abnormalities.
of the chromosome 17 centromere probe were observed in Thousands of probes from throughout the genome are fixed
the same cells. This result is positive for an ERBB2 gene on a solid support and interrogated in a single assay. The
amplification. clones used as targets include cosmids, fosmids, synthetic
oligonucleotides, or bacterial artificial chromosomes (BACs).
nuc ish(D17Z1,ERBB2)×2[60] Unlike aCGH-based microarrays, which perform a direct
comparison between a control sample and a test sample,
single nucleotide polymorphism (SNP)-based arrays deter- Oncology Microarrays

mine relative copy number for a region quantitatively within Similar to constitutional arrays, only the abnormal aberra-
a single genome in comparison to known SNP reference tions are listed in the ISCN. Two examples follow.
controls. See Chap. 18. Microarray analysis of a chronic lymphocytic leukemia
To accommodate varying platforms, microarray nomen- sample shows two microdeletions, a 216 kb deletion
clature specifies nucleotide positions rather than probe types. within band 11q22.3 and a ~1.53 Mb deletion within the
Additional information, such as clone name or accession 13q14.2q14.3 region:
number, gene name, GDB D-number, and type of cloned
DNA, can be included in the final report. arr 11q22.3(108,526,054−108,726,070)×1,
13q14.2q14.3(50,331,912−51,863,519)×1
arr(1−22,X)×2
Microarray analysis of a patient with a myelodys-
arr(1−22)×2,(XY)×1
plastic syndrome showing an ~86.2 Mb deletion of the
long arm of chromosome 5, a 42.8 Mb copy-neutral loss
These represent normal female and male microarray analy-
of heterozygosity (CN-LOH) 7q segment and loss of
sis results. The autosomes are listed first, followed by the sex
chromosome 17:
chromosomes.
If the results are normal using only probes targeted to a specific
arr 5q12.3q32(63,329,099−149,510,083)×1,
chromosome or region, the results are written as follows:
7q31.2q36.3(116,250,176−159,119,707)×2 hmz,
17p13.3q25.3(0−81,195,210)×1
arr(7)×2
Microarray analysis using a microarray comprised of ISCN is a dynamic document that is periodically updated.
clones specific to chromosome 7 shows a normal (diploid) The ISCN Committee met and released a 2013 update [7].
DNA copy number. New terminology was added to accommodate the desc-
For abnormal results, only the relevant regions affected ription of complex oncology microarrays and evolving
are described. For multiple abnormalities, sex chromosome targeted technologies. Four revisions are worth mentioning
aberrations are listed first, followed by the autosomes in here:
ascending chromosome number. Only the band designations 1. For arrays, the genome build should now be specified in
of the aberrant clones are listed. Unlike chromosome nomen- the string:
clature, which is written from the centromere out to the
telomeres, aberrant nucleotides should be listed from pter to arr[hg19]4q32.2q35.19163,146,681−183,022,321)×1
qter for each chromosome to be consistent with the format-
ting of the public genome databases. Multiple nucleotide 2. The triplet “cth” for chromothripsis was added to describe
positions should be listed separated by a comma, or a dash multiple alternating copy number changes (normal, gain,
may be placed between two nucleotide positions to indicate and/or loss) along the length of a chromosome or chromo-
an aberration of the intervening sequence. The parental origin somal segment in neoplastic samples. For example, the
of the abnormality may follow the copy number (×1 mat, ×3 following chromosome 13 aberrations can be described
pat, etc.). There is a space between the copy number and the using the detailed form:
inheritance abbreviation (dn, mat, pat), but a space is not
used if the inheritance abbreviation follows a parenthesis in arr[hg19]13q12.12q12.2(24,226,609−28,672,052)×4,
the detailed system: 13q12.3(29,136,283−30,993,921)×1,13q12.3
(31,377,869−31,803,043)×2,13q13.1q13.2(32,518,057−
arr 4q32.2q35.1(163,146,681−183,022,312)×1 dn 34,768,219)×1,13q13.2(4,771,543−35,030,739)×3,
13q13.2q13.3(35,435,048−35,879,773)×1,13q13.3
arr 4q32.2q35.1(163,002,425×2,163,146,681−183,022,312×1, (36,509,727−36,857,806)×2,13q13.3q14.1(38,167,497−
184,332,231×2)dn 47,219,875)×3,13q14.13q14.3(47,219,983−53,057,363)×1
arr 17p11.2(16,512,256−20,405,113)×3 dn
This may be shortened to:
The specific genome build (human genome assembly [6]) arr[hg19]13q12.12q14.3(24,226,609−53,057,363)cth
may now be added to the array nomenclature if desired:
3. For complex rearrangements seen in high-grade tumor
arr[hg18]6q25.3(157,052,244−157,341,934)×1 samples, the use of “cx” for complex rearrangements by
array may be used for complex cases that cannot be

further characterized. Summary
arr(1−22,X)cx ISCN recommendations provide a strong foundation for
4. A new triplet code has been added for those laboratories communicating consistent and accurate results among cyto-
that use targeted region-specific assays or “rsa.” The rsa geneticists and to physicians worldwide. Valuable teaching
triplet should be used when describing results from tools can be created by supplementing a current copy of ISCN
MLPA, QF-PCR, or bead-based methods. Five examples with illustrations of rare or less frequent cytogenetic abnor-
follow: malities and complex FISH patterns seen in the laboratory.
Despite the fact that ISCN guidelines are merely recommen-
rsa(13,18,21,X)×2 dations, they provide “proof of principle” of how standardized
This is a normal female. concepts keep communication flowing globally, providing a
rsa(13,18,21)×2,(X,Y)×1 means for genetic databases to be compared and contrasted
This is a normal male. to facilitate the application of personalized medicine.
46,XX,rsa 22q11.2(HIRA)×2
Two copies of HIRA were identified after
a region-specific assay. References
46,XY,rsa 8p23.1(GATA4)×1 1. Tjio JH, Levan A. The chromosome number of man. Hereditas.
One copy of GATA4 was identified after 1956;42:1–6.
2. ISCN. In: Shaffer LG, Slovak ML, Campbell LJ, editors. An interna-
a region-specific assay.
tional system for human cytogenetic nomenclature. Basel: S. Karger;
rsa(13)×3 2009.
3. Wyandt HE, Tonk VS, editors. Atlas of human chromosome hetero-
Trisomy 13. morphisms. Dordrecht: Kluwer Academic Publishers; 2008.
4. http://www.genenames.org/guidelines.html. Accessed 13 Apr 2011.
5. http://www.genenames.org/index.html. Accessed 13 Apr 2011.
6. http://www.ncbi.nlm.nih.gov/. Accessed 13 Apr 2011.
7. ISCN. In: Shaffer LG, Schmid MJ, McGowan-Jordan J, editors. An
international system for human cytogenetic nomenclature. Basel: S.
Karger; 2013.
Glossary of commonly used ISCN terms

Term (ISCN abbreviation) Definition
Acentric fragment (a) A segment of a chromosome that lacks a centromere
Additional material Indicates additional material of unknown origin has been added to a chromosome region or band. The “add” triplet
of unknown origin (add) does not imply any particular mechanism, and the additional material may actually replace part of a chromosome arm.
Allele A sequence variant of a gene
Band A part of a chromosome that is distinguishable from its adjacent segments by appearing darker or lighter with one
or more banding techniques
Centromere (cen) The primary constriction that divides the chromosome into the short arm (p arm) and the long arm (q arm); the
region of a chromosome that contains the kinetochore, a microtubule organizing center (MTOC) responsible for
attachment of the sister chromatids to the spindle apparatus at mitosis
Chiasma or chiasmata The point where two homologous non-sister chromatids exchange genetic material during meiosis (sister
(xma) chromatids also form chiasmata between each other, but because their genetic material is identical, it does not
cause any change in the resulting daughter cells). The chiasmata become visible during the diplotene stage of
prophase I.
Chimera (chi) Cell lines originating from different zygotes. A chimera is formed by the merging of two nonidentical twins in
early blastocyst phase or acquired through allogeneic hematopoietic stem cell transplantation.
Chromatid (cht) One of the two identical copies of a replicated chromosome
Chromatid gap (chtg) A nonstaining region of a single chromatid in which there is minimal misalignment of the chromatid
Chromosome (chr) An organized structure of DNA-bound proteins that houses many genes and regulatory elements. Each chromo-
some is made up of DNA tightly coiled many times around histones that support its structure.
Chromosome paint Fluorescent probes stretching over the entire length of a specific chromosome. These probes consist of libraries of
DNA sequences derived from flow-sorted chromosomes.
Clone A cell population derived from a single progenitor cell. A clone comprises two mitotic cells with the same gain or
structural aberration or three cells with the same chromosome loss.
Comparative genomic A molecular cytogenetic method for the detection of copy number changes (gains/losses/chromosomal imbal-
hybridization (cgh) ances) in a patient’s DNA
Composite karyotype (cp) A karyotype containing all clonally occurring abnormalities in a tumor
Constitutional anomaly (c) An abnormality present at conception. The letter “c” in a nomenclature string refers to a constitutional abnormal-
ity that is present in a tumor karyotype.
Deletion (del) A mutation that results in the loss of nucleotides from a DNA sequence or a chromosome
De novo (dn) Genetic mutation that neither parent possesses or transmits to their offspring
Derivative chromosome A structurally rearranged chromosome generated either by a rearrangement involving two or more chromosomes
(der) or by multiple aberrations within a single chromosome. The term always refers to a chromosome that has an
intact centromere.
Dicentric (dic) An aberrant chromosome with two centromeres that forms when two chromosome segments (from different
chromosomes or from the two chromatids of a single chromosome), each with a centromere, fuse, with loss of the
resulting acentric fragments
Double minute (dmin) Extrachromosomal DNA associated with gene amplification and a selective growth advantage in human tumors
Duplication (dup) A segment of a chromosome that is present more than once on that chromosome
Endoreduplication (end) Duplication of the genome without mitosis, giving rise to four-stranded chromosomes at prophase and metaphase
Exchange (e) Refers to either chromatid (chte) or chromosome (chre) exchanges. Exchange is the result of two or more
chromatid or chromosome lesions and subsequent rearrangement of chromatid or chromosome material.
Fission (fis) Centric fission refers to breakage through the centromere resulting in two derivative chromosomes
Fluorescence in situ A technique used to detect and localize the presence or absence of specific DNA sequences on chromosomes
hybridization (FISH) using fluorescent probes that bind with high sequence similarity to the part of the chromosome being interrogated
Fragile site (fra) Heritable chromosomal sites that exhibit gaps or constrictions on chromosomes when exposed to partial
replication stress. Fragile sites are classified as either “common” or “rare,” depending on their frequency.
Heterochromatin, Highly condensed, repetitive DNA found in the centromeres and telomeres that are transcriptionally silent.
Constitutive
Heterozygous (htz) Diploid cell or organism that contains two different variants of a given gene, chromosome, or chromosome
region/arm
Homozygous (hmz) Diploid cell or organism that contains two identical copies of a given gene, chromosome, or chromosome region/arm
Homogeneously staining Intrachromosomal segments of various length and uniform staining intensity after G-banding that house amplified
region (hsr) genes
Idem Latin for the same. Refers to the stemline or most basic acquired aberrations in a subclone of a tumor population. The
terms idem and sl may be used interchangeably if only one additional subclone is present in a tumor population.
Idiogram A diagrammatic representation of a karyotype
Incomplete karyotype (inc) The karyotype present is incomplete, usually because of poor chromosome quality. The term inc is placed at the
end of the nomenclature string, after the description of the identifiable abnormalities.
Glossary of commonly used ISCN terms

Term (ISCN abbreviation) Definition
Insertion (ins) A chromosomal segment is displaced (two breaks) and relocated into a different chromosomal region (necessitating
a third break). The orientation of the inserted segment may be retained in its original orientation or inverted. An
insertion may involve different chromosomes or may be intrachromosomal
Inversion (inv) A chromosomal segment created by two breaks is rotated 180º and reinserted into the same chromsome
Isochromosome (i) An abnormal chromosome with two identical arms
Isoderivative chromosome Designates an isochromosome formation from one of the arms of a derivative chromosome
(ider)
Isodicentric chromosome Designates an isochromosome with two centromeres
(idic)
Karyogram A systematic array (picture or figure) of chromosomes
Karyotype The chromosomal complement of an individual (if constitutional) or tissue (if acquired) or cell line
Landmark A cytological feature of a chromosome that aids in the identification of that specific chromosome, for example,
the centromere, p arm, q arm, telomere, or certain defined bands
Mainline (ml) A quantitative term referring to the most frequent chromosome constitution of a tumor cell population
Marker chromosome (mar) A structurally abnormal chromosome in which no part can be unambiguously identified by conventional banding
techniques
Maternal origin (mat) Derived from the mother
Microarray (arr) An ordered array of microscopic elements on a planar substrate that allows the specific binding of genes or gene
products
Modal number (mn) The most frequent chromosome number in a tumor cell population. The modal number may be expressed as a range.
Mosaic (mos) Two or more cell lines are present
Neocentromere (neo) A functional centromere in a novel (non-centromeric) location. May lack specific classes of deoxyribonucleic
acid (a-satellite DNA) that are usually present in a centromere.
Nucleolar organizing regions A part of the acrocentric short arm that contains tandem copies of ribosomal or rRNA genes in large, clusters (~40
(NOR) copies per gene), present on the stalks of the short arms of chromosomes 13, 14, 15, 21, and 22. NORs are detected
with silver staining.
Paternal origin (pat) Derived from the father
Premature centromere Premature centromere division represents a loss of control over the sequential separation and segregation of
division (pcd) chromosome centromeres because the chromosomes are not attached at the centromere; for example, pcd with
chromatid puffing in areas of constitutive heterochromatin is found in Robert syndrome.
Premature chromosome Results when an interphase cell fuses with a mitotic cell, causing the interphase cell to produce condensed
condensation (pcc) chromosomes prematurely; for example, pcc may be achieved following cell fusion mediated either by fusogenic
viruses or by polyethylene glycol.
Quadriradial (qr) An interchange figure with four chromosome arms
Ring chromosome (r) Results when a chromosome breaks in two places and the ends of the chromosome arms fuse together to form a
circular structure
Reciprocal (rcp) An exchange of material (translocation) between two nonhomologous chromosomes
Recombinant chromosome A structurally rearranged chromosome with a new segmental composition resulting from meiotic crossing-over
(rec) involving a displaced segment and its normally located counterpart in certain types of structural (inversion or
insertion) heterozygotes
Region An area of a chromosome lying between two adjacent landmarks. Regions are numbered outward from the
centromere on both chromosome arms
Robertsonian translocation A translocation involving two acrocentric chromosomes that fuse near the centromere with resulting (inconse-
(rob) quential) loss of the short arms
Sister chromatid exchange An interchange of homologous segments between two chromatids of one chromosome
(sce)
Stemline (sl) A term referring to the most basic clone of a tumor cell population. The stemline is always listed first in the
nomenclature string.
Subtelomeric region (subtel) The chromosomal region just proximal to the telomere (end of the chromosome) comprised of highly polymorphic
repetitive DNA sequences that are typically situated adjacent to gene-rich areas
Telomere (tel) A region of repetitive DNA at the end of a chromosome that protects it from deterioration. In humans, the
telomeres are comprised of a repeating string of TTAGGG, between 5 and 20 kilobases in length, and stain darkly
by T-banding.
Telomeric association (tas) Fusion of chromosomes by their telomeres, which predisposes a cell to genetic instability
Translocation (t) A chromosome abnormality caused by an exchange of genetic material between two chromosomes.
Translocations may be balanced or unbalanced (resulting in loss or gain or material and derivative chromosomes)
Triradial (tr) An interchange figure with three chromosome arms
Uniparental disomy (upd) The condition of having both homologs, a chromosome region, or gene from only one parent
Part II
Examining and Analyzing
Chromosomes
Basic Cytogenetics Laboratory
Procedures 4
Martha B. Keagle and Steven L. Gersen
Introduction The most critical requirement is that living cells capable

of cell division be received by the laboratory. The manner in
The study of chromosomes using traditional cytogenetic tech- which the sample is collected and subsequently handled will
niques requires cells that are actively dividing. Chromosomes greatly influence whether or not the cells will grow and
are individually distinguishable under the light microscope divide, and the quality of the resulting metaphases. Specimen
only during cell division and are best examined during meta- containers must be sterile and must be labeled with the
phase. Metaphase chromosomes can be obtained from speci- patient’s name and a second identifier. The laboratory may
mens that contain spontaneously dividing cells or ones that reject specimens that are improperly labeled or unlabeled.
are cultured and chemically induced to divide in vitro.
Specimens that contain spontaneously proliferating cells
include bone marrow, lymph nodes, solid tumors tissue biop- Specimen Collection and Handling
sies, amniotic fluids, and chorionic villi. If there are not enough
naturally dividing cells for a chromosome analysis, these spec- Sample Requirements
imen types may also be cultured in the laboratory without the
aid of mitotic stimulants. Peripheral blood lymphocytes usu- Peripheral Blood Specimens
ally require the addition of a mitotic stimulant. The choice of Peripheral blood samples should be collected in sterile
specimen for chromosome analysis depends on clinical indica- syringes or vacuum tubes containing preservative-free
tions and whether the diagnosis is prenatal or postnatal. sodium heparin. Vacuum tubes should be discarded if out-
The individual details of culture initiation, maintenance, dated. Peripheral blood cultures can be initiated several days
and cell harvest vary somewhat for the different sample after the blood is drawn; however, for best results, blood
types; however, the general steps and requirements are simi- samples should be set up within 24 h of collection.
lar. These are summarized below. Temperature extremes must be avoided if samples are trans-
ported or stored. Specimens should be kept at room tempera-
Overview of cell culture and harvest ture or refrigerated above 4°C until they can be processed.
Culture initiation → Culture maintenance → Cell harvest Culture medium is sometimes added to small blood samples,
• Living cells • Sterility • Arrest division as these have a tendency to dry up, especially if collected in
• Sterility • Optimal temperature • Swell cells large containers.
• Proper growth medium • Optimal pH • Fix cells A repeat sample should be requested if these requirements
• ± Mitotic stimulant • Optimal humidity • Prepare slide are not met (e.g., the sample is received clotted, on ice, more
• Microbial inhibitors • Optimal time interval • Stain/band than 24 h old). It is not always practicable or possible to
obtain a new sample, and in such cases, the laboratory should
M.B. Keagle, M.Ed. (*) attempt to salvage the original specimen. There may be
Department of Allied Health Sciences, College of Agriculture and enough viable cells for a cytogenetic analysis, though the
Natural Resources, University of Connecticut, 358 Mansfield Road, number and quality of cells may be compromised.
Unit 2101, Storrs, CT 06269, USA
e-mail: [email protected]
Bone Marrow Aspirates
S.L. Gersen, Ph.D.
The collection requirements for bone marrow samples are
Cytogenetics Laboratory, AmeriPath Northeast, 1 Greenwich Place,
Shelton, CT 06484, USA essentially the same as for peripheral blood. Bone marrow
e-mail: [email protected] aspirates should be collected in sterile syringes or vacuum
54 M.B. Keagle and S.L. Gersen
tubes containing preservative-free sodium heparin and

transported at room temperature. The first few milliliters of Culture Initiation
the bone marrow tap contain the highest proportion of cells
and are the best sample for the cytogenetics laboratory. Blood Growth Media
dilutes the bone marrow sample in later taps and reduces the
number of actively dividing cells present in the sample. The All specimens for chromosome preparation are grown and
success of bone marrow culture is dependent on the number maintained in an aqueous growth medium. Some media are
of actively dividing cells. Bone marrow specimens should be formulated for specific cell types (e.g., AmnioMAX™,
processed without delay upon receipt to avoid cell death. Chang Medium®, or Amniochrome™ for amniocytes, giant
cell tumor-conditioned medium for malignancies, PANDIS
Amniotic Fluid Specimens for breast tumors), while others are appropriate for a broad
Amniocentesis can be performed from as early as 10 weeks spectrum of cell types (e.g., RPMI 1640, MEM). All cul-
of gestation until term (see Chap. 12). Fifteen to thirty mil- ture media are balanced salt solutions with a variety of
liliter of amniotic fluid should be obtained under sterile con- additives including salts, glucose, and a buffering system to
ditions and collected in a sterile container approved for cell maintain the proper pH. Phenol red is often used as a pH
culture. For amniocenteses performed earlier than 15 weeks, indicator in many media. If the medium becomes too acidic,
1 mL of fluid is generally drawn for each week of gestation. it will turn yellow, while medium that is too basic becomes
The first few milliliters of an amniotic tap are the most likely pink or purple.
to be contaminated with maternal cells and should not be Commercial media are available either in powder forms
submitted to the cytogenetics laboratory. Samples should be that must be rehydrated, or as ready-to-use aqueous solutions.
transported at room temperature. Temperature extremes and Both complete and incomplete media are commercially
long transport times should be avoided. available, but most commercial media are incomplete.
The amniocentesis procedure has an inherent, albeit small, Incomplete media do not contain all of the nutrients and
risk of miscarriage and should not be repeated unless abso- additives necessary for cell growth. Incomplete culture
lutely necessary. Every effort to salvage samples improperly medium must be supplemented with one or more additives
collected or handled should be made to diminish the need for before being used for cell culture:
a repeat procedure.
L-Glutamine
Solid Tissue Biopsies l-Glutamine is an amino acid essential for cell growth.
Solid tissue sources include skin biopsies, chorionic villi, l-Glutamine is unstable and breaks down on storage to
products of conception, lymph node and solid tumor biopsies, d-glutamine, a form that cannot be used by cells. l-Glu-
and tissue from stillbirths. Products of conception and still- tamine must therefore be stored frozen to retain its stability,
births (and in most cases, tumor biopsies) are one-of-a-kind and it is optimal to add it to the culture medium just prior to
specimens that cannot be recollected, and repeat collection of use. There are some commercially available complete media
chorionic villi increases the risk of miscarriage, although sub- that contain l-glutamine.
sequent amniocentesis is an option here. Microbial contami-
nation is a common problem for many types of solid tissue Serum
samples. Unlike amniotic fluid, blood, bone marrow, and cho- Serum is essential for good cell growth. Too little does
rionic villi, some solid tissue specimens are not sterile prior to not allow for maximum cell growth, but too much can
collection. In addition, viable cells may be few or even non- have a detrimental effect. Fetal bovine serum (FBS) is
existent. These factors threaten the integrity of the sample and preferred; culture medium is generally supplemented
pose problems for the laboratory. with 10–30% FBS.
Small samples should be collected and transported in ster-
ile culture vessels containing growth or tissue culture medium Antibiotics
(not formalin). Sterile saline is not optimal for this purpose but Microbial inhibitors are added to culture media to retard the
should be used if no other option is available. If distance and growth of microorganisms. This is a stopgap measure at best,
timing permit the laboratory to receive and process the sample and should never be relied upon to compensate for sloppy
at once, it may be delivered with no liquid added at all. Larger technique. Good sterile technique is always the best defense
samples may be sent to the laboratory in toto for dissection. against contamination.
Solid tissue samples may be transported and stored on ice until Penicillin/streptomycin, kanamycin, and gentamicin are
culture is established. Storing tissue specimens on ice slows bacterial inhibitors commonly used in tissue culture.
the action of enzymes that degrade the tissue and slows micro- Fungicides routinely used include nystatin and amphotericin
bial growth in the event of contamination. B. Fungicides can adversely affect cell growth and generally
4 Basic Cytogenetics Laboratory Procedures 55
are only used when the potential for contamination out- laboratory. Blood and bone marrow samples consist of single
weighs this potentially negative effect. free-floating cells. For such suspension cultures, sterile cen-
Bacterial contamination of cultures imparts a cloudy trifuge tubes or tissue culture flasks (T-flasks) may be used.
appearance to the culture medium. Fungal contamination The cells from samples such as amniotic fluid, chorionic
presents to the unaided eye as “woolly” masses in the medium, villi, skin biopsies, and other solid tissues need to attach to a
or when observed under an inverted microscope, as branch- surface to grow. Such samples may be grown in T-flasks or
ing hyphae. Mycoplasma and viral contamination can be with an in situ method.
hard to detect and treat. Mycoplasma should be suspected if
the background level of chromosome breaks and rearrange- Flask Method
ments is higher than usual. Cells are grown on the inner surface of T-flasks until ade-
quate numbers of dividing cells are present. Cell growth is
Mitotic Stimulants (Mitogens) monitored using an inverted microscope. To remove the cells
Some cells, particularly mature lymphocytes, do not sponta- from the surface of the culture flask where they have been
neously undergo cell division and must be stimulated to divide growing, the cultures are treated with an enzyme such as
by the addition of an appropriate mitogen to the cell culture. trypsin. This enzymatic treatment releases the individual
Phytohemagglutinin (PHA) is an extract of red kidney beans cells into the fluid environment and permits their collection,
that stimulates division primarily of T-lymphocytes. Cell divi- harvest, or subculture, as needed.
sion starts 48 h after the addition of PHA, with additional waves
of division at 24-h intervals. The culture period for blood speci- In Situ Method
mens is based on this knowledge. For routine peripheral blood Amniotic fluid, CVS, and other tissue samples can be grown
cultures, 72 h is usually optimal. Blood specimens from new- directly on coverslips in small petri dishes, in “flaskettes,” or
borns may require a shorter culture period. T-cell mitogens in slide chambers. Growth of these cultures is also monitored
may also be indicated for bone marrow samples when some with an inverted microscope. They are harvested as “pri-
chronic lymphoproliferative disorders (depending upon the mary” cultures (those that have not been sub-cultured) when
immunophenotype), or well-differentiated T-cell disorders are adequate numbers of dividing cells are present, and cells do
suspected. not have to be enzymatically removed prior to harvest. The
Some hematopoietic studies require stimulation of cells can therefore be analyzed as they grew in situ.
B-lymphocytes, and B-cell mitogens are indicated for bone
marrow samples when some chronic lymphoproliferative Advantages of the In Situ Method
disorders (depending upon the immunophenotype) or mature Over the Flask Method
B-cell disorders are suspected. There are a number of B-cell The primary advantage of using the in situ method is that it
mitogens available, including Epstein–Barr virus, LPS provides information about the colony of origin of a cell.
(lipopolysaccharide from E. coli), protein A, TPA (12-0-tet- This is important when deciding whether an abnormality
radecanoylphorbol-13-acetate), and pokeweed. A cocktail seen in some but not all cells represents true mosaicism (con-
including PHA and interleukin-2 (IL2) has proven successful stitutional mosaicism) or an artifact of tissue culture (pseudo-
as a lymphoid mitogen for bone marrow samples. The syn- mosaicism). True mosaicism is said to be present when there
thetic oligonucleotide DSP-30 has been shown to improve are multiple colonies from more than one culture with the
detection of abnormalities in patients with CLL, often same chromosomal abnormality. Pseudomosaicism is sug-
together with IL2, and may be useful for other B-cell neo- gested if a single colony with all or some cells exhibiting a
plasms as well [1–3]. chromosomal abnormality is found. In such cases, all avail-
able colonies should be studied to rule out the possibility of
Growth Factors true mosaicism. If only a single colony with a potentially
A variety of additional growth factors are commercially viable abnormality is found, it may result in an equivocal
available and are used by some laboratories to achieve opti- diagnosis. Low-level mosaicism cannot be completely ruled
mal cell growth for different sample types. These include out in such cases. Clinical correlation may help clarify the
giant cell tumor extract (GCT) for bone marrow culture and picture. A repeat amniocentesis may confirm the presence of
specially formulated amniotic fluid culture media. true mosaicism but cannot, of course, eliminate the results of
the first study.
No inference can be made about the origin of cells when
Culture Vessels using the flask method, since cells from all colonies are
mixed together after they are released from the growing sur-
Choice of culture vessel depends in part on the growth needs face. It is impossible to tell if multiple cells exhibiting the
of the sample and in part on the individual preference of the same chromosomal abnormality arose from one or multiple
colonies. Thus, two or more cells exhibiting the same optimal temperature for human cell growth is 37°C, and it is
structural abnormality or having the same extra chromosome essential that incubators be maintained at this temperature.
or three or more cells lacking the same chromosome must be Cultures are maintained either “open” or “closed” systems,
treated as potential true mosaics if the flask method is used. depending upon the type of incubator used.
However, it should be noted that the presence of multiple Open systems are those that allow the free exchange of
abnormal colonies in the same in situ culture might also rep- gases between the atmosphere inside the culture vessel and
resent artifact. Guidelines for interpretation of mosaicism are the surrounding environment of the incubator. To facilitate
available for both methods. the exchange of gases, the tops or caps of tissue culture ves-
Another advantage of the in situ method is that there is sels are loosely applied. A CO2 incubator is required for open
usually a shorter turnaround time (TAT), since only primary systems to maintain the 5% CO2 level necessary to sustain
cultures are harvested. Flask cultures are often sub-cultured, the ideal pH of 7.2–7.4. A humidity level of 97% should be
adding days to the culture time. maintained to prevent cell death due to cultures drying out.
This can be accomplished by placing pans of sterile water in
the bottom of the incubator. A major disadvantage of open
Preparation of Specimens for Culture systems is that they are susceptible to microbial contamina-
tion, especially fungi, due to the moist warm surfaces in the
Amniotic fluid specimens, whole blood, and bone marrow incubator. An open system is required for samples grown on
samples arrive in the laboratory as single cells in a fluid envi- coverslips using the in situ method.
ronment. Whole blood or bone marrow can be added directly Closed systems are those in which the culture vessels are
to the culture medium, or the white blood cells can be sepa- tightly capped to prevent exchange of gases. Humidification
rated from the other blood elements and used to inoculate the is self-maintained, and CO2 incubators are not required.
culture medium. Separation of the white blood cells is easily Commercial media are buffered to the appropriate pH neces-
accomplished by centrifuging the sample or allowing it to sary to sustain short-term cultures such as those from blood
rest undisturbed until the blood settles into three distinct lay- and bone marrow samples. Long-term cultures from amni-
ers. The lowest layer consists of the heavier red blood cells, otic fluid and solid tissue specimens require the use of addi-
the top layer consists of plasma, and the narrow middle tional buffering systems to maintain the proper pH over the
layer—the buffy coat—consists of the desired white blood longer culture period. Microbial contamination is not as great
cells. The buffy coat can be removed and used to establish a risk with closed systems.
the suspension culture. In the final analysis, the decision to use an open or closed
Amniotic fluid contains a variety of cells that arise from system, or a combination of both, involves the type of sample
the fetal skin, urinary and gastrointestinal tracts, and the being processed and the preference of the laboratory.
amnion. These are collectively referred to as amniocytes.
Most of the cells in an amniotic fluid sample are dead or
dying and are not suitable for cytogenetic analysis. Amniotic Culture Maintenance and Growth Interval
fluids are centrifuged at low speed (800–1,000 rpm) to
retrieve the small number of viable cells. The cell pellet is Once the culture requirements are met, the cells must be
then used to establish the cultures. The supernatant may be allowed time to grow and divide. The time in culture varies
used for a variety of biochemical tests including a-fetopro- depending upon the cell type involved.
tein (AFP) and acetylcholinesterase (AChE) assays for open Peripheral blood cultures require little maintenance once
fetal defects. the growth requirements have been met. The culture vessels
Solid tissue samples received in the cytogenetics labora- are placed in an incubator for a specified period of time, usu-
tory are usually too large to culture directly and must be dis- ally 72 h.
aggregated before use. To obtain single cells, the sample Likewise, bone marrow cultures need little attention once
must be finely minced using sterile scissors or scalpels, or the culture has been initiated. Bone marrow contains actively
alternately, cell dispersion can be achieved by enzymatic dividing cells and therefore can be harvested directly, with-
digestion of the sample using collagenase and/or trypsin. out any time in culture, or a 24- to 48-h culture time may be
used to increase the mitotic index. Longer culture periods are
generally not advised since the abnormal cancerous cells
Culture Maintenance may be lost over time or be diluted out by normal precursor
cells that may be present. A short growth period usually pro-
After cultures have been initiated, they are allowed to grow vides a more accurate reflection of makeup of the tumor;
under specific conditions of temperature, humidity, and pH however, there are exceptions, as some tumor cells are slow
until adequate numbers of dividing cells are present. The growing, and some mitogens require longer culture times.
Amniotic fluid and solid tissue specimens require longer Mitotic Inhibitor
culture periods and do not grow at predictable rates. Cell
growth is monitored periodically until there are sufficient A mitotic inhibitor must be used to obtain adequate numbers
numbers of dividing cells present, indicating that the culture of cells in metaphase. Colcemid, an analog of colchicine, is
is ready for harvest. An inverted phase-contrast microscope used in most cytogenetics laboratories. Colcemid binds to
is used to visualize the mitotic cells that appear as small, the protein tubulin, obstructing formation of the spindle
refractile spheres. In situ amniotic fluid cultures are gener- fibers or destroying those already present. This prevents sep-
ally harvested at 6–10 days, sometimes earlier. For amniotic aration of the sister chromatids in anaphase, thus collecting
fluid and solid tissue specimens grown using the flask the cells in metaphase. Exposure time to colcemid is a trade-
method, the culture interval may be 2 weeks or more. off between quantity and quality. A longer exposure results
Amniotic fluid and solid tissue specimens cultured with in more metaphases being collected, but they will be shorter
either the in situ or flask method become depleted of required because chromosomes condense as they progress through
nutrients and additives during the culture period. Depleted metaphase. Longer chromosomes are generally preferred for
medium must be removed and replenished with fresh cytogenetic studies. Exposure time to colcemid varies by
medium. This process is called “feeding” the culture and is specimen type.
done on a regular basis throughout the culture maintenance
period dependent upon the number of cells growing, the
length of time in culture, and the protocol of the laboratory. Hypotonic Solution
Exhausted medium becomes acidic and will appear yellow if
the medium contains a pH indicator such as phenol red. A hypotonic solution is added to the cells after exposure to
colcemid. The hypotonic solution has a lower salt concentra-
tion than the cell cytoplasm, allowing water to move into the
Cell Harvest cell by osmosis. This swells the cells and is critical for ade-
quate spreading of the chromosomes on the microscope
After the cell cultures have grown for the appropriate period of slide. Timing is crucial, as too long an exposure will cause
time and there is a sufficient number of dividing cells, the cells the cells to burst. Too short an exposure to hypotonic solu-
are harvested. Harvest is the procedure of collecting the divid- tion will not swell the cells sufficiently, which results in poor
ing cells at metaphase, their subsequent hypotonic treatment spreading of the chromosomes.
and fixation, and the placement of the chromosomes on glass There are a variety of acceptable hypotonic solutions
slides so they may be stained and microscopically examined. including 0.075 M potassium chloride (KCl), 0.8% sodium
The basic steps of cell harvest are the same for all specimen citrate, dilute balanced salt solutions, dilute serum, and
types, with minor variation. An example is shown in Fig. 4.1. mixtures of KCl and sodium citrate. Morphology of the
Fig. 4.1 Overview of peripheral blood cell culture and harvest for chromosome analysis. This procedure, with minor variations, is utilized for all
specimen types
chromosomes is affected by the hypotonic solution used. gist must have an arsenal of techniques to effectively deal
The choice of hypotonic solution is based on specimen type with these variables.
and laboratory protocol. Some labs use slide drying chambers that control airflow,
humidity, and temperature to standardize several of the
important variables in slide preparation.
Fixative Fixed cells from in situ cultures are not dropped because
they are already attached to a coverslip or other solid surface.
A solution of three parts absolute methanol to one part gla- The coverslips are dried under conditions that favor optimal
cial acetic acid is used to stop the action of the hypotonic chromosome spreading (see Chap. 7) and are checked with a
solution and to fix the cells in the swollen state. This fixative phase-contrast microscope for metaphase quality and number.
also lyses any red blood cells present in the sample. The After slides are prepared, they are aged overnight at 60°C
fixative must be prepared fresh before use since it readily or 1 h at 90°C to enhance chromosome banding. There are
absorbs water from the atmosphere, which adversely affects also techniques that allow chromosomes to be “aged” by
chromosome quality and staining. brief exposure to ultraviolet (UV) light.
Slide Preparation Chromosome Staining and Banding
The final step of the harvest procedure is slide preparation. A Prior to the 1970s, human chromosomes were “solid” stained
well-prepared slide has sufficient numbers of metaphases using orcein or other stains with an affinity for chromatin.
that are not crowded on the slide, metaphases that are well The chromosomes were classified according to their overall
spread with minimal overlapping of the chromosomes, and length, centromere position, and ratio of the short arm to
no visible cytoplasm. long arm. Solid stains provided limited information. Simple
Fixed cells from suspension cultures are dropped onto aneuploidies could be recognized, but structural aberrations
glass slides to allow for subsequent staining and analysis. were difficult to characterize and, in some cases, impossible
When the swollen, fragile cells hit the glass slide, the fixative to detect. In addition, it was not possible to specifically iden-
spreads across the slide and begins to evaporate. The surface tify individual chromosomes. See Chap. 1.
tension of the fixative exerts a downward pressure on the A large number of banding and staining techniques have
cells, and the cells become squashed between the slide and since been developed. These can be divided into two broad
the meniscus of the fixative. As the fixative evaporates, the categories: those that produce specific alternating bands
cell membranes are stretched further and further, and the along the length of each entire chromosome, and those that
cells become flatter and flatter, taking up more surface area stain only a specific region of some or all chromosomes.
on the slide. The longer evaporation takes, the more spread Methods that produce specific alternating bands along the
the cells and chromosomes become. The rate of slide drying length of the chromosomes create unique patterns for each
is therefore of major importance in producing good-quality individual chromosome pair. This property allows for the posi-
metaphase spreads. Variables that hasten evaporation (like tive identification of the individual chromosome pairs and
heat and dryness), will decrease spreading, while those that permits characterization of structural abnormalities. These
slow evaporation, (like cold temperature and increased banding techniques answer many questions by facilitating the
humidity) will enhance spreading. numerical and structural examination of the entire karyotype.
A number of variables affect the rate of evaporation of Those techniques that selectively stain specific regions of
fixative from the slide, and thus influence the spreading of chromosomes are used in special circumstances when a
chromosomes, and the overall quality of the slide prepara- particular piece of information cannot be answered using a
tion. Ambient temperature and humidity; the length of the routine banding method. These special stains are typically
hypotonic treatment; the height from which the cells are utilized to obtain such specific data.
dropped; the use of wet versus dry slides; the use of cold
versus room temperature versus warm slides; the use of
steam, airflow, or flaming the slides; and the angle at which Techniques That Create Bands Along
the slide and/or pipette is held all affect spreading of chro- the Length of the Chromosomes
mosomes. Test slides should be made and checked under a
phase-contrast microscope for metaphase quality and An important measurement associated with these methods is
adjustments made if they are not optimal. The concentra- the level of banding resolution obtained. As chromosomes
tion of the cell suspension can also be adjusted if the cells condense during mitosis, sub-bands begin to merge into
are too dense or too dilute on the test slide. Every technolo- larger landmarks along the chromosome. Obviously, as this
Fig. 4.2 G-banding (Giemsa banding). Note the light and dark bands Fig. 4.3 Q-banding. The fluorescence banding pattern is essentially
along the length of each chromosome (Image provided by Alma the same as with G-banding. Note, however, the bright fluorescence on
Ganezer) the long arm of the Y chromosome (arrow)
progresses, the ability to visualize subtle abnormalities is that actually utilize stains other than Giemsa, such as Wright’s
reduced. Chromosomes with a greater number of visible and Leishman’s stains.
bands and sub-bands (higher resolution) are therefore more
desirable. Laboratories accomplish this in two ways: by opti- Q-Banding (Quinacrine Banding)
mizing the banding and staining procedures themselves so Q-banding is a fluorescent technique and was the first banding
that a maximum number of sharp, crisp bands is produced, method developed for human chromosomes (see Chap. 1).
and by choosing (and in some cases manipulating cultures to Certain fluorochromes, such as quinacrine dihydrochloride,
produce) cells with longer, less-condensed chromosomes. will bind to DNA and produce distinct banding patterns of
Cytogenetic nomenclature (see Chap. 3) utilizes approxi- bright and dull fluorescence when excited with the proper
mations of the number of bands present per haploid set of wavelength of light. Because adjacent A-T pairs are necessary
chromosomes, estimates of the number of light and dark to create binding sites, the brightly fluorescing regions are A-T
bands one would arrive at by counting these in one of each rich. The Q-banding pattern is similar to the G-banding pat-
chromosome (the definition of a haploid set). Minimum esti- tern with some notable exceptions. In particular, the large
mates usually begin at approximately 300 bands. Well- polymorphic pericentromeric regions of chromosomes 1 and
banded, moderately high-resolution metaphases are usually 16, and the distal long arm of the Y fluoresce brightly; the
in the 500- to 550-band range, while prometaphase cells can distal long arm of the Y chromosome is the most fluorescent
achieve resolutions of 850 or more bands. site in the human genome. There are also Q-band polymorphic
regions at the centromeres of chromosomes 3 and 4 that can-
G-Banding (Giemsa Banding) not be appreciated with G-banding. Q-banding is therefore
G-banding is the most widely used routine banding method useful to confirm the presence of Y material or when studying
in the USA. GTG banding (G bands produced with trypsin the cited polymorphic regions. See Fig. 4.3.
and Giemsa) is one of several G-band techniques. With this Most fluorescent stains are not permanent and require the
method, prepared and “aged” slides are treated with the use of expensive fluorescence microscopes and a darkened
enzyme trypsin and then stained with Giemsa. This produces room. Q-banding is therefore not conducive to routine work
a series of light and dark bands that allow for the positive in most laboratories and has essentially been supplanted by
identification of each chromosome (Fig. 4.2). The dark bands fluorescence in situ hybridization (FISH) technology.
are A-T-rich, late-replicating, heterochromatic regions of the However, for an example of the clinical application of
chromosomes, while the light bands are C-G-rich, early- Q-banding, see Chap. 20, Fig. 20.3.
replicating, euchromatic regions. The G-light bands are bio-
logically more significant because they represent the active R-Banding (Reverse Banding)
regions of the chromosomes, while the G-dark bands contain R-banding techniques produce a banding pattern that is the
relatively few active genes. There are also G-banding techniques opposite or reverse of the G-banding and Q-banding patterns.
Fig. 4.4 R-banding (reverse banding). The light and dark bands are the opposite of those obtained with G-banding. R-banding can also be per-
formed with fluorescent staining (Image courtesy of Dr. Sylvie Szpiro-Tapia)
There are fluorescent and non-fluorescent methods. The long arm of the Y chromosome. C-band-positive areas contain
C-G-rich, euchromatic regions stain darkly or fluoresce highly repetitive, late-replicating sequences of a-satellite DNA.
brightly, while the A-T-rich heterochromatic regions stain The function of constitutive heterochromatin is not understood,
lightly or fluoresce dully. The euchromatic, R-band-positive but it is stable and highly conserved evolutionarily.
regions are the more genetically active regions of the chro- With CBG banding (C-bands by barium hydroxide, using
mosomes. Many human chromosomes have euchromatic ter- Giemsa), the DNA is selectively depurinated and denatured
minal ends that can be difficult to visualize with standard by barium hydroxide, and the fragments are washed away by
G-band techniques, since the pale telomeres may fade into incubation in a warm salt solution. Constitutive heterochro-
the background. R-banding is a useful technique for the eval- matin resists degradation and is therefore the only material
uation of these telomeres. R-banding is typically used as an left to bind with the Giemsa stain. The result is pale, almost
additional procedure in many countries but is the standard ghost-like chromosomes with darkly stained areas around
method for routine banding in France (Fig. 4.4). the centromeres, at the pericentromeric polymorphic regions
of chromosomes 1, 9, and 16, and at the distal Y long arm
(Fig. 4.5). C-banding is useful for determining the presence
Techniques That Stain Selective of dicentric and pseudodicentric chromosomes, and also for
Chromosome Regions studying marker chromosomes and polymorphic variants.
C-Banding (Constitutive Heterochromatin Banding) T-Banding (Telomere Banding)

C-banding techniques selectively stain the constitutive hetero- T-banding is an offshoot of R-banding that results in only the
chromatin around the centromeres, the areas of inherited poly- terminal ends or telomeres of the chromosomes being stained.
morphisms present on chromosomes 1, 9, 16, and the distal A more harsh treatment of the chromosomes diminishes
Fig. 4.5 C-banding. This technique stains the constitutive heterochro-

matin found in each chromosome (hence the term C-banding) and is
useful for clarification of polymorphisms. Note the large heterochro-
matic regions in some of the chromosomes (Image provided by Alma
Ganezer)
staining except at the heat-resistant telomeres. There are

fluorescent and non-fluorescent T-banding techniques. Fig. 4.6 NOR staining (silver staining). This procedure identifies
active nucleolar organizer regions, found on the stalks of acrocentric
Cd Staining (Centromeric Dot or Kinetochore chromosomes. Silver nitrate produces dark staining in these areas.
Staining) Arrow indicates an abnormal chromosome with satellites at the ends of
both arms (Image provided by Alma Ganezer)
This technique produces a pair of dots at each centromere,
one on each chromatid. These are believed to represent the
kinetochores or the chromatin associated with them. The dots hybrid cells. The human chromosomes stain pale blue, while
are specific to the centromeric region and are not the same as the rodent chromosomes stain magenta.
C-bands. Only active or functional centromeres will stain
with Cd staining, in contrast to C-banding that will stain inac- NOR Staining (Silver Staining for Nucleolar
tive as well as active centromeric regions. Cd staining can be Organizer Regions)
used to differentiate functional from nonfunctional centrom- This technique selectively stains the nucleolar organizer
eres and to study Robertsonian translocations (centromere to regions (NORs) located on the satellite stalks of the acrocen-
centromere translocations of acrocentric chromosomes), ring tric chromosomes. These regions contain the genes for ribo-
chromosomes, and marker chromosomes. somal RNA and can be stained with silver nitrate.
Theoretically, there are ten NORs per cell, one for each acro-
G-11 Banding (Giemsa at pH 11) centric chromosome. However, not all will usually stain at
This technique specifically stains the pericentromeric regions any one time because the silver stains the activity, not pres-
of all chromosomes, the heterochromatin regions of chromo- ence, of rRNA genes. NOR staining is useful for the
somes 1, 9, 16, and the distal Yq, and the satellites of the identification of marker chromosomes and rearrangements
acrocentric chromosomes. An alkaline treatment of the chro- or polymorphisms involving the acrocentric chromosomes.
mosomes causes loss of the Giemsa binding sites. Optimal See Fig. 4.6.
results are achieved at pH of 11.6. At this high alkaline pH,
only the azure component of Giemsa binds with the majority of DAPI/DA Staining (4,6-Diamino-2-Phenole-
the chromosomes, staining them light blue. The eosin compo- Indole/Distamycin A)
nent of Giemsa binds specifically to the heteromorphic regions This stain combines DAPI, a fluorescent dye, with distamy-
cited previously, staining them magenta. G-11 banding is used cin A, a non-fluorescent antibiotic. Both form stable bonds
for delineating these heterochromatin polymorphisms. preferentially to similar, but not identical, A-T-rich, double-
G-11 banding also has research applications. It is used to stranded regions of DNA. Used together, DAPI/DA fluoresces
differentiate between human and rodent chromosomes in certain A-T-rich areas of constitutive heterochromatin in the
C-band regions of chromosomes 1, 9, and 16, the distal Yq, The peak prometaphase index occurs 5–6 h later, and this is
and the short arm of chromosome 15. Prior to the develop- when the harvest is performed.
ment of fluorescence in situ hybridization techniques, this To achieve cell synchrony, (+) amethopterin or methotrex-
was the only stain that differentiated between satellite regions ate (MTX) can also be used, and BrdU (5-bromodeoxyuridine),
of any of the acrocentric chromosomes. an analog of thymidine, can be used to release the block.
DAPI/DA is used to identify rearrangements of chromo-
some 15; to confirm variations in the polymorphic regions of
chromosomes 1, 9, and 16 and distal Yq; and to study marker Chemical Elongation
chromosomes with satellites.
Ethidium bromide (EB) can be added to cultures prior to har-
Fluorescence In Situ Hybridization (FISH) vest to achieve longer chromosomes. Ethidium bromide acts
The development of fluorescence in situ hybridization tech- by intercalating between the bases of DNA, thus preventing
nology represents an important advancement in cytogenet- or slowing its contraction. This results in the collection of
ics. FISH is a marriage of classical cytogenetics and long, if not truly prometaphase, chromosomes. The proce-
molecular technologies and has a large number of applica- dure is technically very simple and is used routinely on blood
tions (see Chap. 17). While many laboratories still utilize and bone marrow cultures.
traditional special stains in select circumstances, FISH tech- The major drawback to using EB is that it is highly muta-
niques have replaced special stains in many laboratories. genic. Extreme care must therefore be taken when utilizing
this reagent.
Newer, less toxic reagents that produce similar results
Chromosome Elongation Studies have recently become available, including Chromosome
Resolution Additive (CRA).
Chromosomes are routinely examined during metaphase, In previous decades, before the introduction of molecular
when they are at their most contracted state. While this is analysis for fragile X syndrome (see Chap. 19), the diagnosis
often sufficient for chromosomal analysis, small structural of this disorder was made in the cytogenetics lab, using special
abnormalities may not be detected in chromosomes of meta- culture conditions. Among these was the inclusion of FUdR,
phase length. In such cases, longer, less-contracted prophase described previously. Laboratories observed that one by-
or prometaphase chromosomes are needed. Historically, product of this procedure was longer chromosomes. Although
these were referred to as high-resolution cytogenetic studies, the exact mechanism is not known, the addition of FUdR to
but with the advent of arrays (see Chap. 18), they are no lon- blood cultures 24 h prior to harvest does in fact seem to produce
ger truly high resolution, so the term has gone into disfavor. chromosomes of greater length, and this technique is used in sev-
To achieve longer chromosomes, the cells can be synchro- eral labs. One consideration, however, is that this can facilitate
nized and harvested earlier in the cell cycle, or chemical the expression of folate-sensitive fragile sites (see Chap. 14).
elongation techniques can be used to prevent condensation Some laboratories employ an amniotic fluid harvest tech-
of the chromosomes. nique that includes overnight exposure to colcemid. Many
have also found that the addition of BrdU to these cultures
also increases chromosome length, probably by replacing
Cell Synchronization Techniques thymidine with a larger base, thereby reducing chromosome
condensation.
Randomly dividing cells can be synchronized with knowl-
edge of the average timing of the stages of the human cell
cycle. The cells are blocked and then released at the appro- Culture Failure
priate time so that a large percentage of cells accumulate in
prophase or prometaphase at the time of harvest. There are All culture failures must be investigated. The circumstances
several protocols for generating such synchronization. of the failure should be recorded as a part of an ongoing
One method involves the addition of FUdR quality assurance program (see Chap. 6). A record of failure
(5-fluorodeoxyuridine) to peripheral blood cultures prior to rates for each specimen type in the laboratory must be kept
harvest. FUdR is an inhibitor of thymidylate synthetase, as a baseline so that deviations from the norm can be detected.
which plays an important role in the folic acid pathway. Folic It is important to isolate the reason(s) for a culture failure so
acid is required for incorporation of thymidine during DNA that steps can be taken to prevent future similar failures.
synthesis. The addition of FUdR blocks cell division at the Some culture failure is unavoidable, but adherence to strict
G1/S border. After 17 h, the accumulated cells are released standards and rigorous investigation of all failures should
from the block by the addition of a high level of thymidine. keep this number to a minimum.
There are many possible origins of culture failure. It can stimulants. The laboratory is not always made aware of these
be due to improper specimen collection or transport, improper confounding factors. Bone marrow samples that have been
laboratory technique, or the condition of the sample. There contaminated with blood may not have adequate numbers of
are general sources of failure that apply to all sample types spontaneously dividing cells present. For this reason, it is
and specific ones that pertain to one or more of the sample important that the cytogenetics laboratory receive the first
types. few milliliters of the bone marrow tap. Bone marrow samples
Errors in sample collection and handling include failure are notorious for producing poor-quality metaphases. There
to submit an adequate amount of sample, collection under are sometimes adequate numbers of metaphases, but the
non-sterile conditions resulting in microbial contamination, chromosomes are so short and so poorly spread that analysis
use of an inappropriate collection vessel or medium, failure is difficult or impossible. In addition, metaphases of poor
to use an anticoagulant, use of an inappropriate or expired quality often represent an abnormal clone.
anticoagulant, delay in transport, and improper storage The failure rate of solid tissues may be quite high and is
before and/or during transport of the sample. often due to the samples themselves. In the case of products
In the laboratory, errors can occur at any step from culture of conception or stillbirths, the sample may not contain via-
initiation to staining. Failure to follow proper protocol can ble cells, or the wrong tissue type may have been collected.
cause loss of a culture. This is one reason for establishing Additionally, microbial contamination is a frequent contrib-
multiple cultures for all samples and harvesting them at dif- uting factor, since many solid tissue samples are not sterile
ferent times. Faulty media, sera, or other reagents can also prior to collection.
result in culture failure. It is therefore important to test all
new lots of media and sera for sterility and ability to support
cell growth before using these on patient samples. It is also Preservation of Cells
important to maintain a log of lot numbers of all reagents
used and the date each was put into use to help identify the Cells do not survive indefinitely in tissue culture. After a
source of any problem. During the culture period, improper period of time, they become senescent and eventually die. At
temperature, CO2 level, or pH of the culture can have delete- times, a sample may need to be saved for future testing, to
rious results. The temperatures and CO2 levels of all incuba- look at retrospectively, or because it is unusual or interesting
tors must therefore be monitored and recorded at least daily, and might be of some value in the future. In such cases, the
and samples should be split and grown in separate incubators cells need to be kept alive and capable of division long term
in the event an incubator malfunctions. In general, all equip- or indefinitely.
ment used in the laboratory must be monitored at regular Cultured cells can be kept alive by cryopreservation, the
intervals and maintained to prevent malfunction. storage of cell in liquid nitrogen. The freezing process is
Lack of viable cells or unsuitable cell type can compro- critical to cell survival. Rapid freezing will cause cell death
mise amniotic fluid samples. Samples from patients with due to formation of ice crystals within the cells. Improper
advanced gestational age (20 weeks or greater) may consist freezing can also denature proteins, alter the pH, and upset
primarily of mature nondividing cells or dead cells. Some electrolyte concentrations. The cells must be cooled slowly
samples consist principally of epithelial cells, which typi- so that water is lost before the cells freeze. The addition of
cally produce few metaphases of poorer quality than the 10% glycerol or dimethyl sulfoxide (DMSO) to the storage
desired fibroblasts. medium lowers the freezing points and aids in this process.
Amniotic fluid samples are usually clear yellow in One-milliliter aliquots of the sample in storage medium are
appearance. A brown fluid indicates prior bleeding into the placed in cryogenic freezing tubes. The samples are then
amniotic cavity, which may suggest fetal death or threat- slowly frozen under controlled conditions at a rate of 1°C per
ened miscarriage. In such samples, there may be few if any minute to a temperature of −40°C. The sample can then be
viable cells present. Bloody taps containing large numbers rapidly frozen to about −80°C. Alternately, the samples may
of red blood cells can be problematic. The physical pres- be placed in a −70°C freezer for 1–4 h. After this initial
ence of large numbers of red blood cells can prevent the freezing has been accomplished, the cells are stored in the
amniocytes from settling on and attaching to the growth liquid phase at about −190°C.
surface of the culture vessel. In addition, the red cells uti- Thawing of the sample is also critical. Rapid thawing is
lize nutrients in the culture medium, thereby competing necessary to prevent the formation of ice crystals.
with the amniocytes. B-lymphocytes can be transformed so that they will pro-
Patient factors can influence the success of peripheral liferate indefinitely in tissue culture by exposing them to
blood and bone marrow samples. Disease conditions, immu- Epstein–Barr virus (EBV). These immortalized lymphoblas-
nosuppression, and use of other drugs can affect both the toid cell lines do not become senescent and can therefore be
number of lymphocytes present and their response to mitotic maintained indefinitely in culture.
begin with the microscope, where selection of appropriate

Chromosome Analysis metaphases begins the process. Although technologists are
trained to recognize well-spread, high-quality cells under
Selection of the correct specimen for chromosome analysis low-power magnification, they must also remember to exam-
and additional tests is not always straightforward, and the ine some poor-quality metaphases when analyzing hemato-
submission of an inappropriate sample to the laboratory can logical samples, as these often represent abnormal clones.
create frustration for both patient and clinician. Under high power, the chromosome morphology and band-
This was not always as complex an issue as it is today. In the ing resolution are evaluated. If these are appropriate, the num-
1970s, prenatal diagnosis involved an amniotic fluid specimen, ber of chromosomes is counted, and the sex chromosome
often obtained at exactly 17 weeks of gestation, for chromo- constitution is typically determined. The microscope stage
some analysis and alpha-fetoprotein testing. Other tests were coordinates of each metaphase are recorded, and in many lab-
available but rare. The cytogenetic contribution to hematology/ oratories, an “identifier” of the cell is also noted. This is typi-
oncology essentially involved whether a bone marrow speci- cally the position of one or more chromosomes at some
men was “positive or negative” for the “Philadelphia chromo- reference point(s) and serves to verify that the correct meta-
some.” Constitutional chromosome analysis from peripheral phase has been found should there be a need to relocate a cell.
blood implied that the patient had to be an adult or a child. Any other characteristics of the metaphase being examined,
Today’s prenatal caregivers and their patients must choose such as a chromosome abnormality or quality of the banding
between traditional amniocentesis, early amniocentesis, chor- and chromosome morphology, are also noted.
ionic villus sampling, or, sometimes, percutaneous umbilical In the United States, certifying agencies such as the
blood sampling. A decision must be made concerning whether College of American Pathologists (CAP) require that a mini-
ploidy analysis via FISH is warranted, and acetylcholin- mum number of metaphases be examined for each type of
esterase is often a factor in the diagnosis of certain open fetal specimen, barring technical or clinical issues that can some-
lesions, but AFP and AChE cannot be performed on all sam- times prevent this (see Chap. 6). There are also requirements
ple types. Many disorders can be also diagnosed by biochem- for a more detailed analysis (typically band-by-band) of a
ical or molecular methods, and ethical dilemmas surround the certain number of cells, as well as standards for the number
potential to diagnose, prenatally, late-onset disorders such as of metaphases from which karyograms are prepared.
Huntington’s disease. Screening for increased risk or predis- Regulations notwithstanding, it is clearly a good laboratory
position to developing certain cancers or other diseases has practice to analyze every chromosome completely in several
created new moral and ethical pitfalls. Each collection method cells and even more important to check all chromosomes in
may ultimately affect the number of cells available for chro- certain situations, such as when analyzing cancer specimens.
mosome analysis, and all of these issues can play a role in the Depending upon the results obtained and/or initial diagnosis,
timing and choice of sampling procedure. additional cells may be examined in order to correctly iden-
Today, the cytogenetics laboratory provides indispensable tify all cell lines present (see Chap. 6 for additional discus-
information for the diagnosis, prognosis, or monitoring of sion of guidelines and standards).
patients with a wide variety of hematological disorders and Once the appropriate number of mitotic cells has been
other neoplasms, using not only bone marrow but also, in examined and analyzed, a representative sample must be
some cases, blood, lymph node biopsies, or tumor tissue or selected for imaging and ultimate preparation of karyograms.
aspirates. Treatment decisions often rest on the results of a Today, computer imaging and automated production of karyo-
chromosome analysis, but some tissue types are only appro- grams have virtually replaced traditional photography and
priate under certain conditions, and an incorrect selection manual arrangement of chromosomes (see Chap. 7). Many
here can delay a vital diagnosis. laboratories also image additional cells to be included as ref-
A blood sample today could be from a patient with leuke- erences in the patient chart. Ultimately, summary information
mia or from a fetus rather than a child or an adult suspected (patient karyotype, banding resolution, number of cells exam-
of having a constitutional chromosome abnormality. These ined, analyzed, imaged, etc.) is recorded in the patient’s file
must all be handled differently, and the information they pro- and is used in the clinical report (see Chap. 6).
vide is unique in each circumstance. The final steps of the process typically involve a clerical
review of all relevant clinical, technical, and clerical data,
examination of the patient’s chart and karyograms by the labo-
Procedure ratory director (often preceded by the supervisor and/or other
senior laboratory personnel), and generation of the formal
After all of the appropriate laboratory manipulations and clinical report. In addition to the appropriate physician and
staining procedures have been performed, there are several patient demographic information, this should include the
steps involved in the clinical analysis of chromosomes. These number of metaphases that were examined microscopically,
the banding resolution obtained for the specimen, the number Rooney DE, editor. Human cytogenetics: constitutional analysis. 3rd
of cells that were analyzed in detail, the number of karyograms ed. Oxford: Oxford University Press; 2001.
Rooney DE, Czepulkowski BH, editors. Human cytogenetics: a practi-
prepared, the patient’s karyotype, and the clinical interpreta- cal approach, volume I constitutional analysis. New York: IRL
tion of the results, including, where appropriate, recommenda- Press, Oxford University Press; 1992.
tions for additional testing and/or genetic counseling. Verma RS, Babu A. Human chromosomes. New York: McGraw-Hill,
Inc.; 1995.
The American College of Medical Genetics (ACMG) Standards and
Guidelines for Clinical Genetics Laboratories. ACMG 2009 Edition/
Summary Revised 01/2010; www.acmg.net/StaticContent/SGs/Section_E_
2011.pdf.
The purpose of this chapter is to provide a general overview of
the many steps involved from receipt of a sample in the cytoge-
netics laboratory to the generation of a patient report and to
References
impress upon the reader the labor-intensive nature of this work.
While the basic procedure is always the same, there are cultur- 1. Dicker F, Schnittger S, Haferlach T, Kern W, Schoch C.
ing and processing variations that are sample type-dependent, Immunostimulatory oligonucleotide-induced metaphase cytogenet-
choices of methodology that are diagnosis-dependent, and ics detect chromosomal aberrations in 80% of CLL patients: a study
microscopic analysis decisions that are results-dependent. All of 132 CLL cases with correlation to FISH, IgVH status, and CD38
expression. Blood. 2006;108:3152–60.
of these in turn depend upon individuals with the appropriate 2. Strucsi S, Gervais C, Helias C, Herbrecht R, Audhuy B, Mauvieux
expertise and dedication to patient care. L. Stimulation of B-Cell Lymphoproliferations with CpG-
Due to the nature of this chapter, individual citations were Oligonucleotide DSP-30 Plus IL-2 Is More Effective than with
not always practical. In addition to the references cited TPA to Detect Clonal Abnormalities. Poster presented at American
Society of Hematology 50th Annual Meeting and Exposition,
below and the authors’ personal experience, the following 2008.
were used as supplemental sources of information: 3. Meloni-Ehrig A, Meck J, Christacos N, et al. Stimulation of B-cell
mature malignancies with the Cpg-oligonucleotide DSP30 and IL2:
Barch MJ, Knutsen T, Spurbeck JL, editors. The AGT cytogenetic labo- which malignancies respond best? Poster presented at American
ratory manual. Philadelphia: Raven-Lippincott; 1997. Society of Hematology 51st Annual Meeting and Exposition, 2009.
The Essentials of Light Microscopy
5
Nathan S. Claxton and Stephen T. Ross
The Microscope Optical Train and Conjugate

Introduction
Planes
Light microscopy is an indispensible tool in the cytogenetics
A knowledge of conjugate focal planes helps in the under-
laboratory, both for routine analysis and for techniques such
standing of how the various components of the microscope
as fluorescence in situ hybridization (FISH). Modern tools,
work in concert to produce the best possible images. There
such as digital imaging and advances in image analysis, have
are two sets of conjugate focal planes, typically referred to as
aided the microscopist in drawing conclusions from micro-
the “image” plane and “aperture” plane. When aligned properly,
scope images. A clear understanding of optical systems and
the specimen, field diaphragm, and detector (eye or camera)
the trade-offs involved in imaging is essential to maximizing
are focused together so that a sharp image of the specimen
the quality of such images. This chapter covers the basic
is projected onto the detector. The “aperture” components
principles and applications of the light microscope, including
(the light source, condenser aperture, and back focal plane of
brightfield and contrasting techniques, and an introduction to
the objective) are also focused to each other in a reciprocal
the fluorescence microscope.
manner relative to the image plane. Light rays focused in one
set of conjugate planes are nearly parallel in the reciprocal
set of conjugate planes. The microscope cutaway in Fig. 5.1
Brightfield Microscopy shows conjugate planes in a modern upright microscope.
The modern compound microscope is the most important
Köhler Illumination
diagnostic tool in the cytogenetics laboratory and is designed
Proper centering and focusing of the condenser is necessary
to enhance the observable detail of specimens by magnifying
for the full and even illumination that is essential for good
images, resolving structures, and applying various contrasting
resolution and high-quality imaging. Köhler illumination,
techniques. In transmitted light brightfield microscopy, spec-
developed in the late nineteenth century by August Köhler,
imens are typically mounted on glass slides and light is
completely defocuses the inherently uneven light source
passed through them to illuminate and resolve structures not
(such as an incandescent filament) in the image plane resulting
visible to the naked eye. While some specimens may be
in a very even field of illumination, while focusing the light
viewable in their natural state, optical dyes and stains are
source in the aperture plane for optimal brightness and reso-
often used to add contrast to typically transparent and color-
lution. Setting Köhler illumination requires a lamp with a
less cellular features.
collector lens to focus light at the front aperture of a focus-
able and centerable condenser. A simple method for setting
basic Köhler illumination follows:
1. Using a low-magnification objective, place a specimen on
the stage and bring it into focus. Remove the specimen.
2. Close down the field aperture, typically located at the base
of the microscope, so that the shutter blades are visible in
the field of view.
N.S. Claxton (*) • S.T. Ross, Ph.D. 3. Rack the condenser up or down until the field aperture
Product and Marketing Department, Nikon Instruments Inc.
1300 Walt Whitman Road, Melville, NY 11747-3064, USA blades come into sharp focus and center it in the field
e-mail: [email protected] of view.
68 N.S. Claxton and S.T. Ross
Fig. 5.1 Conjugate planes in the optical microscope (cutaway). In this

cutaway of a modern microscope, “image” planes are marked in black
while “aperture” planes are marked in red, denoting the two conjugate
sets of light planes (Figure used by permission of Michael W. Davidson
of the National High Magnetic Field Laboratory and MicroscopyU.com)
Fig. 5.2 Conjugate planes in the optical microscope (light paths).
4. Reopen the field diaphragm to the field of view, whether The ray-trace shown in red illustrates the path taken by light focused at
eye or digital detector. or originating from an aperture plane. The ray-trace shown in yellow
illustrates the path taken by light focused at or originating from an
This aligns the illuminating components into precise image plane (Figure used by permission of Michael W. Davidson of the
physical locations resulting in optimal illumination. Köhler National High Magnetic Field Laboratory and MicroscopyU.com)
illumination is also the starting point for the proper operation
of various advanced contrasting techniques and should be
performed each time the microscope is used and for indi- An adjustable rheostat, located on the microscope body
vidual objectives. Figure 5.2 shows the conjugate planes of a or the external power supply, regulates the voltage deliv-
microscope aligned for Köhler illumination in both the illu- ered to the bulb and adjusts the intensity of the light. With
minating and image-forming light paths. incandescent bulbs, such voltage adjustment also changes
the color temperature, and this can cause major changes to
Transmitted Light Source the hue or color property of the image, particularly when
The transmitted light source for brightfield microscopy is combined with digital imaging. Neutral density filters
usually located in an external housing or the microscope (NDs) can be used to control the light intensity without
base and is most commonly an incandescent tungsten-halo- changing the color balance by attenuating light evenly
gen bulb. Some newer microscopes have integrated light- across the entire spectrum. While digital cameras have
emitting diodes (LEDs). The housing reflects as much light greater ability than film to adjust for changes in color tem-
as possible toward the collector lens, which then directs perature by automatically “white balancing,” which digi-
light into the microscope condenser. In some cases, the tally shifts the hue of colors in relation to each other, the
light source is manually centered and focused, but most use of NDs to adjust light intensity reduces this need.
modern housings automatically center the bulb. Halogen Unlike incandescent bulbs, LEDs have a constant color
bulbs produce heat and often require some amount of temperature regardless of voltage adjustment.
ventilation, while LEDs operate at much cooler tempera- Halogen bulbs emit a continuous spectrum of light that
tures and have extended life. extends from about 300 to 1,400 nm. The collector lens for
5 The Essentials of Light Microscopy 69
the lamp typically blocks ultraviolet (UV) light, while a condenser that delivers that light. For optimal resolution, the
separate infrared (IR) filter may be used to block IR light that NA of the condenser should closely match or exceed the NA
can cause eye strain and high background on digital images. of objective lens.
LEDs tuned for white light do not emit UV or IR light, thus The simplest common condenser is known as an Abbe
reducing eye strain for the operator and eliminating the need condenser named for its inventor, Ernst Abbe. While Abbe
for extra UV- or IR-blocking filters. A neutral color-balancing condensers are available with a variety of NAs, they do not
filter, typically NCB11, is often placed in the light path to have significant correction for optical aberrations. The Abbe
adjust the color temperature of the incandescent light nearer condenser can be used for basic inspection of routine
to that of daylight, while LEDs are often pre-tuned to this brightfield samples but may not be suitable for critical or
color balance as a manufacturing specification without the high-detail investigations, such as in cytogenetics.
need for an additional filter. Aplanatic condensers are corrected for spherical aberra-
Other filters may be used to increase the visual contrast tion, which is an optical imperfection characteristic of lenses
in cytogenetics specimens. For instance, contrast in with curved surfaces in which light rays passing near the lens
G-banded chromosomes can be improved using a simple periphery focus to a different point than rays traveling
green glass filter that absorbs light of all colors except green. through the center of the lens, leading to a reduction in sharp-
Depending on the correction level of the microscope optics, ness. Correction for this aberration may be accomplished for
performance may be improved with monochromatic green individual wavelengths of light. The performance of apla-
light. In this case, a more efficient green interference filter is natic condensers is best using green light, and this is assumed
better at producing monochromatic green light for the best in the optical design since aplanatic condensers are not
imaging conditions. Interference filters reject unwanted corrected for chromatic aberrations.
wavelengths by reflecting and causing destructive interfer- Chromatic aberration in microscopy generally refers to
ence. A green interference filter (often labeled GIF) can be axial chromatic aberration, in which light of differing wave-
differentiated visually from a green glass filter by its unique lengths does not focus to the same point. Achromatic con-
reflective property, which often produces an orange or yel- densers are corrected for axial chromatic aberrations to bring
low tint when viewed at an angle. Interference filters, includ- blue and red light to the same focus as green light, but they
ing those used for fluorescence, have very thin-layered are not corrected for spherical aberration. Aplanatic-
coatings on their surfaces, and great care should be taken achromatic condensers are corrected for both spherical and
when cleaning them. chromatic aberrations.
The effective NA of the condenser can be adjusted by
Field Diaphragm, Condenser, and Aperture opening or closing the aperture diaphragm, thus increasing
Diaphragm or reducing the angle of light entering the specimen and
The field diaphragm, typically located after the light source objective. Since higher NA relates to higher resolving power,
and its associated filters, is an adjustable iris-type diaphragm it may seem counterintuitive to purposely reduce the NA of
that defines the total area of illumination. It should be opened the condenser by restricting the diaphragm. However, as the
just past the field of view, whether to the eye or camera sensor, aperture diaphragm is closed down and the NA is reduced,
to fully illuminate the specimen while reducing stray light. visual contrast and depth of field are both increased even as
Proper Köhler illumination will focus the image of the field ultimate resolving power is decreased. Visual contrast lost
diaphragm in the specimen plane. by opening the aperture can often be restored by processing
The microscope condenser gathers and focuses light from digital images. A clear understanding of this interplay
the source and passes it through the specimen, providing full between contrast and resolution helps the user navigate
and even illumination. The condenser assembly contains an challenging samples and extract the maximum amount of
adjustable diaphragm in its front focal plane known as the information.
aperture diaphragm and may also house various light condi-
tioners used in advanced contrasting techniques.
The maximum angle of incidence for light rays in the Objective Lenses
cone of light that the condenser can deliver is determined by
the numerical aperture (NA) of the condenser. This optical The objective lens is the major determinant of magnification
property of lenses ultimately determines the resolving power and resolution and is perhaps the most significant single
of the optical system, which is the limit of its ability to sepa- component of the optical train with regard to observational
rate fine details. While the objective lens may be the most capability. Most manufacturers offer a wide selection of
prominent component that affects magnification and resolution, objectives with various magnifications and NAs and with
the effective NA (and thus resolving power) of the objective varying optical design considerations for aberration correc-
collecting transmitted light cannot exceed the NA of the tion and application. Objectives typically have markings on
the barrel, indicating magnification, NA, aberration correc- 2.5 times per 27.6 mm, the physical pixel size of the detector
tion, working distance, immersion medium, and coverslip must be smaller than 27.6/2.5 or ~11 mm. However, if the
correction. magnification of the objective were only 40×, the resolved
size at the detector is 276 nm × 40 or 11.04 mm, which would
Resolution vs. Magnification require a pixel size smaller than 11.04/2.5 or ~4.4 mm.
The two main functions of objectives are to resolve detail
and magnify the image. It is a common misconception that Objective Types
magnification determines resolution when, in fact, the Another major consideration in objective selection is the
resolving power of a lens is a function of numerical aperture. level of correction for optical aberrations. Common types
The ability to resolve detail is directly tied to the numerical of objectives include achromat, plan, fluorite, and apochro-
aperture of the objective such that higher NAs translate to mat. While the specific details of correction naming con-
higher resolving power, while magnification makes those ventions are not completely standard across all manufacturers
resolved details easier to observe. Magnification increases in the industry, these serve as a good general description of
size but not resolution. the type of optical corrections engineered in the lens.
As waves of light encounter very small objects, particu- Plan lenses are corrected for flatness of field so that the
larly of a size approaching the wavelength of the light, they periphery of the field of view lies in the same focal plane as
are bent around the object. This property of light is described the center. Non-plan lenses may exhibit field curvature in
as diffraction. It can also refer to the way light waves spread which the edges of the image are out of focus, while the center
as they pass through small apertures. As light is passed is in focus. Flatness of field is very important for digital
through a small object of interest on a microscope, such as a imaging, though in practice, even non-plan lenses may be flat
chromosome, the light that holds the information about that in the field of view of the camera sensor, which is often
object may propagate at very steep angles relative to the smaller than the full visual field of view. The plan designa-
optical axis. NA describes an objective’s ability to collect tion is often combined with other corrections, such as plan
this highly diffracted light. The more highly diffracted the achromat, plan fluorite, or plan apochromat.
light that is collected, the better that object can be resolved Modern achromat objectives are corrected for spherical
by the lens. Since NA describes light-collecting ability, it aberration in a single wavelength (typically green light) and
also indicates relative brightness of the image produced, axial chromatic correction in two colors (typically blue and
which is of major importance in light-challenged applica- red). If color information is not necessary, an achromat
tions such as fluorescence. objective will perform best with monochromatic green light
The method of observation (visual vs. imaging system) due to the correction for spherical aberration in this wave-
should be considered when selecting magnification. For length range.
visual inspection, magnification selection is dependent on Fluorite objectives (also called FL, fluor, fluar or semi-apo),
whether relevant resolved details can be observed by the whose name is derived from the calcium fluoride crystal
human eye. For digital imaging, the required level of called fluorite or fluorspar from which the lenses were origi-
magnification needed to accurately record resolved details nally manufactured, are corrected for spherical aberration in
depends on the physical pixel size of the detector. In general, at least two colors and chromatic aberration in at least two
higher NAs call for smaller pixels, which are usually a con- colors.
sequence of a greater number of pixels on the detector, but Apochromat objectives (often abbreviated apo) are the
this need is mitigated as magnification increases. The Nyquist most highly corrected lenses and are corrected for spherical
sampling theorem states that to accurately record and recon- aberration in at least three colors as well as chromatic aber-
struct a continuous analog signal using discrete units (pixels rations in at least two colors. Many modern apo objectives
in this case), the sampling rate, or frequency of the digital exceed these specifications by correcting spherical and chro-
data point in time or space, must be at least two times the matic aberration in four or more colors.
smallest observable signal.
A common rule of thumb for digital cameras is that effec- Immersion Objectives and Oils
tive pixel size in the specimen plane should be 2.5 times the While the speed of light in a vacuum is a constant, the speed
maximum resolution of the objective. For example, to deter- of light as it travels through a medium is defined by its refrac-
mine the resolution of an objective with a 1.3 NA, assuming tive index (RI or n). This is expressed as a ratio of the speed
long green light of 589 nm, the Rayleigh criterion for of light in a vacuum over the speed of light in the medium.
resolution (0.61l/NA) gives a result of ~276 nm for resolv- Thus, the refractive index of air is very nearly 1, while the
able detail. Magnifying this result by 100× results in a refractive index for crown glass, a common optical lens
resolved size at the detector of 27.6 mm. In order to sample material, is about 1.5.
As light passes from a medium of one refractive index to objectives to observe fine detail, will significantly degrade
a medium of differing refractive index at an angle, the light resolution. To correct for this, some objectives are fitted with
will change speed and direction. If light passes from a higher correction collars that adjust internal lens spacing as they are
refractive index to a lower one, the light is bent toward the rotated. Similarly, some objectives have correction collars to
interface. In microscopy, this means that if light exiting a correct for other optical path length differences such as the
specimen and coverglass (n » 1.5) at a high angle passes thickness of a plastic culture dish or when imaging deep into
through air (n » 1.0), it will be bent further away from the a thick sample.
objective and thus may not be collected, resulting in a loss of Other objectives are designed to be used without a cover-
resolving power. However, by filling the space between the glass. These are marked as “NCG” (no coverglass) on the
glass coverslip and the objective with oil having a refractive outer barrel of the objective.
index similar to glass, this loss of light and thus information
can be avoided.
Refractive index also directly relates to numerical aper- Eyepieces
ture in that the effective NA of an objective cannot be greater
than the refractive index of the medium through which light The eyepieces of the microscope further increase the
passes. This means that for a “dry lens,” the theoretical limit magnification of the image and project it to a point where it
of NA is 1.0 (in practice, often 0.90 or 0.95). Thus, these can be comfortably viewed. Magnification is typically listed
lenses, usually available in magnifications of 40× to 100×, on the eyepiece along with another important specification
provide relatively good resolving power without the use known as the field number, which defines the field of view. To
of oil. determine the field of view, the field number as listed in mil-
An NA higher than 1.0 may be needed to observe the limeters is divided by the objective magnification (and any
finest details. For an objective to achieve this higher NA, an intermediate magnification). Eyepieces are also available in
immersion medium with a refractive index higher than that high-eyepoint versions to allow the use of eyeglasses or a
of air must be used. For standard oil immersion objectives, more comfortable viewing position or with individual diopters
the theoretical NA limit is about 1.5 (in practice, often 1.4– to correct for focus without the need for corrective lenses.
1.49). The same is true in transmitted light microscopy for Additionally, various markers such as crosshairs, pointers, or
the delivery of light to the sample by the condenser. In order measuring reticles can be positioned in the field plane of the
to achieve the same high NAs, the condenser must be oiled eyepiece so that they appear in focus with the specimen.
so that light does not pass through air between the condenser
front element and the glass slide.
Since oils may have varying optical or chemical proper- Beam Splitter
ties, the use of the particular type of oil specified by the
objective manufacturer for that lens is recommended. Oils of Microscopes that have digital imaging capabilities include a
different brands should not be mixed without consulting the beam splitter to direct light either toward the eyepieces or the
manufacturer. As immersion oils dry, they can become very detector. The beam splitter may send 100% of the available
gummy or sticky and sometimes degrade optical coatings or light in either direction or in some cases will split some
cements. Therefore, oil objectives should always be wiped percentage of the light to both the eyepieces and detector at
clean with optical tissue after use. the same time for simultaneous visual inspection and imaging.
Coverglass Correction and Correction Collars

If a coverglass (coverslip) is used, the optical design of the The Microscope Stage and Coordinate Location
objective must take into consideration the thickness of this
glass. Most standard objectives are corrected for a 0.17-mm- The microscope stage provides a flat, level surface for the
thick coverslip, and this property is marked on the outer microscope slide and a means of affixing the slide to the
casing of the objective. stage. Controls on a mechanical stage allow the microscope
In coverglass parlance, a #1.5 coverglass has a target slide to be moved in x- and y-axes. Mechanical stages
thickness of 0.17 mm. Manufacturing tolerances of standard usually have a coordinate grid on each axis to precisely iden-
coverslips generally allow that a #1.5 coverslip may vary in tify the location of an object on the slide. The microscope
thickness from 0.16 to 0.19 mm. This small variation can stage can also be moved in an up-down manner (z-axis) by
induce spherical aberration that, when using high NA using the coarse- and fine-focus controls.
Coordinate Location changes in amplitude, visual contrast can be enhanced using

Recording accurate coordinates is essential for documenta- phase contrast microscopy. Through a rather different mech-
tion of cytogenetic findings. In most instances, notation of anism, another technique known as differential interference
the x and y coordinates are used for this purpose. contrast (DIC) converts optical path length gradients into
amplitude changes observed as visual contrast.
Vernier Grids and England Finders®
When a metaphase is to be relocated at a microscope other Phase Contrast
than that used for the original analysis, a system of coordi- There are two major obstacles that must be overcome in
nate conversion between the two microscopes needs to be phase contrast microscopy. First, the specimen information
employed. is too dim compared to the background, and second, small
Microscopes of the same manufacturer and model can phase shifts must be converted to intensity differences. Light
often have their stages aligned so that the coordinates of one that interacts with the specimen is diffracted, while light that
scope can be used at another. Vernier grids or England passes through without interacting, termed zero-order light,
Finders® allow for easy conversion of coordinates between contains no specimen information but adds overall bright-
similar microscopes whose stages cannot be aligned or when ness to the resultant image (background). The first step is to
the microscopes are made by different manufacturers. This reduce the intensity of zero-order non-diffracted light. Since
technique provides a printed grid whose value is read at one this zero-order light is much higher in amplitude than higher
microscope and then simply relocated at the second. orders of diffracted light, attenuating its intensity helps in the
visualization of the higher-order diffracted light.
To accomplish this, illuminating light is focused into an
Microscope Slides, Coverslips, and Mounting annulus in the condenser front focal plane resulting in a
Media ring of parallel illuminating light exiting the condenser
front element. Figure 5.3 shows the placement of the com-
The microscope slides, coverslips, and mounting media play ponents and the light path in phase contrast microscopy.
a significant role in the contrast and resolution of an image. Any light that is not diffracted by the specimen, and thus
Microscope slides and coverslips should be made from high- contains no information, will enter the objective as parallel
quality glass to allow light to pass with the least generation rays, meaning it will be focused at the back aperture of the
of optical aberrations. A microscope slide with a thickness of objective. By placing a ring-shaped phase plate in the back
1.0 mm is well suited for cytogenetics microscopy. Coverslip aperture of the objective, this non-diffracted zero-order
thickness can be 0.17–0.18 mm, depending upon the recom- light (surround light, shown in yellow in Fig. 5.3) can be
mendation of the microscope manufacturer. It is important to blocked, typically by 60–90%. Light that is diffracted by
note that high numerical aperture lenses have a very low the specimen will be defocused at the back aperture of the
tolerance to variance of slide, mounting medium, and cover- objective and will be largely unaffected by the phase plate
slip thickness (± 0.05 mm for NAs greater than 0.7). Images (shown in red in Fig. 5.3).
that cannot be brought into good Köhler illumination are As light is diffracted by the specimen, a phase shift is also
often a sign of a specimen whose thickness has exceeded the introduced that is typically a retardation of approximately ¼
capacity of the microscope lenses. wavelength. In positive phase contrast, the phase plate in the
objective also advances the phase of light passing through it
by approximately ¼ wavelength, resulting in a total phase
Brightfield Contrasting Techniques difference of ½ wavelength between diffracted and zero-
order light. The diffracted light then destructively interferes
In brightfield microscopy, samples that are thin or transparent with any remaining zero-order light to produce intensity
are often stained to enhance visual contrast. There are times, variations that are observed as visual contrast, where phase
however, when it is desirable to observe samples without objects appear darker than the background. In negative phase
staining, such as when monitoring living cells in culture or contrast, the zero-order light is retarded with respect to
checking the quality of chromosome spreading prior to diffracted light, leading to phase objects appearing brighter
staining. There is almost no detectable visual contrast when than the background. See Fig. 5.4 for an example of the
light passes through cells and subcellular structures, since images created using the two variations of phase contrast.
there is little to no absorption, but the speed of light does In practical application, the phase ring in the objective
change as it passes through them resulting in a phase shift. and the condenser annulus must be aligned so that they over-
The human eye and digital cameras only detect changes in lap along the optical axis. For proper phase contrast opera-
intensity (amplitude) of light and cannot easily detect phase tion, it is essential to start with Köhler illumination. Since
shifts. By converting these very small phase shifts into large the size of the phase ring in the objective varies with NA and
Fig. 5.3 Light path in a phase

contrast system. After the
illumination light passes through
the condenser annulus and
specimen, any non-diffracted
zero-order light, termed the
surround light and marked in
yellow, is largely blocked and
phase-shifted by the phase ring in
the objective. This light represents
mostly background signal with no
information. Light that is diffracted
by the specimen, marked in red,
remains mostly unaffected by the
objective phase ring (Figure used
by permission of Michael W.
Davidson of the National High
Magnetic Field Laboratory and
MicroscopyU.com)
allowing living cells or unstained chromosomes to be

easily observed.
Since the phase ring is typically deposited on a glass plate
in the objective, specific phase contrast objectives must be
used. In some specialized systems, the phase ring is located
in a conjugate aperture plane outside the objective so that
phase objectives are not needed, but this is not typical.
Differential Interference Contrast

In differential interference contrast (DIC), constructive and
destructive interference between light rays that traverse
slightly different optical path lengths creates visual contrast
in the image. DIC allows for detailed visualization of trans-
parent specimens with several advantages over phase con-
trast, including the absence of the halo artifact sometimes
associated with phase contrast, as well as the ability to create
Fig. 5.4 Positive and negative phase contrast systems. Representative high-quality images even through relatively thick specimens.
images created by positive and negative phase contrast systems (Figure Since plane-polarized light is required for this technique,
used by permission of Michael W. Davidson of the National High
Magnetic Field Laboratory and MicroscopyU.com)
a polarizer is placed between the light source and condenser.
The plane-polarized light, which is vibrating in only one
direction, passes through a birefringent prism in the con-
denser known as a Nomarski-modified Wollaston prism.
magnification, a properly sized condenser annulus must be This splits the beam into two beams that are vibrating
selected. The objective will be marked on the outer barrel perpendicular to each other (often termed the ordinary and
with an indication of the proper condenser annulus, such as extraordinary wavefronts; see Fig. 5.5). The two beams travel
Ph1, in which case a condenser annulus also marked Ph1 slightly different optical path lengths induced by specimen
should be used. To align the fixed phase ring to the adjustable refractive index and thickness, and are recombined by a
condenser annulus, the aperture plane must be visualized second prism behind the objective. As a result of phase shifts
using an eyepiece telescope or Bertrand lens built into the in the beams with different optical path lengths, constructive
eyepiece tube. The objective phase ring will appear as a dark and destructive interference create light and dark areas
circle and should overlap the illuminating ring from the particularly along the edges of optical path length gradients.
condenser annulus. When the phase contrast microscope is A second polarizer (termed the analyzer) is in a crossed
properly aligned, very small changes in phase can be detected orientation to the first and blocks unmodified background
as they are rotated relative to each other as a minimum of

intensity. Since the required shear angle, or distance between
the two beams following splitting or “shearing” by the
Wollaston prism, may vary according to objective magni-
fication, the condenser prism should be matched to the objec-
tive (check marking on the objective barrel). There are
several methods that may be employed to control the level of
contrast, including physically moving the objective prism
relative to the optical axis (introduction of bias retardation)
or using a rotating polarizer attached to a fixed quarter-
wavelength retardation plate (termed de Sénarmont
compensation).
For the best DIC imaging, strain-free microscope optics
that are manufactured without material stresses, which could
create strain-induced birefringence, should be used to avoid
artifacts that can be caused by polarized light. In addition,
imaging through birefringent or strained materials like the
extruded plastic used for culture dishes can also produce
poor results.
Epifluorescence Microscopy
The basic principle of fluorescence microscopy is that light

of a particular wavelength can be efficiently absorbed by a
fluorescent dye (also termed a fluorophore or fluorochrome)
and emitted at a longer wavelength. The absorbed energy
from incident photons raises the fluorophore molecule to an
excited state. As the fluorophore returns to the ground state,
this energy is emitted as a photon. The emitted photon is
usually of longer wavelength than the originally absorbed
photon. This change in wavelength is described as the
Stokes shift.
Epifluorescence capabilities can often be added to modular
brightfield microscopes with the addition of an epifluores-
cence illumination system. Originally used in the cytogenetics
Fig. 5.5 Wavefronts in a Nomarski DIC system. Ordinary wavefronts laboratory to observe Q-banded chromosomes (see Chap. 4
(red arrows) showing vibration across the page are shown together
with extraordinary wavefronts, represented by blue circles that are
and Fig. 4.3), epifluorescence microscopes are now utilized
essentially arrows going into and out of the plane of the page to show in a number of molecular cytogenetic techniques such as
perpendicular vibration to ordinary wavefronts. In the center optical fluorescence in situ hybridization (FISH), comparative
axis of the system, both wavefronts propagate together. On the sides of genomic hybridization (CGH), and multiplex FISH (M-FISH)
the optical axis, one wavefront becomes advanced or retarded relative
to the other, represented by either the blue trailing the red or the red
or spectral karyotyping (SKY). See also Chap. 17.
trailing the blue (Figure used by permission of Michael W. Davidson of
the National High Magnetic Field Laboratory and MicroscopyU.com)
The Fluorescence Microscope
light, darkening the field, while passing elliptically polarized Unlike transmitted light brightfield microscopy, epi-illumi-
light that represents the specimen information. The result is nation microscopes deliver and collect light from the same
a shadow-mask effect that appears as a seemingly three- side of the sample. A light delivery path is introduced behind
dimensional pseudo-relief, although it is not indicative of an the objective lens, which focuses light onto the sample and
actual topographical structure. collects light that returns. This illuminator may include aper-
Following Köhler illumination, the polarizer and analyzer ture and field diaphragms for the epifluorescent light path, as
should be crossed for maximum extinction, which is observed well as various filters. Epifluorescence techniques typically
require relatively intense light that is filtered from white light

to a specific bandwidth.
Fluorescence Light Sources

While halogen bulbs can be used, arc lamps such as mercury
vapor, metal halide, or xenon are the most common light
sources for epifluorescence due to their higher intensity in
various wavelengths optimal for widely used fluorophores.
These lamps produce an electric arc between two electrodes
in a gas-filled envelope and operate under high pressure and
temperature for efficiency and brightness. This requires
ample ventilation. They are often contained in a housing
at the rear of the microscope that may also include bulb
alignment controls, adjustable reflectors, and an adjustable
collector lens.
Many modern microscopes forgo the adjustable attached
housing in favor of remote housing that uses a pre-centered
bulb and delivers light to the illuminator through a liquid-
filled light guide and collimating light adapter. These light
sources eliminate the need for manual alignment of the bulb
while integrating the power supply and often use extended-
life bulbs. These self-contained boxes may also integrate an
iris and neutral density or other filters.
Light-emitting diodes (LEDs) are gaining popularity as Fig. 5.6 Light path of a fluorescence filter cube. The “cube” contains
light sources for fluorescence work and enjoying the advan- three optical elements: an excitation filter, a dichroic mirror, and an
tages of very long lifetimes, low heat output, and the ability emission filter. Excitation light is first filtered by the excitation filter and
to switch on and off very quickly, potentially eliminating the is reflected toward the sample by the dichroic mirror. Returning light is
collected by the objective and sent back toward the dichroic mirror.
need for a physical shutter. However, since they are tuned by Longer wavelength light produced by fluorescence passes through
the manufacturer to a specific wavelength range with some while any back-reflected excitation light is blocked. Light passing
bandwidth, they are not as flexible as a white-light source if through the dichroic mirror is then filtered by the emission filter (Figure
the desired wavelength range changes. used by permission of Michael W. Davidson of the National High
Magnetic Field Laboratory and MicroscopyU.com)
The intensity of arc lamps is not generally directly con-
trollable by voltage adjustment, so neutral density filters
become important for attenuating brightness. Infrared filters any added fluorophores, producing nonspecific emission.
are also often employed since even though excitation light The typical fluorescence filter set consists of three filters: the
is defined to a spectral band by an excitation filter (see excitation filter, the dichroic mirror, and the emission or
“Fluorescence Filters”, next section), IR light may inadver- barrier filter, in a single housing commonly referred to as a
tently pass through these interference filters. Sometimes “cube” (illustrated in Fig. 5.6).
called “heat filters,” the IR filter helps reduce background The full-spectrum light from the source passes through
signal on a digital detector that is caused by detector sensitiv- the excitation filter, which defines the spectral range that is
ity to IR light and helps to extend the life of multicoated efficiently absorbed by a particular fluorophore. The light is
fluorescence bandpass filters. then directed toward the back of the objective by reflecting
off a dichroic mirror placed at a 45° angle to the optical axis.
Fluorescence Filters A standard dichroic mirror is reflective to wavelengths below
Through the use of filters, the fluorescent property of a certain cutoff and transmissive to wavelengths longer than
fluorophores and the resulting Stokes shift are exploited to the cutoff range. The objective focuses excitation light onto
allow for efficient illumination of the sample and collection the specimen where fluorophores are illuminated and emit
of the emitted longer wavelength light, while blocking the light of longer wavelength as defined by the Stokes shift.
collection of any returning illumination light, resulting in This longer wavelength light is collected by the objective
high specificity and signal-to-noise ratio. In this case, the and passes through the dichroic mirror and then through the
“signal” is the emitted light, while the “noise” might be exci- emission filter. Any scattered or reflected excitation light
tation or stray light, as well as autofluorescence, which is the does not pass through the dichroic mirror due to its reflective
property of some tissues and materials to fluoresce without property to the excitation wavelength. The emission filter
defines a spectral band specific for the emission of the target Special considerations may need to be made depending on
fluorophore while blocking background autofluorescence of the wavelengths used for fluorescence microscopy. In the
other wavelengths and light from any other fluorophores and case of UV or IR excitation and emission, objectives
secondarily blocks any excitation light that may have leaked specifically made to transmit those wavelength ranges may
through the dichroic mirror. be needed. Beam splitters used with fluorescence should
Since excitation and emission bandwidths are discretely direct 100% of the available light to the detector.
defined, careful planning can allow multiplexing of multiple Background signal can greatly obscure the information of
fluorophores on a single sample. Multiband filters can allow interest. To reduce this, low-autofluorescence oils should be
for simultaneous viewing of multiplexed fluorophores, which used with oil immersion objectives. The transmitted con-
can also be recorded by a color digital camera. However, for denser and light source do not play a role in epifluorescence
reasons of efficiency and specificity, individual fluorescent and should be defocused and blocked. Stray room light can
colors are usually captured sequentially as “channels” by a also contribute to background, so critical fluorescence imaging
monochromatic digital camera using single bandpass filter is often performed in a darkened room.
cubes. These channels can then be “pseudocolored” and
overlaid in analysis software to produce a multispectral Acknowledgments The authors extend special thanks to Michael W.
image. Davidson of the National High Magnetic Field Laboratory and
MicroscopyU (www.microscopyu.com) for figures and information
and also acknowledge Christopher McAleer, author of the chapter in
Fluorescence Considerations previous editions, for providing the general scope and framework of
Since the amount of light emitted by fluorophores is rela- the material.
tively dim compared to brightfield microscopy, high NA
optics are greatly preferred for fluorescence microscopy.
When performing multispectral fluorescence, chromatic References
correction may also be of great importance to ensure that
different fluorophores focus to the same plane. Phase con- 1. Inoue S, Spring KR. Video microscopy, the fundamentals. 2nd ed.
New York: Plenum Press; 1997.
trast objectives are not ideal for dim fluorescence, as the
2. Nikon Microscopy U. www.microscopyu.com
phase plate will result in a significant reduction of brightness. 3. Molecular Expressions. www.micro.magnet.fsu.edu
Quality Control and Quality Assurance
6
Martha B. Keagle
Introduction There are many steps that occur between obtaining a

specimen for chromosome analysis and the generation of a
Upon receiving news that results of a chromosome analysis final clinical report. After collection of the specimen itself,
are abnormal (and even sometimes that they are normal), a accessioning, culturing, harvesting, slide preparation and
patient will frequently ask: “How do I know that the lab staining (probe hybridization for fluorescence in situ hybrid-
didn’t make a mistake? How do I know that the sample they ization [FISH]), microscopic analysis, electronic imaging,
reported on was really mine? How can I be certain that this is karyogram production, creation of a final report, and actual
all correct?” Most would be surprised to learn of the myriad reporting of results are the path that specimens follow as they
of checks and balances that exist in clinical cytogenetics progress into and out of the cytogenetics laboratory. During
laboratories. Based on the consensus of professionals and on this process, many variables can subject a specimen or data
common sense, The American College of Medical Genetics to a variety of conditions that must be managed for a proper
(ACMG) Standards and Guidelines for Clinical Genetics diagnosis to ultimately be reached.
Laboratories are the basis for oversight by regulatory agen- Central to any QA/QC program is the laboratory’s stan-
cies and are intended to prevent clinical and clerical errors dard operating procedure (SOP) manual. This often formi-
[1]. These comprise the area of laboratory medicine known dable document contains the policies and procedures that
as quality assurance and quality control (QA/QC). They are must be followed in order for the laboratory to perform chro-
supplemented by both total quality management (TQM) and mosome analysis. It includes requirements of physical space
complete quality improvement (CQI) programs that seek to and mechanical systems, specimen requirements and collec-
minimize errors when the laboratory interfaces with refer- tion procedures, transport requirements, personnel experience
ring physicians and their patients. and credentials, and safety and protection for personnel.
The nature of clinical cytogenetics is such that it includes It includes sections on training and compliance with the vari-
both quantitative and qualitative components of tests. Some ous regulatory agencies that monitor and inspect laborato-
aspects are generic to practices in laboratories of any kind, ries, and, finally, it may contain a section pertaining to quality
while others are specific to cytogenetics laboratory tests. assurance and quality control. The majority of these issues
A proper QA/QC program requires that policies for valida- pertain to the analytic component of testing.
tion of protocols and reagents, training and credentials of indi- With the rapid growth of knowledge and expansion of
viduals performing cytogenetic analysis, sample identification, genetic testing, the laboratory has become increasingly
safety for laboratory staff, and other compliance issues must involved in ensuring that the pre-analytic and post-analytic
all be in place. Laboratories are inspected periodically by aspects of testing are also designed to ensure the appropriate
various state and national entities, and most have institutional use of tests and their results. These commonly include issues
and internal regulations and guidelines as well. of analytical test validation, documentation of clinical valid-
ity, interpretation of test results, and educational materials
that allow the laboratories’ clients to interface with it. These
aspects are commonly encompassed in a complete quality
improvement program.
M.B. Keagle, M.Ed. (*) Entire books could be written that address each of these
Department of Allied Health Sciences, College of Agriculture and
issues in detail; entire chapters could be devoted to labels
Natural Resources, University of Connecticut, 358 Mansfield Road,
Unit 2101, Storrs, CT 06269, USA alone! Such detail is beyond the scope of this book, however.
e-mail: [email protected] This chapter will provide an overview of the ways in which
78 M.B. Keagle
laboratories deal with many of these steps in order to ensure instructions for specimen collection and transport. Thus,
proper patient care. quality assurance and quality control begins by interactions
with the health-care providers who will collect and submit
specimens for chromosome analysis.
Pre-analytical Testing Components
Collection Protocol
Before a test specimen arrives in the laboratory, there are a A collection protocol from the cytogenetics laboratory is of
number of things that must be done correctly to ensure that critical importance, as it establishes the collection guidelines
an accurate and useful test result is provided. Laboratories for individuals who are not intimately familiar with the oper-
often develop and provide materials to their clients to guide ating procedures of the laboratory. A collection protocol
them in understanding when to test, what to test, and how to should include:
order tests. Often considered outside of the day-to-day func- • Ideal volume of specimen for collection.
tioning of the laboratory, these are important to ensuring safe • Suitable transport containers, anticoagulants, or media.
and effective testing. • Transport temperature and the maximum permissible
transport time to ensure optimum specimen growth.
• Confirmation of the identification of the patient from
Test Validation whom the specimen was collected.
• Specimen container labeling and requisition form
Prior to initiating testing, there should be evidence of clinical requirements.
validation of the test. This may be done by the laboratory • Laboratory hours, phone numbers, contact individuals,
developing the test or may be apparent from the scientific and after-hours procedures.
literature and merely documented. With the advent of the Once established, it is important to keep copies of this
1992 modifications to the 1988 Clinical Laboratory protocol anywhere a specimen might be collected, including
Improvement Amendments (“CLIA ‘88”) regulations, labo- a hospital’s general laboratory, departmental clinics and
ratories are required to validate all tests being introduced operating room suites, and outpatient clinics and referring
into service whether they were newly developed or long used physician’s offices. It is also a good idea to routinely discuss
in other laboratories [2]. Further, all new tests must be revali- collection protocols with the appropriate individuals, espe-
dated every 6 months. Approaches to validation vary for cially those who do not frequently submit samples to the
quantitative versus qualitative tests. Classical concepts such laboratory. Regular interaction helps promote a complete
as sensitivity (the ability to detect a target when it is present) understanding of collection requirements, as well as general
and specificity (the ability to not detect a target when it is not expectations for samples submitted for cytogenetic analysis.
present) are common measures of analytical validity for It also provides an opportunity to discuss questions, con-
quantitative tests. These are most often applied to FISH (par- cerns, or suggested improvements of collection or submis-
ticularly when interphase based) and microarray tests, (see sion procedures.
Chaps. 17 and 18) but also are important when mosaicism is
under consideration. Requirements for validation may vary Specimen Labeling and Requisition Forms
with the regulatory status of a product. When a test is Accurate specimen identification is one of the most impor-
approved by the US Food and Drug Administration (FDA), tant policies to implement. Specimen labels should include
the laboratory is expected to demonstrate that the test oper- at least two sources of identification, such as patient name,
ates within the performance characteristics described by the date of birth, or a unique patient-specific number, for proper
manufacturer. When tests are not FDA approved or have identification in the event of a labeling error.
been modified, the laboratory is expected to demonstrate The requisition form is equally important, as it supplies the
their validity independently. For the more qualitative classi- laboratory with the patient and clinical data associated with
cal chromosome analysis, laboratories commonly validate the specimen. When Medicare is to be billed for laboratory
their ability to process particular specimen types, to perform tests and the physician believes that a portion of the laboratory
particular tests, or to detect a particular abnormality by test- charges may not be covered, the requisition (or an accompany-
ing samples from individuals with those abnormalities. ing document) must also include an advanced beneficiary
notification (ABN), which informs the patient that he or she
will be billed should Medicare deny payment. Certain states or
Specimen Submission other regulatory agencies also require that informed patient
consent be part of, or accompany, the requisition form.
Specimens are almost always collected by individuals who For obvious reasons, it is desirable to have a properly
rely upon the laboratory to provide a requisition form and completed requisition (paper or electronic) accompany each
6 Quality Control and Quality Assurance 79
specimen submitted to the laboratory, but it is also important Assessing the Condition of the Specimen
for the laboratory to develop a policy for dealing with speci- and Requisition
mens that are not accompanied by a requisition, or for requi- After receipt of the specimens in the laboratory, the individual
sitions that have not been filled out completely. Of special responsible for accessioning specimens must check the sample
importance are those requests for chromosome and/or FISH and requisition for the appropriate labels, transport reagents
analysis that are made verbally with the laboratory. In these (medium, anticoagulants, etc.), specimen condition (color, clot-
instances, it is important for the laboratory to obtain written ted, adequate sample size, transport temperature, etc.), and date
or electronic authorization for the study. The provision of of collection. When a problem is detected, the individual should
sufficient clinical information to ensure that appropriate tests follow the laboratory procedure for informing the “submitter”
and analyses have been requested is a valuable cross-check. of the specimen and take appropriate actions. Problems with the
specimen and action taken might also be documented.
Rejection Criteria
It is very important for individuals to clearly understand the Accession Numbers and Patient Database
minimum requirements for submission of a specimen for It is important to assign a unique identifier to each specimen
chromosome analysis, FISH, or arrays, and what circum- as it enters the laboratory, distinguishing it from other speci-
stances would prevent a laboratory from performing analy- mens, as well as from a patient’s previous studies. The lab
sis. The collection protocol and requisition forms should number, patient data, and clinical information are then often
clearly state these requirements. Although extremely rare, transferred into a logbook or electronic database, creating a
circumstances can arise that prevent a laboratory from patient record that can be tracked and cross-referenced
accepting a specimen for analysis. against previous and/or future studies. In addition, other data
In the event of a problem with a sample, the laboratory can be entered into a database record as a study progresses,
should make immediate contact with the individual submit- allowing the laboratory to track:
ting the specimen, either to obtain clarification of the speci- • Culture conditions
men identity or to discuss potential difficulties in obtaining a • Results
result. In most instances, both parties will elect to proceed, • Turn around times (TATs)
knowing that the success of the analysis may be impacted. In • Dates of specimen receipt, processing, and report
some instances, the problems are insurmountable, and a • Individual(s) issuing reports
repeat sample is needed. When this occurs, it is a require- • Cytogenetic results versus the findings of patients with
ment for the lab to carefully document the reason for rejec- similar histories or abnormalities (interpretation of results)
tion or failure, as well as disposition of the specimen in the • Culture failures, labeling errors, transcription errors, mis-
patient report and appropriate log. diagnoses, and actions taken
• Incidence of submission problems
Electronic databases need to be managed within the labo-
Analytical Testing Components ratory to ensure the accuracy of the data as well as patient
confidentiality.
The analytical phase of testing includes the actual processing Once a specimen has been logged into the cytogenetics
and analysis of the specimen. Although specimen accession- laboratory, it must be prepared for cell culture. This may
ing are often considered pre-analytical, it is included here include notification of appropriate individuals of its receipt,
because labeling and tracking of specimens through a test is creation of culture records and container labels, and creation
among the most common causes of error in clinical labora- of a patient folder or file for paper records. If the sample is
tory testing. This phase usually ends when a laboratory test not set up immediately, it needs to be stored under appropri-
result is apparent. ate conditions.
There should be a system for identifying specimens that
require special handling such as an accelerated study, a pre-
Specimen Accessioning liminary report, or a completion by a certain date to meet
anticipated turn around times. These requirements should be
Once a specimen has been received, an accession process is clearly indicated on all appropriate forms and/or computer
used to log it into the laboratory and to prepare it for analy- fields, and all individuals involved with the study should
sis. During this time an accession and/or laboratory number be notified.
is assigned to a specimen, relevant patient and clinical data
are entered into a logbook and/or database, and the culture Specimen Labels
and analysis requirements for the studies requested are The accuracy of any laboratory result requires correct speci-
identified. men labeling. After the initial accessioning process, a number
80 M.B. Keagle
of items need to be labeled, including a culture worksheet; to manufacturer or from lot to lot (such as serum), prepurchase
culture flasks, tubes, or Petri dishes; microscope slides; a testing of multiple lots can ensure that the highest-quality
microscope analysis worksheet; metaphase prints; karyo- reagent is available to the laboratory. The methods of QC
grams; FISH images; and reports. The laboratory labeling testing should be appropriate to the reagent and method
policy should allow patient identification to be cross-checked being tested and may include parallel testing of the current
in the event of a labeling error. validated reagents/devices against the new lots of reagents/
devices using nonclinical control specimens or reference
materials. It is also important to track the history of protocol
Specimen Culture, Harvesting, Slide Preparation, modifications, allowing a comparison of past culture tech-
and Staining niques and successes. The format of a culture protocol should
comply with the requirements of the agency used for labora-
All equipment and supplies used for culture and harvesting tory accreditation.
of cells, preparation of slides, and banding and staining of
chromosomes should be monitored in order to provide high- Equipment Maintenance
quality analyses. Consistency and reliability of laboratory procedures cannot
be accomplished without well-maintained equipment, and
Cell Culture there are many regulations that reflect this.
Whenever possible, duplicate or independently established Refrigerators, freezers, and water baths should be closely
cultures should be created for all samples, and these should monitored daily for temperature and cleaned following reg-
be placed in separate incubators, each equipped with its own ular schedules. Centrifuges should be monitored for accu-
power, CO2 source (if utilized), and emergency alarm. A rate speed semiannually. Laminar flow hoods should be
backup procedure must also be created that ensures that cul- cleaned before and after use and be equipped with an anti-
tures will be maintained in the event of a power (emergency bacterial light or cover to prevent contamination during
generator) or CO2 (automatic gas tank supply change) periods of nonuse. Biological safety cabinets also should be
failure. checked and certified annually for airflow and bacterial con-
Precautions to prevent contamination should be taken tamination, and pH meters should be cleaned and calibrated
when a specimen is added to culture medium, a culture is regularly. Balances should be kept clean of laboratory
transferred between containers, or reagents are added to a reagents and calibrated regularly to ensure proper weight
specimen culture. Working with specimens within the area measurements. Ovens need to be monitored daily for tem-
of a laboratory designated for biological hazardous materials perature. Trays for slide preparation and storage should be
and using sterile technique in laminar flow hoods will greatly kept clean to reduce chemical contamination of staining
reduce the risk of bacterial contamination of the specimen reagents.
and exposure of staff to biohazards. In addition, using latex Incubator temperature and gas (CO2) concentration should
gloves, cleaning work surfaces with alcohol before and after be monitored continuously and documented daily. Incubators
use, and exposing container openings, pipettes, or other mea- should be on a regular cleaning schedule and, as discussed
suring devices to a flame will reduce the likelihood of earlier, should also be equipped with separate power and gas
contamination. sources, as well as emergency alarms. Incubator gas and
Working with one specimen at a time and disposing of all power supplies should also have a backup in the event of a
used pipettes or containers that come into contact with a failure, and the laboratory should maintain an emergency
specimen (before moving onto the next) will greatly reduce plan in the event of complete incubator failure. Records of
the likelihood of cross contamination or improper equipment monitoring and maintenance should be docu-
identification. It is also important to note that the transfer of mented in an equipment log.
reagents into a culture should be performed using a fresh Automated harvesting procedures are used by many cyto-
pipette when there is any risk of contact with a specimen or genetics laboratories as a way of increasing laboratory pro-
specimen aerosol. ductivity and improving consistency (see Chap. 7). However,
automation does not imply “carefree.” Laboratories that uti-
Culture Protocols lize such technology must strictly follow the manufacturer’s
Cell and tissue culture begins with a protocol that outlines recommended operational guidelines and closely monitor
tested and reproducible steps to produce cells and metaphase the equipment for acceptable performance. A procedure for
chromosomes for analysis. The quality control of new reagent the use of automated equipment that details the procedural
lots and changes in established protocols should be com- steps for operation, appropriate reagents, calibration and
pleted prior to their use with patient specimens. For critical cleaning requirements, and preventive maintenance must be
reagents that may be of variable quality from manufacturer prepared. It is also important for individuals operating the
equipment to receive proper training before using it on • Harvesting method (centrifuge tubes vs. in situ process-
clinical specimens. ing) (see Chap. 4)
• The humidity and temperature of the laboratory or drying
Harvesting, Slide Making, and Staining chamber utilized (see Chap. 7)
The transition from cell/tissue culture to microscopically • The number of fixations and the method of fixing the
analyzable chromosomes is achieved by harvesting the divid- specimen
ing cells (which involves mitotic arrest, osmotic swelling of • The slide temperature
cell membranes, and fixation), spreading of the chromo- • Wet or dry slides? How much water?
somes on microscope slides, and staining the chromosomes • The angle of the slide during specimen application
with one of various methods which produce an appropriate • The method of applying the specimen
banding pattern (see Chap. 4). Each of these steps must be • The method of drying the slide
optimized to facilitate correct diagnoses. • The slide-aging technique
Each of these factors can significantly contribute to the
Protocols success of slide preparation. As these can be variable from
After cells have been successfully cultured, the techniques of day to day and between individuals, close observation and
harvesting, slide making, and banding/staining will deter- documentation of technique may allow the highest proficiency
mine the ultimate quality of the metaphase chromosomes of these skills.
available for analysis. Following validated protocols is very
important for these procedures, but frequent modifications Banding and Staining
may be required to address changing laboratory conditions. While slide preparation and aging are important factors con-
It is important to note that these procedures can be especially tributing to the lab’s ability to successfully stain a specimen,
sensitive to individual technique, particularly fixation and adjustments to solution concentrations, the time slides are
slide making, and that mastery of these skills requires indi- left in the staining solution, etc., can also influence success-
viduals to observe and document minor variations in proce- ful staining of cytogenetic samples. Careful preparation of
dure or laboratory conditions that improve or detract from reagents and documentation of adjustments made to staining
chromosome morphology. procedures help the laboratory personnel to refine their
New protocols, procedural changes, introduction of new techniques.
reagents, reagent concentrations, microscope slides, etc. The shelf life and storage conditions of banding and stain-
must be validated under controlled conditions. The method ing reagents are important considerations and should also be
of validation should be one that is appropriate for the reagent documented in a staining log. As reagents arrive in the labo-
or technique being tested and may include parallel testing of ratory, lot numbers should be recorded and compared with
current versus new, testing on nonclinical control specimens, previous lots used. Reagent containers should be labeled
or direct analysis using reference materials. It is also impor- with the reagent name, quantity, concentration, storage
tant to track the history of harvesting, slide preparation, and requirements, date received, and expiration date. Reagents
staining protocol modifications in order to allow a compari- that require refrigeration should have minimum and maxi-
son of past techniques to present successes. Documentation mum permissible temperatures documented, and these
of proactive and reactive factors from these procedures is should not be exceeded. Existing supplies of reagents should
important to ensure quality metaphase chromosomes, as well be rotated so that they are depleted before new supplies are
as to identify and track problems that reduce specimen used.
quality. Although good specimen staining is critical for optimal
microscope analysis, it is also necessary to consider the
Slide Preparation microscope on which a specimen will be analyzed and the
The chromosomes present in harvested metaphases must be staining requirements of the recording medium. When a
spread apart so that they can be microscopically analyzed. laboratory has a variety of microscopes, each may have a
They must lie flat so that staining is uniform and all chromo- light source, contrast or interference filters, objectives, or
somes are in a single plane of focus, and they must be aged other lenses that produce images with a unique set of visual
(literally or artificially) in order for most banding and stain- characteristics. Additional variables, such as excitation and
ing procedures to work properly. barrier filters, are introduced with the use fluorescence
Even when all else has gone well with the tissue culture microscopy, and features such as the numerical aperture of
and harvesting procedures, poor slide preparation can result lenses or bulb intensity may be critical (see Chap. 5).
in scarce, poorly spread, or improperly aged metaphase Individual preference is also an important factor in identify-
spreads for staining and microscope analysis. The following ing a staining intensity that is well suited for microscope
variables should be considered: analysis.
82 M.B. Keagle
When accessing the quality of banding in G-banded images, a protocol should establish the policies for the storage of
it is important to identify staining intensities that produce: microscope slides and retention of images, both during anal-
• Chromosome pale ends that contrast well against back- ysis and once analysis has been completed.
ground areas
• A wide range in mid-gray intensity Personnel Requirements
• Dark bands in close proximity that appear as distinct The experience level, credentials, and workload of each tech-
bands nologist are all important considerations, and the laboratory
Comparing the requirements of the individual performing must be appropriately staffed to allow for complete, accu-
the microscope analysis against the requirements of the rate, and timely results of all samples received. When possi-
recording media and documentation of ideal conditions in a ble, it is often recommended to split the analysis of a
staining log will help laboratories gain control of the many specimen between two individuals in some way, increasing
variables of a staining procedure. the potential for detection of a subtle abnormality.
Establishing goals for individuals or groups to meet, such
as turn around time and the number of cases to be completed
Specimen Analysis in a week, is an important aspect of effective laboratory man-
agement. The quality of analysis should not, however, be
Any chromosome analysis begins by identifying the specific sacrificed in the attainment of these goals, and performance
requirements for the specimen type being examined. monitors should include frequent statistical analysis of fail-
Following this, the basic steps are: the microscope analysis ure rates and percentage of abnormal cases.
itself (location of metaphase spreads suitable for analysis,
counting the chromosomes and determining the sex chromo- Microscopy
some complement, and analysis of the band pattern of the A significant part of quality microscopy lies in the training
individual chromosomes), imaging of the metaphase spreads, an individual receives on the components of a microscope
preparation of karyograms, and documentation and reporting and their proper use. Any protocol for microscopy should
of results. The procedure begins with a protocol that must be therefore include training of personnel in the use of micro-
accessible and thoroughly understood by all individuals per- scopes, quality checks to identify equipment in need of ser-
forming chromosome analysis. An analogous process is vice or adjustment, and identification of individuals in need
required for FISH studies. of additional training.
The selection of microscopes for analysis and documen-
Analysis Protocols tation of results (image production) is also a very important
An analysis protocol must identify the general requirements consideration. It is not unusual for a laboratory to have
for each specimen type. The protocol should identify normal microscopes of various quality grades, and users need to
parameters and normal variants and should distinguish understand the limiting factors of any given scope. “Newer”
between true abnormality and artifact. The number of cells does not necessarily imply “better,” and many “veteran”
from which chromosomes are to be counted and the sex microscopes produce excellent images. It is often the resolu-
chromosome complement identified and analyzed in detail tion of the objective (lens), not extraneous accessories, that is
(band-for-band) must be clearly stated, including whether the key to image clarity. Also, good images are more likely
each type of examination is to occur at the microscope, on an to come from well-prepared microscope slides. Controlling
image, or via a karyogram. A protocol should set standards the slide preparation process and using a microscope with
for the selection of suitable metaphase spreads, as well as the the appropriate lenses and features will promote quality
number of cultures (and colonies, when applicable) from cytogenetic analysis and image documentation. For addi-
which cells should be examined. When an abnormality is tional details on microscopy, see Chap. 5.
detected, the appropriate steps to take should be specified.
Other things, such as an appropriate banding resolution level, General Analysis Requirements
maximum allowable number of overlapping chromosomes, Analysis requirements have evolved as a mix of “conven-
random chromosome loss, and dealing with metaphases in tional wisdom” and statistically validated needs for specific
close proximity, might also be included. types of studies. Professional organizations have developed
A protocol should identify the procedures used to docu- consensus-based standards for different types of analyses
ment each metaphase, as well as the data to be recorded on a (e.g., The American College of Medical Genetics (ACMG)
microscope analysis worksheet, requirements for imaging, 2009/revised 2010 [1]), and regulatory bodies have typically
the number of cells to create karyograms from, the number used these as guides when specifying minimum require-
of individuals who should take part in performing the analy- ments for each sample type processed for chromosome
sis, and the individual who should verify the results. Finally, analysis. Individual laboratory protocols and individual
state requirements frequently augment these. It should be Chorionic Villus Samples

noted that minimum requirements are just that; the standard Many laboratories examine cells from both “direct” (uncul-
of care frequently requires more rigid guidelines. It must tured) and cultured preparations, but in clinical use, uncul-
also be remembered that most listed standards apply to tured preparations should only be used if a culture technique
chromosomally normal samples. Once an abnormality has of 48 h or more is also used. Band resolution should be as
been discovered, it is important to confirm its presence or high as possible.
absence in each cell examined and to identify additional For cultured preparations, the chromosome count and sex
procedures that may be necessary for correct diagnosis. It is chromosome complement should be determined for at least
also important to realize that a patient’s clinical indications 20 cells, distributed as widely as possible from at least two
may dictate that analytical resolution should be higher than independent cultures. At least two karyograms should be
the stated minimums. prepared. If more than one cell line is present, at least one
The following are some general guidelines for constitu- karyogram must be prepared from each.
tional chromosome studies for different specimen types: For combined direct/cultured preparations the chromo-
some count and sex chromosome complement should be
PHA-Stimulated Blood (Non-neoplastic Disorders) determined for a minimum of 20 cells, at least 10 of which
At least two cultures should be established for each sample. are from the cultured preparations. Additional cells should
The chromosome count and sex chromosome complement be examined when mosaicism is detected, particularly when
should be determined for at least 20 cells. If a mosaic sex there are discrepancies between the direct and cultured prep-
chromosome abnormality is suspected and confirmed in the arations, which are often an indication of confined placental
20-cell analysis, no additional counts are required; however, mosaicism (see Chap. 11).
if not confirmed in the standard 20-cell evaluation, a mini- At least five metaphases (four from cultured material, if
mum of ten additional metaphases should be examined. At possible) should be completely analyzed, and at least two
least five metaphase cells should be completely analyzed, karyograms should be prepared. If more than one cell line is
and at least two karyograms should be prepared. If more than present, at least one karyogram must be prepared from each.
one cell line is present, at least one karyogram must be pre- A resolution of at least 450 bands should be obtained if
pared from each. A minimum band resolution of 550 should possible.
be the goal for constitutional studies, but greater resolution
may be required for focused studies of specific chromosome Percutaneous Umbilical Blood Sampling (PUBS)
pairs. A minimum of two cultures from fetal blood should be estab-
Although guidelines for the diagnosis of fragile X syn- lished if there is adequate sample, and harvests at 48 and
drome via cytogenetic analysis did at one time exist, current 72 h are recommended. At least five metaphase cells should
standard of care now involves analysis via molecular meth- be completely analyzed, and at least two karyograms should
ods (see Chap. 19). be prepared. If more than one cell line is present, at least one
karyogram must be prepared from each. The fetal origin of
Amniotic Fluid, In Situ Method the sample should be confirmed.
The chromosome count and sex chromosome complement
should be determined for one cell from each of at least 15 Solid Tissue (Non-neoplastic Studies)
colonies. As many colonies as possible should be examined At least two cultures should be established for each
when a true mosaic condition is detected or, in some cases, to sample. The chromosome count and sex chromosome com-
clarify pseudomosaicism. Cells must originate from at least plement should be determined for at least 20 cells from at
two independent cultures (from more than one sample least two independent cultures. If a mosaic sex chromosome
syringe or tube, when possible). At least five metaphases abnormality is suspected and confirmed in the 20-metaphase
from independent colonies should be completely analyzed, analysis, no additional counts are required; however, if not
and at least two karyograms should be prepared. If more than confirmed in the standard 20-cell evaluation, a minimum of
one cell line is present, at least one karyogram must be pre- ten additional metaphases should be examined. At least five
pared from each. The band resolution should be as high as metaphases should be completely analyzed, and at least two
possible and should not be less than 450. karyograms should be prepared. If more than one cell line is
present, at least one karyogram must be prepared from
Amniotic Fluid, Flask Method each.
The chromosome count and sex chromosome complement The following are some general guidelines for analysis
should be determined for at least 20 metaphases from at least of acquired chromosome abnormalities for different speci-
two independent cultures as described earlier. Other require- men types:
ments are the same as for the in situ method.
84 M.B. Keagle
Bone Marrow and Unstimulated Peripheral Blood the stemline and one of each sideline present, if any, should
Bone marrow is usually the sample of choice for the study of be prepared.
premalignant and malignant hematologic conditions. If an For follow-up studies of patients who have had a
adequate bone marrow aspirate cannot be obtained, an hematopoietic cell transplant for whom donor and recipient
unstimulated peripheral blood study may yield satisfactory cells cannot be differentiated by G-banding, 20 cells should
results if the circulating blast count is greater than 10–20%. be analyzed fully for previously identified clonal abnormali-
Guidelines vary by the type of study, and the reader is ties and for any newly acquired abnormalities. Two karyo-
referred to the ACMG Standards and Guidelines (ACMG grams of the stemline and one of each sideline should be
2009/revised 2010) for more specific information [1]. In produced.
general, thorough, for initial diagnostic studies, examination
of 20 consecutive cells from unstimulated cultures is recom- Lymph Nodes
mended, when possible. Metaphase selection should not be The number of cultures established should be based on the
based on good chromosome morphology. All 20 cells should apparent cellularity of the sample, but if lymphoma is part of
be fully analyzed. Attempts should be made to count and the differential diagnosis, the cultures should include at least
identify structural abnormalities in cells skipped because of one unstimulated, 24-h suspension culture. Selection of cells
poor morphology. for analysis should not be based on good chromosome mor-
If one abnormal clone is present, two karyograms should phology. Twenty metaphase cells should be analyzed when-
be created. If more than one related abnormal clone is pres- ever possible. Since lymphomas can have very complex
ent, two karyograms from each stemline and one from each karyotypes, and thus can be very labor intensive, some labo-
sideline should be prepared. If unrelated clones are present, ratories may choose to perform an abbreviated study (usually
two karyograms from each stemline and one from each per- at least ten cells) when the abnormal clone has been charac-
tinent sideline should be generated. If only normal cells are terized. Documentation guidelines are the same as for neo-
present, two karyograms should be made. If both normal and plastic blood and bone marrow specimens. If there is a
abnormal cells are present, only one karyogram from the confirmed diagnosis of lymphoma and only normal meta-
normal cell line is required. phases are seen on an analysis of 20 cells, additional analy-
For follow-up studies of patients with previous G-band sis/scoring by G-banding or FISH for specific
studies who have not received an allogeneic hematopoietic lymphoma-related chromosomal abnormalities may be
cell transplant, 20 cells should be analyzed. If all cells are appropriate.
normal, additional cells may be evaluated by G-banding or
FISH for a specific abnormality. Solid Tumors
For follow-up studies of patients who have had a Cytogenetic evaluation of solid tumors can be performed to
hematopoietic cell transplant for whom donor versus recipi- establish a diagnosis, to assess prognosis, or for patient man-
ent cells can be distinguished by sex chromosome comple- agement. Since cytogenetic abnormalities in pediatric tumors
ment or cytogenetic polymorphisms, 20 cells should be are often disease-specific and may have prognostic value,
analyzed (note: FISH or molecular methods are more sensi- cytogenetic evaluation is recommended whenever sufficient
tive than G-banding for determining engraftment status and tissue is available. FISH analysis may also be used as a pri-
should be used in preference to G-banding in such cases). mary or secondary method of evaluation when a rapid diag-
If only donor cells are present, two karyograms should be nosis is needed. Cytogenetic analysis of adult tumors should
prepared. be based on diagnostic and prognostic value.
If donor and recipient cells are present, all recipient cells Culture methods should be chosen based on the type of
of the 20 should be analyzed fully for previously identified tumor submitted. Non-small cell round tumors grow better
clonal abnormalities and for any newly acquired abnormali- as attached cell cultures (flask or in situ), and small cell round
ties. Analysis or scoring of additional recipient cells may be tumors (SCRT) are usually best grown in suspension culture,
indicated, depending upon the number of recipient cells in but SCRTs will also grow in monolayer; thus, if sufficient
the initial 20 cells. For the recipient cells, two karyograms of material is available, both types of culture should be
the stemline and one of each sideline, if present, should be established.
prepared. For donor cells that have been previously docu- Analysis of 20 metaphase cells or sufficient numbers to
mented, one metaphase image should be captured; if donor characterize all abnormal clones and subclones should be
cells have not been previously documented, two karyograms performed. If there are only normal cells, or if there is one
should be generated. abnormal clone, two karyograms should be made. If there is
If only recipient cells are present, 20 cells should be ana- more than one related abnormal clone, two karyograms of
lyzed fully for previously identified clonal abnormalities and the stemline and one of each sideline should be prepared. If
for any newly acquired abnormalities. Two karyograms of unrelated clones are present, two karyograms from each
stemline and one from each related sideline should be made. Metaphase FISH results should be confirmed by at least
If both normal and abnormal cells are present, one karyo- two persons with experience evaluating FISH samples; one
gram should be prepared from the normal cells, plus karyo- may be the laboratory director. At least two images should be
grams from the abnormal clones as previously described. maintained by the laboratory.
Supplemental FISH studies can be used to provide rapid The following are some general guidelines for interphase/
results to aid in making the differential diagnosis, for planning nuclear fluorescence in situ hybridization:
therapy, for appraising prognosis, or for ruling out cryptic
aberrations in solid tumors. FISH can also be used longitudi- Applications for interphase FISH include detection of
nally once a baseline FISH pattern has been established in the numerical abnormalities, duplications, deletions, chromo-
initial study. FISH is also useful when there are inadequate somal rearrangements, constitutional sex chromosome com-
cells for G-band analysis in cases where there is a suspected plement, mosaicism (with proper caution), and gene
diagnosis for which a recurring abnormality is known, or amplification. The specificity and limitations of the probes
when conventional cytogenetics studies are normal. used must be considered when interpreting results.
The following are some general guidelines for metaphase Interphase FISH only provides information about the
FISH (including repeat sequence and whole chromosome probe(s) in question and does not replace a full cytogenetic
probes): evaluation. If the suspected abnormality can also be detected
with conventional cytogenetics, confirmatory chromosomal
Applications for metaphase FISH include evaluation/ analysis should be performed, although the interphase FISH
identification of marker chromosomes and material of result may be issued as a preliminary finding. If the inter-
unknown origin attached to or within a chromosome, phase FISH study is for disease monitoring, adjunctive con-
clarification and characterization of structurally rearranged ventional cytogenetics may not be necessary.
chromosomes, detection of losses or gains of chromosome Regulatory requirements for different interphase FISH
segments (including microdeletions), and detection of mul- probes vary based on the origin of the probes and their FDA
tiple cell lines. approval status (refer to the ACMG Standards and Guidelines
Metaphase FISH is considered adjunctive to conventional for specific information [1]).
cytogenetics in most circumstances; it only provides infor- Probe localization and analytic validation, including sen-
mation about the probe(s) in question and does not replace a sitivity and specificity, must be established for all interphase
full cytogenetic evaluation. FISH probes (refer to the ACMG Standards and Guidelines
Regulatory requirements for different metaphase FISH for further details [1]). Establishment of databases and
probes vary based on the origin of the probes and their FDA reportable reference ranges and biannual calibrations are
approval status (refer to the ACMG Standards and Guidelines required.
for specific information [1]). Whole chromosome probes and probes for whole chro-
Probe localization and analytic validation, including sensi- mosome arms are not appropriate for interphase FISH analy-
tivity and specificity, must be established for all metaphase sis. When multiple probes are used concurrently, different
FISH probes (refer to the ACMG Standards and Guidelines fluorochromes should be used to enable their differentiation.
for further details [1]). In a laboratory that has attained 98% Care must be taken when interpreting results of repeated
sensitivity during its internal test validation, a minimum of sequence probes studies since, infrequently, some individu-
five cells should be evaluated when characterizing non-mosaic als have small numbers of the sequence target. Normal results
marker chromosomes or unknown chromosomal regions of interphase FISH for detection of microdeletions or micro-
within or attached to a chromosome. For non-mosaic microde- duplications in which the probe is not specifically for the
letions, a minimum of 10 cells should be evaluated. If any gene in question need to be interpreted carefully, and a dis-
discordant cells are found, additional cells from a second claimer should be included in the written report.
slide should be examined. A minimum of 50 nuclei should be scored (split between
Since the probes in whole chromosome cocktails are not two independent readers); many laboratories routinely examine
always uniformly distributed along the entire length of chro- 200 or more nuclei for each probe, especially for oncology
mosomes, caution must be used when interpreting results in diagnoses. Additional nuclei may need to be scored if there is
target regions of small size. Care must also be taken when significant discrepancy between the two readers, if a result
interpreting negative results of repeated sequence probes does not meet the laboratory’s established reporting ranges, or
studies since, infrequently, some individuals have small if mosaicism for a constitutional abnormality is suspected.
numbers of the target sequence. Additionally, results of Interphase FISH results should be confirmed by at least
metaphase FISH for confirmation of microdeletions in which two persons with experience evaluating FISH samples; one
the probe is not specifically for the gene in question need to may be the laboratory director. At least two images should be
be interpreted carefully. maintained by the laboratory.
86 M.B. Keagle
The following are some general guidelines for multi- a particular karyotypic abnormality. The laboratory should
target FISH tests: also have a written protocol to determine whether any regions
covered by the array represent known regions of copy num-
FISH tests developed to analyze several chromosome loci in ber variants. The finding of abnormal copy number in such
a single test format provide information only on the specific regions should be characterized by another method (FISH,
probe loci used and are not a substitute for complete karyo- parental studies, quantitative PCR, etc.).
typic analysis. Abnormalities detected by multi-target FISH Follow-up studies (FISH, chromosome analysis, arrays,
should be confirmed by another method when possible etc.) of biological parents or other family members may be
(G-banding, locus-specific FISH, etc.). indicated when an abnormality is detected by arrays to rule
Regulatory requirements for different interphase FISH out balanced rearrangements, inherited duplications or dele-
probes vary based on the origin of the probes and their FDA tions, or for interpretation of findings of uncertain clinical
approval status (refer to the ACMG Standards and Guidelines significance.
for specific information [1]).
Probes used in multi-target FISH tests must be validated Analysis Worksheets
for localization, sensitivity, and specificity. Established data- Laboratories routinely use some form of worksheet to docu-
bases and reportable reference ranges and biannual calibra- ment microscopic analysis data. This is the technologist’s
tions are required for multi-target probes that include working document but becomes part of the patient’s perma-
interphase FISH probes. nent laboratory chart, and as such serves as an additional
The following are some general guidelines for microarray clinical and clerical cross-check.
analysis (constitutional studies): The analysis worksheet typically includes patient data
(patient name, laboratory accession, and case numbers), indi-
CGH- and SNP-based arrays can be used to detect copy cation for study, and specimen type. The identification of each
number gains and losses resulting from aneuploidies and slide examined should be verified, and previous studies might
unbalanced structural chromosomal abnormalities. They will be noted. The technologist(s) performing the analysis and the
not detect balanced structural rearrangements, some ploidy date of analysis should be recorded. The microscope being
changes, or single gene abnormalities. CGH-based arrays used is often indicated, and microscopic coordinates are
will not detect uniparental disomy, but this can be detected recorded for each metaphase examined, along with other data
with SNP-based arrays. Additionally, array CGH may not (slide number, culture of origin, banding method, and identifiers
detect low-level mosaicism for aneuploidies and unbalanced for relocating the cell). The number of chromosomes and the
structural rearrangements. sex chromosome complement is typically noted, along with
Arrays can be used as adjuncts to conventional cytogenet- other relevant data such as quality of banding, abnormalities,
ics and targeted FISH or as a primary diagnostic test. polymorphisms, chromosome breakage, whether the cell was
There are various types of arrays that can be used in the analyzed and/or imaged, which cells should be considered for
clinical laboratory (FDA approved, for investigational use karyotyping, etc. Finally, a summary of the results, including
only, for research use only, and “home brews”), some of the patient’s karyotype, can be included, along with documen-
which can be purchased commercially, and others that are tation that the entire case has been reviewed for clerical accu-
developed within the laboratory itself. Each type may require racy prior to release of the final report.
different levels of validation depending upon their intended
use (refer to the ACMG Standards and Guidelines for a more
complete discussion [1]). The laboratory must also demon- Imaging Systems and Karyogram Production
strate expertise in array performance and analysis.
The laboratory should have a laboratory procedure man- During or upon completion of the microscopic analysis,
ual that includes written protocols for DNA extraction, label- selected metaphases must be imaged and karyograms must
ing, and quantification; obtaining adequate DNA quality and be created and printed.
quantity; proper fragmentation; and fluorescent labeling. Historically, photomicroscopy was used to capture
These should be documented in each patient record. The images of metaphases, darkroom techniques were used to
laboratory should also have protocols for confirmation of make photographic prints, and the chromosomes were cut
abnormal or ambiguous array findings (G-band karyotyping, out with scissors and manually arranged on the karyo-
FISH, PCR, etc.). gram form.
Both male and female control samples should be estab- Today, electronic tools are used to record microscope
lished, and the laboratory should have guidelines indicating images and create karyograms. Saved images document the
how they are to be used in same-sex and opposite-sex com- findings and allow the chromosomes to be reanalyzed as nec-
parisons. Controls usually have normal karyotypes, but there essary. Understanding how to operate, optimize, and main-
are circumstances when it is appropriate to use controls with tain the materials and equipment used in the imaging process
is necessary in order to achieve optimum results from any formed the microscopic analysis cannot prepare or review
sample. the karyograms for that patient assures that multiple indi-
Computer-driven imaging systems are essentially the dig- viduals see every case (an exception to this rule can be
ital equivalent of photography. Instead of photographing a made for abnormal cases, particularly for oncology sam-
metaphase, it is electronically captured in digital form, and ples, where the technologist who performed the analysis
instead of developing film and using filters to produce prints has a better feel for the abnormalities that are present and
with the appropriate contrast and background, the image is the risk of missing an abnormal karyotype is no longer a
electronically enhanced to achieve a similar appearance, and concern). Preparation of karyograms by appropriately
a printer provides a hard copy if needed. Finally, images are trained individuals who are not technologists as described
stored not as photographic negatives, but as digital files on earlier also accomplishes this. When review by the labora-
optical disks, DVD-R, or other digital storage media. Many tory supervisor or another senior individual followed by
laboratories store images on a server. An understanding of final review by the laboratory director is added, a well-
the theory and hardware, and appropriate training, is requi- designed protocol can ensure that at least four or more
site to utilizing an imaging system properly and efficiently trained “pairs of eyes” examine chromosomes from every
(see also Chap. 7). patient, increasing the likelihood of detecting a subtle
abnormality or clerical error.
Karyogram Production A special consideration in this area involves the use of the
The final laboratory manipulation required for chromosome computerized imaging system to prepare patient karyograms.
analysis is typically generation of the ordered arrangement Pattern-recognition software has improved to the point that
of chromosomes known as a karyogram (while the term many sophisticated systems can now arrange the chromo-
karyotype was historically used to refer to both the nomen- somes with little or no human input (see Chap. 7). This, of
clature describing the chromosomal complement and the course, creates a quality concern. Laboratories using such
systematized array of the chromosomes, in 2005 the ISCN tools can deal with the issue by putting in place protocols
committee recommended that the term karyogram be used that require appropriate review of all computer-generated
for the systematized array of chromosomes and that the word karyograms. When properly monitored, such systems can
karyotype be restricted to describing the nomenclature of the increase laboratory efficiency by markedly reducing the time
chromosome complement. See also Chap. 3). required for karyogram production.
If there was ever a perfect example of the value of training
in laboratory medicine, it is this process. A bright individual
with a modest comprehension of the theory behind cytoge- Retention of Case Materials
netics and essentially normal pattern recognition and motor
skills can be taught the normal human chromosome Slides used in diagnosis and stained by a permanent method
identification well enough to perform this task in about a (e.g., G-, C-, or R-banding; NOR staining) must be retained
week. Yet, the comment most often made by visitors to a by the laboratory for at least 3 years. Retention of slides
cytogenetics lab is typically: “These chromosomes all look stained using fluorescent methods is at the discretion of the
alike. How do you tell them apart? I’d never be able to do laboratory director.
that.” In reality, all that is required is a sufficient number of Residual original patient samples, such as cell pellets,
images for repeated attempts, plus sufficient patience on the should be kept until sufficient metaphase preparations have
part of the individuals doing the training. By making attempt been made to complete the analysis. Processed patient sam-
after attempt (and receiving the appropriate corrections each ples should be retained until the final report is signed, and
time), the novice eventually begins to recognize certain pairs many laboratories keep them for longer periods of time. Some
and then eventually all pairs. Mastery of the subtleties laboratories do not discard cell pellets from abnormal cases.
sufficient to perform actual microscopic analysis, of course,
requires much more training, but in many laboratories, tech-
nicians, lab aides, interns, or students are often employed to Post-analytical Testing Components
generate karyograms. When such adjunct personnel are used,
a good rule to follow is that no such individual be permitted Laboratory Reports
to create karyograms for an entire case without supervision
or review by a trained technologist. Reporting the results of chromosome analysis and/or FISH
Karyogram production is one method laboratories can can have a direct impact on the diagnosis and treatment of a
use to divide analyses between two or more technologists. patient; thus, it is important to establish a reporting proce-
A guideline that specifies that the technologist(s) who per- dure that:
88 M.B. Keagle
• Summarizes the findings of the laboratory • Indication of additional work performed (e.g., to resolve
• Cross-checks the findings against the various specimen possible mosaicism), correlation to previous studies, con-
labels for labeling errors trols used, etc.
• Interprets the test results, where appropriate • Interpretation of results written for a non-geneticist physi-
• Establishes a reporting process to outside individuals so cian (including clinical correlation, discussion of abnor-
that the data, the individual issuing the report, the indi- mal results, recommendations for additional genetic
vidual receiving the data, and the report date are properly studies or genetic counseling)
documented • Information about any preliminary reports
• Signature of qualified laboratory director
Preliminary Reports • Date of final report
Although potentially risky, preliminary results are some- Once completed, final reports can be generated electroni-
times released by a laboratory before the full chromosome cally or on paper. If a preliminary report was provided, any
analysis has been completed. Preliminary reports are often variations from it should be stated. Once the final report has
issued verbally once enough data has been collected to for- been completed, a record should be kept of the individuals to
mulate a likely indication of the final result, or once the data whom a report was issued, as well as the date(s) of issue. In
already available is clinically critical and must be communi- most instances, a report is typed or printed electronically by
cated to a physician (this might also pertain to FISH analysis a computer program and filed in a patient folder. Patient fold-
that has been completed while results of chromosome analy- ers are retained in the laboratory or filed in an outside facil-
sis are still pending, in which case a preliminary report could ity. Whether stored within a laboratory or at an off-site
be in writing). Once verified, it is important to follow an facility, it is important to have access that allows prompt data
established procedure for reporting preliminary results. retrieval.
Individuals reporting the data should be qualified to interpret
the preliminary findings and to give an indication of the pos-
sible outcome once a complete study has been conducted. It Quality Assurance
is important for this individual to document the microscope
analysis data, patient and cytogenetic data reported, date of Laboratories can experience a variety of difficulties with the
the report, and individual receiving preliminary data. It is samples themselves. Some of these are inevitable and there-
also vital to impress upon the person receiving the report fore are not preventable (insufficient volume, wrong sample
what may change once the study is completed. type, no living or dividing cells present, etc.), while others
may be due to improper collection or transport of the speci-
Final Reports men before it reaches the laboratory, incorrect labeling, or
The final report summarizes and interprets the results of the errors in handling and processing in the laboratory itself.
study. Some states and regulatory agencies also require a Any of these can result in an incorrect diagnosis, or in failure
statement describing the limitations of chromosome and/or to reach one at all. It is therefore very important for improve-
FISH analyses, and many laboratories choose to include such ment of overall laboratory quality to investigate and docu-
statements, whether required or not. A protocol for the cre- ment all problems that arise, thereby determining ways to
ation of final reports should include a checklist to insure that prevent similar occurrences in the future.
all appropriate procedures have been completed and that all It is also important to monitor specific types of laboratory
data is clerically correct. The final report should include the test outcomes in order to judge a laboratory’s performance.
following: This is most commonly done when a laboratory can expect a
• Case identification (including patient name, date of col- particular distribution of outcomes. In studies of products of
lection and receipt by the laboratory, laboratory accession conception (POCs), for example, review of distribution of
number, sample source, name of physician or authorized results can alert laboratory personnel of potential problems
agent who ordered the test) with tissues provided and dissected for study (e.g., if the
• Reason for study male: female ratio is not close to 1). In leukemia and cancer
• Number of cells in which chromosomes were counted, testing, there may be subsets of cases for which there is an
analyzed, and karyogrammed expectation of study success and abnormality detection rates.
• Culture methods when significant to the cytogenetic For instance, among patients entered in national cancer coop-
findings erative group studies, there is usually a group-wide expecta-
• Banding method, band resolution, and ISCN description tion based on prior performance of laboratories in the
of analyzed cells group.
Specimen Failures whenever possible. There should also be appropriate

backup power, redundant CO2, and alarm/warning sys-
The inability of a laboratory to provide a diagnostic result is tems in place, and all major equipment should be on a
typically due to one of two basic reasons: cells from the sam- preventative maintenance schedule. Nevertheless, unusual
ple did not grow in culture, and therefore no mitotic cells hardware problems do occur.
were produced, or a problem occurred in one of the many • Reagent failure. There are rare but unfortunate examples
post-culture steps, rendering the processed material useless. of supplies that are supposedly quality-controlled by the
The purpose of this section is not to convince the reader that manufacturer being released (unknowingly) for purchase
problems are inevitable, but rather to impress upon the reader by laboratories without actually meeting the appropriate
the amount of care and attention to detail that is required, and criteria. Improperly cleaned water storage tanks have poi-
the critical role quality assurance plays in the cytogenetics soned entire lots of culture medium, and syringes made
laboratory. with natural rubber stoppers have periodically resulted in
amniotic cell death on contact. Again, with proper pre-
Culture Failure cautions in place (testing all supplies before use and
As described in Chap. 4, the basic procedure for producing dividing all cultures between two lots of everything),
chromosomes for analysis from any tissue type requires liv- these risks can be minimized.
ing cells that can somehow be coaxed into active division. • Human error. While also unlikely, it is always possible for
Without mitosis, there can be no chromosomes to process a technologist to inadvertently prepare culture medium
and examine. incorrectly, forget to add the appropriate mitogen, or uti-
There are several possible reasons for cell culture failure: lize equipment improperly.
• The sample did not contain any living cells. In some cases, Every culture failure must be documented and the cause
this is clinically not surprising; it is frequently the case investigated to the extent possible. The laboratory should
with products of conception obtained after a fetal demise, keep records of these, along with periodic measurements of
or in necrotic or aplastic bone marrow samples. At other culture failures for each specimen type, as a way of detecting
times, the cause can be deduced (such as a delay in sam- an increasing trend before it becomes a serious problem.
ple transport or exposure of the specimen to extremes of
temperature during transport when an outside reference Post-culturing Errors
lab is used). In other instances, however, no explanation is There are few things as frustrating to those working in a
readily available. In these cases, the entire path the speci- cytogenetics laboratory as having seemingly good cell cul-
men followed between the point of collection and deliv- tures or routine blood cultures produce no usable metaphases.
ery to the laboratory is suspect and must be investigated. While these are admittedly rare events, they do occur and, as
• An inappropriate specimen is submitted to the laboratory. with all culture failures, must be fully investigated and docu-
This may involve a peripheral blood with no circulating mented. Some examples are:
blasts having been collected instead of bone marrow • Harvesting errors. As outlined in Chap. 4, there are a
(without blasts, there are no spontaneously dividing cells variety of steps in the harvest procedure, and each pro-
present and the unstimulated cultures used for many vides the potential for error. If Colcemid® is not added, an
hematopoietic disorders will not produce metaphases). It insufficient number of mitotic cells can be the result. If
might be due to the wrong collection tube being used, or fixative is added before the hypotonic solution (unfortu-
to products of conception being placed in formalin and nately an easy thing to do but a mistake a good technolo-
then sent to the lab. The specimen and the way it is col- gist makes only once), cells will not swell, and chromosome
lected must match the intended application of chromo- separation is impossible. If a centrifugation step is omit-
some analysis. ted, all cells except those that have settled due to gravity
• An insufficient specimen is submitted to the laboratory. will be removed via aspiration or pour-off. Other errors,
Specimen descriptions such as “2 mL of extremely bloody such as adding the wrong hypotonic solution, making any
amniotic fluid” or “0.5 mL of watery bone marrow” fre- of the reagents incorrectly, or using incorrect timing, can
quently accompany cultures that fail. However, all such also render a harvest unusable. Finally, a catastrophic
samples should be submitted to the laboratory, which will event that results in the loss of all material (e.g., spillage
do everything it can to generate a result, no matter how or breakage of a rack of tubes) will, of course, result in
unlikely this may seem. loss of usable material.
• The laboratory suffers a catastrophic equipment failure. • Problems with robotic harvesters. As described in Chap.
With proper precautions in place, this is unlikely. 7, many types of cultures are now amenable to harvesting
Specimens should be divided, and multiple cultures on a robotic device. Although the motivation for using
should be initiated and placed in separate incubators, such a machine is often to free up technologist time for
90 M.B. Keagle
other vital functions, it is not a good practice merely to Misdiagnosis

load the cultures onto the harvester, press the start button,
and walk away. Solution bottles must be filled with the While perfection is always a goal in medicine, realistically it
proper reagents, lines must be free of clogs, and the is never achieved. Every laboratory discipline strives to elim-
computer program must be functioning correctly. All of inate all mistakes, but given the fact that human beings are
these must be verified before a technologist leaves the involved, all lab areas have “acceptable” error limits. A small
machine alone, and it is a good laboratory policy to make cytogenetics laboratory processing 2,000 samples per year
periodic checks up until the cultures are in fixative. that achieves a 99.97% accuracy rate (far in excess of the
• Slide-making and drying errors. It has often been said performance of the typical excellent pathology lab) will
that clinical cytogenetics is part art and part science. make six diagnostic errors in a 10-year period.
Producing high-quality metaphases during the slide- Misdiagnosis in the cytogenetics laboratory can occur in
making process is one example. This procedure is three ways: as the result of incorrect specimen labeling
described in Chap. 4 and also discussed in Chap. 7; (described earlier), by incorrect interpretation of a chromo-
suffice it to say that if not done properly, the laboratory’s some abnormality, or by failing to identify abnormality that
ability to correctly analyze a patient’s chromosomes can is present. Despite the many “pairs of eyes” that typically see
be compromised. each specimen in most laboratories, as described previously
• Banding/staining errors. Banding and staining are exam- in this chapter, some errors occasionally still manage to
ples of the art of cytogenetics. Correct “aging” (actual or get all the way through such a system undetected.
artificial via baking slides in an oven) and timing of each The consequences of an incorrect interpretation of a chro-
step in this process is critical to producing well-banded mosome abnormality can range from negligible to serious.
chromosomes (see Chap. 4), and a failure to interpret Because of the less than optimal chromosome morphology
results and adjust parameters accordingly can ruin even often produced by bone marrow aspirates or solid tumors, or
the best of preparations. to the complex abnormalities frequently present in such sam-
• Miscellaneous accidents or human error. Although each ples, it is often difficult, if not impossible, to correctly iden-
of the basic post-culture steps has been covered, there are tify every chromosomal change that may be present. It is not
still unusual things that can occur at any point in the pro- uncommon for a laboratory to receive serial bone marrow
cess, from wiping the wrong side of a slide to breaking it aspirates from a patient only to discover that, due to improved
completely. resolution in the current sample, an abnormality can be more
accurately characterized and that either a previous interpreta-
tion was not quite correct, or an abnormality initially
Labeling Errors described as uninterpretable can now be described. This is
typically of little clinical consequence and can easily be
The result of a labeling error can range from an incorrect addressed in the current clinical report. On the other hand,
laboratory number appearing on a report to the misdiagnosis misidentification of, or failure to detect, a disease-specific
of a specimen. Collection containers, requisition forms, com- rearrangement can lead to incorrect therapy and potentially
puter databases, culture flasks or dishes, culture worksheets, disastrous results.
microscope slides, and microscope analysis worksheets are Incorrect identification of a constitutional chromosome
all places where specimen labeling errors can occur. abnormality is less common than it once was, since most
Regardless of the outcome, labeling errors lead to improper such changes can be confirmed or further characterized
identification of or assign incorrect/inaccurate information to via FISH or microarrays (see Chaps. 17 and 18). Many
a specimen and are therefore a significant concern of any structural rearrangements are family-specific, but predict-
laboratory. Processing specimens one at a time using con- ing the phenotype likely to result from an unbalanced
trolled, standardized procedures serves to greatly reduce the aberration is never an exact science. Nationwide
likelihood of labeling errors. Nevertheless, it is important to proficiency tests often result in numerous similar but dif-
remember that people make mistakes, and the laboratory ferent interpretations of the abnormalities presented in
must therefore implement a system that cross-checks the any given challenge, demonstrating that “getting it right”
accuracy of the labels assigned to a patient, as well as the can be subjective in the field of cytogenetics.
data collected from a cytogenetic study. Each step that cre- Failure to identify a chromosome abnormality that is, in
ates the possibility for misidentification should have a cross- fact, present can be a serious issue if the inaccuracy is ulti-
check built into it, and some form of overall clerical review mately discovered, but such is not always the case. As dis-
of a patient chart is frequently carried out before results are cussed earlier, an abnormality may be detected in one bone
released. marrow aspirate but not in a prior one, particularly if there is
a difference in quality between the two. If the same labora-
tory receives both specimens, this can be detected and inter- country to country and even from state to state in the USA.
preted correctly, and while it may not make a therapeutic Federally, the Food and Drug Administration (FDA) regu-
difference, it is possible that the referring physician(s) may lates manufacturers of devices, some reagents, some soft-
comment that the patient’s treatment would have been differ- ware, and testing kits sold to laboratories. Though the FDA
ent had the abnormality been detected earlier. However, it is has suggested that the regulation of laboratories is within the
not uncommon today for different labs to be used for serial purview of its federal mandate because the laboratories make
studies, and in the scenario presented earlier, the initial diag- some of their own reagents, there is no precedent for their
nostic failure might never be revealed. involvement at this level. The majority of direct laboratory
Perhaps the most serious example of a missed diagnosis is oversight is focused on laboratory practices including per-
an unbalanced chromosome abnormality that is not detected sonnel requirements, general quality control and assurance,
in a prenatal sample. Failure to identify a balanced rearrange- and quality control and assurance specific to the area of prac-
ment could have consequences for the extended family, usu- tice. Clinical cytogenetics is among the areas with specialty-
ally by resulting in the failure to identify other family specific requirements under the Clinical Laboratory
members who are at risk for carrying it (see Chaps. 9 and 21) Improvement Amendments of 1988 (CLIA ’88) regulations
but (fortunately) rarely has an impact on the current (CFR§493.1276) [2].
pregnancy. Failure to identify an unbalanced abnormality,
however, will very often result in the birth of an abnormal Accreditation, Inspections, and External
child, and should the parents believe that they would have Proficiency Testing
interrupted the pregnancy had the abnormality been identified Under CLIA ’88, every laboratory performing moderate- to
prenatally, a lawsuit can result. The outcome of such cases high-complexity testing (i.e., every cytogenetics laboratory
often depends on whether the laboratory’s methods, quality in the USA) must enroll in US Department of Health and
systems, and results measure up to what is considered to be Human Services-approved external inspection and testing
the standard of care (i.e., everything covered in this chapter) programs [2]. Almost all clinical laboratories in the USA do
and whether or not the abnormality “should have been so under the auspices of the CLIA-deemed program of the
detected.” The latter often involves presenting uninvolved College of American Pathologists (CAP) [3]. This accredit-
professionals with the karyograms to determine whether or ing organization inspects laboratories and provides the
not they can identify the abnormality (a biased process, since American College of Medical Genetics and Genomics
these individuals obviously must know that something is (ACMG)/CAP proficiency testing survey program, accord-
wrong) and soliciting their opinions as “expert witnesses” ing to CLIA requirements, several times a year [3]. A lab’s
concerning whether or not the laboratory should have caught ability to perform and be reimbursed for testing depends
the abnormality, or whether it was too subtle to detect. upon successful participation in each aspect of this process,
Regardless of the nature of the error that is detected, it is and repeated failure can lead to loss of accreditation. As of
important to determine the cause of the problem and to put this time, no areas of genetic testing have mandated perfor-
into place the necessary changes to minimize recurrence. mance requirements for these proficiency testing programs.
CAP sends a team, typically from another laboratory, to
inspect each facility every other year; during off years, the
The Laboratory Quality Assurance Program laboratory must conduct and report the results of a self
inspection. Proficiency testing and interlaboratory compari-
An organized process of review, communications, and staff son programs vary according to specialty; in cytogenetics,
education is required to realize the benefits of a laboratory the proficiency tests generally consist of four unknown cases
system that tracks and monitors its functions, performance, in the form of banded metaphase preparations and sufficient
and problems. While at times this may involve subsets of the clinical information for the lab to make a diagnosis. A fifth
laboratory personnel, it is often part of the ongoing training unknown case, in the form of a peripheral blood sample, is
and continuing education program that should be available to also frequently submitted, but there are obvious logistical
the entire staff. and medical challenges of this procedure; there are enough
cytogenetics laboratories in the USA that care must be taken
not to exsanguinate the individual (typically a carrier of a
Oversight rearrangement) who has volunteered to be the test subject!
Depending on the probe type, proficiency testing for FISH
In addition to the numerous steps already described, cytoge- can involve slides to be hybridized, images to be interpreted,
netics labs, like all other clinical laboratories, are subject to or both.
many external guidelines, inspections, and tests that are State requirements can be quite variable. Several require
designed to ensure and improve quality. These vary from participation in the CAP programs. One of the more rigorous
92 M.B. Keagle
programs is administered by the New York State Department Graduates of NAACLS-accredited programs are eligible
of Health, which conducts its own inspections and proficiency to sit for the American Society for Clinical Pathology Board
tests of all labs in the USA that process specimens from of Certification (ASCP BOC) examination in cytogenetics
New York State residents [4]. This body also has its own immediately upon graduation from the program using the
certification process for clinical laboratory directors. education route. On-the-job-trained technologists can qual-
ify to take the ASCP BOC cytogenetics examination through
Laboratory Staff Qualifications other routes that require combinations of educational achieve-
Many US states require, either formally or informally, that ment and work experience in a cytogenetics laboratory.
the individual who signs chromosome analysis reports (typi-
cally the director of the cytogenetics lab) be board-certified State Licensure
in clinical cytogenetics by the American Board of Medical Credentialing requirements vary by state, and some states
Genetics (ABMG), a body that is recognized by both the have their own licensing requirements for cytogenetics labo-
American Board of Medical Specialties and the American ratories and personnel. Laboratories in those states and/or
Medical Association. It is similarly approved by the US laboratories receiving specimens from residents of those
Department of Health and Human Services under the CLIA states, and personnel working in them, must abide by the
’88 regulations [2] as among the boards required of labora- state requirements.
tory directors. Such certification is awarded to a doctor (M.D.
or Ph.D.) who passes a comprehensive examination in gen- International QA/QC
eral genetics as well as a specialty exam (in this case in clini- There are cytogenetics laboratories worldwide, and a com-
cal cytogenetics). Both exams must be passed for an prehensive discussion of the quality control/quality assur-
individual to be board-certified. Diplomates certified during ance and personnel issues in other countries is not feasible
or after 1993, when maintenance of certification became a here. Those interested are encouraged to contact professional
requirement, must recertify after 10 years. Those certified cytogenetics and clinical laboratory organizations in the
prior to 1993 are not required to recertify but are encouraged countries of interest. However, as a close neighbor, a few
to do so. words about personnel requirements in cytogenetics labora-
Many technologists, supervisors, and even directors in tories in Canada are included.
clinical cytogenetics labs in the USA learned to perform
chromosome analysis on-the-job, and such experience was Canada
all that was needed in order to find employment. While many In Canada, technologists working in cytogenetics and molec-
cytogenetic technologists are still on-the-job trained, a bac- ular laboratories are required to have earned a Bachelor of
calaureate degree is now required, and postbaccalaureate Science (B.Sc.) degree and to have graduated from a post-
certificate and degree programs in cytogenetics exist in sev- degree diploma program that includes extensive clinical
eral colleges, universities, and laboratories in the USA. laboratory experience. There are only two such programs in
Qualified individuals can become certified as technolo- Canada: one at the British Columbia Institute of Technology
gists in cytogenetics by passing the national certification (BCIT) in Burnaby, British Columbia, and the other at the
examination in cytogenetics given by the American Society Michener Institute in Toronto, Ontario.
for Clinical Pathology (ASCP) Board of Certification (BOC) The Canadian Society of Medical Laboratory Science
[5]. Candidates who pass the examination, which includes (CSMLS) is the credentialing/certification organization in
both didactic and practical components, can use the creden- Canada [7]. In all provinces except Ontario, candidates take
tial CG(ASCP)CM. Initial certification is valid for 3 years. a combined cytogenetics/FISH/molecular examination, and
Recertification via the Certification Maintenance Program successful candidates receive the credential MLT (clinical
(CMP) requires 36 units (hours) of continuing education genetics). In Ontario, instead of a combined credential, tech-
every 3 years and is a requirement of continued certification. nologists can become certified in cytogenetic/FISH or
Formal cytogenetics education programs can attain accred- molecular technology. There are no longer on-the-job routes
itation by the National Accrediting Agency for Clinical for certification in Canada. Canadian certification does not
Laboratory Sciences (NAACLS) by meeting specified stan- have a requirement of continuing education, thought it is
dards that include minimum qualifications of the program usually encouraged by employers.
director, didactic and clinical faculty, and curricular There is no uniformity in the credentials needed to be a
requirements that include didactic and clinical components cytogenetics laboratory director in Canada, although many
[6]. Programs seeking NAACLS accreditation are required to are certified by the Canadian College of Medical Genetics
complete a comprehensive self-study and site visit. Programs (CCMG) [8]. Candidates for this certification must have suc-
are reaccredited periodically by similar requirements. cessfully completed a CCMG-accredited training program.
Related Topics tion is an example of a nonclinical indication for study,

and specimens submitted solely for this purpose are there-
This chapter has covered most issues involved in the genera- fore refused by some laboratories. Because of the obvious
tion of clinical results in the cytogenetics laboratory. difficulties faced by all involved with such issues, a writ-
However, no such work would be complete without making ten policy, created by an internal ethics committee, can be
mention of the ancillary QA/QC that must also be dealt with extraordinarily helpful and is recommended.
on an ongoing basis: Laboratories can also be faced with (often unique) ethi-
• Safety. In past decades, laboratory design and protocols cal dilemmas on a case-by-case basis; these require thought
put the specimen first and the technologist second. Mouth and discussion to allow the laboratory to address them in
pipetting was common, even with potentially toxic what it deems to be the most appropriate manner.
reagents (e.g., Giemsa stain is frequently dissolved in • Health Insurance Portability and Accountability Act of
methanol). Gloves were not used, and “medical waste” 1996 (HIPAA): The HIPAA privacy rules created new
was any garbage can that had come in contact with a spec- requirements for health-care providers to protect the pri-
imen. Cytogenetics labs often reeked of acetic acid (used vacy and security of individually identifiable health infor-
in fixing samples; see Chap. 4), and laminar flow hoods mation. This is defined as information that is created or
(“sterile hoods”) were constructed with no separation received by a health-care provider that relates to the past,
between the specimen and the technologist and utilized a present, or future physical or mental health or condition
back-to-front horizontal flow of filtered air. The sample of an individual; the provision of care to an individual; or
was protected from microbial contamination as air blew past, present, or future payment for the provision of health
over it directly into the technologist’s face! The reader is care to an individual or information that identifies an indi-
reminded that hepatitis existed long before the human vidual. They went fully into effect on April 14, 2003.
immunodeficiency virus (HIV). The requirement to comply is triggered when the medi-
Hoods in use today (“biological safety cabinets”) fea- cal geneticist of the institution at which he or she practices
ture split vertical airflow and protective glass windows. electronically transmits health information for billing or
Pipetting devices are typically required, and in the USA, other purposes. Once required to comply, it applies to all
material safety data sheets (MSDS) for every reagent used information including that in nonelectronic form. There
in the laboratory must be available to all employees. are three main areas of requirement. These include the
Acceptable concentrations of all volatile reagents are monitoring and control of the uses and disclosures of pro-
maintained via ventilation systems and are monitored, tected health information (PHI), providing patients with
and universal precautions govern every process that certain rights with respect to their PHI, and establishing
involves contact with patient samples. Most laboratory and implementing certain administrative policies and pro-
inspections include a safety component. All laboratories cedures to ensure maintenance of privacy. Not all rules
should have general and specific laboratory, chemical, apply equally to clinicians and laboratories. For instance,
biological, and, if needed, radiation safety programs. because the laboratory is considered to have an indirect
Many also include ergonomics (including, but not limited treatment relationship with the patient, it is considered
to, correct hand, arm, and body position for working at a exempt from the consent requirements that require distri-
microscope, hood, or computer) as part of their safety bution of a Notice of Privacy Practices and from obtaining
program. the acknowledgment. Clinicians may extend their protec-
• Reference laboratories. Not every cytogenetics lab per- tion under HIPPA to a third party, such as a laboratory, by
forms every type of test on every type of sample. Some entering into a business associate agreement. If the labora-
specimens require additional non-cytogenetic testing. tory is only analyzing specimens, such an agreement
Some laboratories experience backlogs or other similar should not be necessary. However, part of the American
difficulties, which require that some samples be sent to Recovery and Reinvestment Act of 2009 known as the
another lab to enable them to “catch up.” For these rea- Health Information Technology for Economic and Clinical
sons, proper record keeping and other regulations exist to Health Act (HITECH) has a subtitle that deals with privacy
ensure proper handling and timely reporting of results for and security issues associated with electronic transmission
such specimens. Reports must clearly indicate where the of PHI and extends those aspects of HIPAA (including
testing took place. updated civil and criminal penalties) to business associates
• Ethics policies. While most laboratorians who perform (in this case, a laboratory) of entities covered by these pro-
prenatal testing consider themselves to be “pro-choice” visions (i.e., clinicians).
regarding a patient’s right to make informed decisions, There is a wide range of information that may be con-
many feel compelled to contribute only clinically relevant sidered “individually identifiable,” including names,
results. Prenatal analysis for gender identification/selec- Social Security numbers, geographic subdivisions smaller
94 M.B. Keagle
than a state, etc. Care must also be taken in the use of

References
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of Technology, for his assistance in preparing the section on Canadian
personnel.
Instrumentation in the Cytogenetics
Laboratory 7
Steven L. Gersen
ries: robotic harvesters, environmentally controlled drying

Introduction chambers, and computerized imaging systems, which can
also include automation of certain microscopy steps. There
All laboratory procedures were essentially manual at one have also been devices developed to eliminate some of the
time. Primitive centrifuges were hand operated, and the ear- manual steps involved in performing chromosome banding
liest microscopes (examples of “new technology” in their and staining and fluorescence in situ hybridization (FISH)
own time) utilized operator-positioned mirrors to gather sun- studies, and instrumentation and computing power are
light or candlelight before the discovery of electricity and the required for microarray analysis. It should be pointed out
invention of the electric light bulb. Today, however, the typi- that some cytogenetics laboratories use many if not all of
cal clinical laboratory is dominated by technology, comput- these types of devices, while most use one or a few.
ers, and automated instrumentation. These have improved
laboratory practice in three basic ways:
• Automation of tasks, which can free up technologist time, Robotic Harvesters
thereby improving efficiency and reducing costs
• An increase in the speed, accuracy, and reproducibility at As described in Chap. 4, harvesting of mitotic cells for cyto-
which tasks can be performed genetic analysis involves exposing the cells to a series of
• Performance of tasks that cannot be accomplished manually reagents that separate the chromosomes, fix them, and pre-
Previous editions of this book made a point of emphasiz- pare them for the banding and staining process. This tradi-
ing the manual nature of chromosome analysis and drew a tionally involves pelleting the cells by centrifugation between
distinction between cytogenetics and other aspects of clini- steps, in order to aspirate one reagent and add another.
cal laboratory medicine when it came to automation. In cer- However, the in situ method of culture and harvest of amni-
tain ways, this has not changed. Chromosome analysis still otic fluid (and other) specimens requires that the cells remain
requires a great deal of labor; cultures must be initiated, cho- undisturbed in the vessel in which they were cultured.
rionic villus samples and products of conception require Reagents are therefore removed and added without the need
technologist time and manipulation at a dissecting micro- to collect the cells in a tube that can be centrifuged. Thanks
scope, and even where technology exists, human interaction to improvements in technology both processes lend them-
with that technology is often required; whether through a selves to automation.
microscope or on a computer screen, visual examination of Webster defines a robot as “…an automatic apparatus or
chromosomes by human beings is necessary. Nevertheless, device that performs functions ordinarily ascribed to human
technology has advanced considerably in recent years, and beings….” In this context, those functions are aspiration of
many steps in the process of preparing and examining chro- the growth medium from the centrifuge tube or culture dish,
mosomes have now been automated to some degree. addition of a hypotonic solution, and, after an appropriate
Such instrumentation can assist with specimen processing incubation time, removal of the hypotonic solution and addi-
and chromosome analysis and falls into several basic catego- tion of several changes of fixative, each with its own dura-
tion. For suspension cultures, a centrifugation step is also
necessary before each aspiration. What is required, then, is a
S.L. Gersen, Ph.D. (*)
device that can aspirate and dispense liquids (and, for sus-
Cytogenetics Laboratory, AmeriPath Northeast,
1 Greenwich Place, Shelton, CT 06484, USA pension cultures, perform the necessary centrifugation),
e-mail: [email protected] monitor the timing of each step, and control these steps
96 S.L. Gersen
Fig. 7.1 Multiprep robotic harvester. This device was designed

specifically for cytogenetics laboratories, with enhancements such as Fig. 7.2 Hanabi-PII metaphase chromosome harvester (Photo cour-
automatic fixative mixing, integral fume extraction, multiple dispens- tesy of Transgenomic, Inc.)
ing, and aspiration probes to reduce the risk that blockage will ruin a
harvest, and onboard programming, which eliminates the need for an
external computer, reducing bench space requirements (Photo courtesy Robotic processors are also available for the blood/bone
of Genial Genetic Solutions) marrow harvest procedure. Because these devices must be
capable of both liquid handling and centrifugation, they
correctly regardless of the number of cultures or tubes being require a substantial amount of space and are designed to be
processed at any one time, i.e., some form of computer con- freestanding rather than benchtop units (Fig. 7.2).
trol that can be “told” how many specimens there are and
where on the device they are.
The initial approach to automation of certain in situ harvest- Drying Chambers/Slide Makers
ing steps was modification of existing computer-controlled
horizontal liquid handling devices that utilized a probe that Again, as described in Chap. 4, the typical end product of the
moved along its X and Y axes. This is no longer necessary; cytogenetic harvest is a centrifuge tube with fixed cells, both
laboratories can now choose from among several instruments mitotic and nonmitotic. Spreading of chromosomes is
designed specifically for harvesting specimens for chromo- achieved by placing one or more drops of this suspension on
some analysis a number of microscope slides and is controlled by the height
For in situ cultures, liquid handling devices have been from which the suspension is dropped, the temperature and
replaced by those built specifically for cytogenetics laboratories; condition of the slide, and any number of manipulations
one such instrument is programmed by the user directly, with- while the slide is drying (including the ambient conditions in
out the need for an external computer. This, along with the the laboratory). Results are monitored with phase contrast
machine’s vertical design, dramatically reduces its footprint, microscopy, and any slide that is not satisfactory can be
conserving valuable bench space (Fig. 7.1). discarded and replaced; trained individuals can determine
7 Instrumentation in the Cytogenetics Laboratory 97
the adjustments necessary to improve drying and spreading.

Provided that such adjustments are made properly and quickly,
running out of cell suspension is generally not a problem.
This is not always true, however, particularly with small
volume bone marrow aspirates, and it is never the case with in
situ culture and harvesting, typically utilized for prenatal
diagnosis. Most cytogenetics laboratories initiate four to six
in situ cultures from each amniotic fluid or CVS sample (see
Chap. 12), depending on the condition of the specimen upon
receipt. Regulations and good clinical sense require that cells
from at least two of these are examined, and in many cases,
three or more cultures are required. When one considers that
at least one culture or some other form of backup should be
retained against an unexpected need for additional testing, it
becomes evident that every culture dish must produce useable
metaphases. The concept of discarding one and trying again,
possible in many cases when making slides from cell suspen-
sions, does not apply. Further complication is introduced by
the fact that the physical force generated by dropping the cells
onto a glass slide is not available when in situ processing is Fig. 7.3 Benchtop drying chamber. Initially developed for the culture
used, and so spreading of chromosomes is accomplished of insect cells (which are grown at room temperature, and so the incuba-
solely by the manner in which the cultures are dried. tor must be capable of cooling as well as heating), this chamber has
been modified to control humidity as well, and fans have been installed
As the 3:1 absolute methanol:glacial acetic acid fixative used to allow for control of airflow over coverslips or slides (Photo courtesy
in cytogenetics laboratories dries, it “pulls” the cell membrane of Percival Scientific, Inc.)
across the slide or coverslip with it, allowing the chromosomes
of mitotic cells to separate. If this process is viewed with a phase Several companies have developed self-contained cham-
contrast microscope, the metaphases appear to open much like bers specifically for the purpose of drying in situ cultures; an
a flower blossom. Clearly, the ambient temperature and humid- example is shown in Fig. 7.3. The advantages to this type of
ity, as well as air flow over the cells (and possibly, as suggested hardware are its ability to maintain conditions, quick recov-
by some studies, the barometric pressure), all affect the rate of ery time after opening the chamber to insert or remove dishes,
drying and the ultimate quality of the chromosome preparation and potential for external venting if necessary. The disadvan-
[1, 2]. When utilizing in situ processing, controlling these tage is the necessity to remove the fixative prior to placing
parameters is the only way to control chromosome spreading. the dishes in the chamber, creating the potential for drying to
In fact, of greatest importance is not merely controlling begin under noncontrolled conditions if there is any delay in
conditions, but maintaining them with a high degree of con- getting the dishes into the chamber.
sistency. With each change in any one parameter, drying and A variation on this theme is shown in Fig. 7.4. Here, the
spreading of chromosomes changes; once the correct combi- entire drying process, including aspiration of fixative, can take
nation is achieved, it is of paramount importance that it be place inside a freestanding chamber. The technologist sits at the
maintained throughout the entire harvest. This is true regard- unit and manipulates the processing with a glove-box approach.
less of the specimen type or harvest method. The advantage here is that the drying process takes place under
There have probably been almost as many solutions to this controlled conditions from the instant the fixative is removed;
situation as there are cytogenetics laboratories. Some constructed there is no need to rush to get dishes or slides into the chamber.
enclosed chambers in which air flow, humidity, and temperature The drawback to this concept is the large size of the unit, and a
could be varied, although these were typically prone to failure somewhat more cumbersome and limiting setup; removing one
whenever the air-conditioning broke down since it is easy to or more cultures for examination (an absolute requirement) can
warm the air inside the chamber but almost impossible to cool it. be more intrusive to the workflow.
Some labs designed climate-controlled rooms; these frequently A benchtop device that combines advanced computer
functioned well, but the drawbacks here were the need to main- control capability for precise control and monitoring of con-
tain conditions while properly venting out fixative fumes (an ditions with the ability to perform aspiration inside the cham-
engineering challenge, but certainly possible) and the potential to ber is also available (Fig. 7.5).
expose the technologist to uncomfortable if not unhealthy condi- These condition-controlled chambers are gaining in pop-
tions. Such rooms were also costly to build. ularity in cytogenetics laboratories, and some use them not
The availability of specialized equipment has all but elim- only for in situ processing but for routine slide making as
inated the need for such homegrown solutions. well due to the consistency they provide. For this reason,
Fig. 7.6 Hanabi metaphase spreader. Temperature, humidity, and
airflow are set and rapidly stabilized so when chromosome preparations
are placed on microscope slides, they are dried in a consistent and
reproducible manner. Multiple slides can be created simultaneously
(Photo courtesy of Transgenomic, Inc.)
Fig. 7.4 Floor model drying chamber (Photo courtesy of Thermotron
Industries)
Fig. 7.5 Monalisa® ambient conditions chamber. Exact specifications

for multiple programs, to accommodate different tissue types, are soft-
ware-controlled via a laptop computer for ease of operation (Photo Fig. 7.7 Hanabi-PIV automated slide maker (Photo courtesy of
courtesy of elja, Inc.) Transgenomic, Inc.)
devices designed specifically for slide making are now also Chap. 17) utilize specimen types that are not processed for
available (Fig. 7.6), and one of these actually automates the chromosome analysis, such as slides cut from paraffin blocks
entire slide-making process (Fig. 7.7). or made from bladder wash/urine samples. Such sample
types require deparaffinization or other pretreatment before
any FISH procedure can be performed. While not difficult or
Instrumentation for FISH complicated, these procedures are repetitive and time con-
suming. Fortunately for the laboratory, devices that automate
While fluorescence in situ hybridization (FISH, see Chap. 17) such steps are available (Fig. 7.9). These devices also offer
represents one of the most exciting and clinically significant the laboratory the flexibility of performing other FISH pre-
developments in cytogenetics, most of the steps involved treatment procedures, and they can even be programmed to
in preparing samples for analysis are unremarkable and perform certain routine cytogenetic or cytological proce-
often repetitive and therefore lend themselves to automa- dures, making them more cost efficient for certain institu-
tion. When one considers the FISH sample volume that tions. This can be significant, as these instruments are not
many cytogenetics laboratories receive, any device that can inexpensive.
reduce the labor component of the process becomes
indispensable.
The entire FISH process can be performed automatically Hybridization
(Fig. 7.8), but laboratories also have the option of utilizing
instruments that are specifically designed for different aspects As with any DNA hybridization procedure, FISH requires a
of the procedure. series of heating and cooling steps to facilitate denaturation
and renaturation/hybridization of probe and target DNA.
Analogous to the thermocyclers utilized for the polymerase
Pretreatment chain reaction (PCR) in the molecular genetics laboratory,
devices are available that permit a technologist to add FISH
For many applications of FISH, the only thing one must do probes to a sample slide, close the cover, initiate a prepro-
to prepare a sample for analysis is make one or more addi- grammed series of temperature changes, and walk away.
tional slides or, in some cases, destain a slide that has already These instruments can handle a modest number of slides at
been examined so as to be able to interpret the results of one time, and store several user-de fi ned programs for
hybridization to already-analyzed metaphases. However, analytical flexibility. Newer models include fluid handling
some applications of the technology (e.g., ERBB2 analysis capabilities so that various pretreatment steps can also be
for breast cancer or FISH for bladder cancer recurrence; see performed prior to hybridization (Fig. 7.10a, b).
The drawback to these devices is that large volume or
frequent use of probes that require different programming
necessitate the purchase of more than one unit. Some have,
however, come down in price in recent years.
Fig. 7.9 VP 2000 Processor. This device automates various laboratory

Fig. 7.8 Xmatrx™ automated FISH processing system (Photo protocols, such as a pretreatment or deparaffinization step prior to per-
courtesy of Abbott Molecular, Inc.) forming a FISH assay (Photo courtesy of Abbott Molecular, Inc.)
100 S.L. Gersen
Fig. 7.10 ( a ) Thermobrite StatSpin ® programmable temperature Inc.). ( b ) Thermobrite® Elite Automated Laboratory Assistant. This
controlled slide processing system. Up to 12 slides can be placed in device adds an automated fluidic system to facilitate pretreatment
the device, which can be programmed to heat and cool as required of different specimen types (Photo courtesy of Iris Sample
for various FISH protocols (Photo courtesy of Abbott Molecular, Processing)
Enrichment
Eliminating the need for actively dividing cells notwithstand-

ing, FISH assays can still suffer from difficulties in detecting
abnormal cell populations if these are present in small num-
bers. Techniques to enrich the population of target cells prior
to performing FISH are gaining popularity and are expected
soon to become standard of care for certain neoplasms (see
also Chap. 17). One approach to enrichment is to chemically
couple magnetic beads with an antibody to a specific cell
surface marker. Target cells can then be magnetically sepa-
rated from a specimen prior to a FISH assay. Instruments to
automate this process are now available; an example is shown
in Fig. 7.11.
Automated Imaging Systems
Introduction Fig. 7.11 RoboSep instrument for automating the magnetic enrich-
ment of a target cell population prior to performing FISH or other pro-
The original method of imaging chromosomes was photo- cedures (Photo courtesy of Stemcell Technologies, Inc.)
micrography. A photograph of metaphase chromosomes
was taken, the film was developed and photographs were
printed in a darkroom, and the chromosomes were cut out situ hybridization (FISH), interphase FISH, and comparative
and arranged to form a karyogram. Though it was a stan- genomic hybridization (CGH) can also be attributed to
dard technique for many years, this process increased the automated imaging (see Chap. 17).
already time-consuming nature of clinical cytogenetics, Although the primary application of an imaging system
and it has for all intents and purposes been replaced by in many cytogenetics laboratories used to be the production
digital imaging. of karyograms, the use of automated imaging systems for
Automated imaging systems dramatically reduce the time FISH rapidly gained popularity, and some laboratories
it takes to produce a karyogram and therefore can be seen perform more FISH imaging than chromosome imaging.
as one of the most important developments in automation Automated metaphase finding and fluorescent spot counting
of the cytogenetics laboratory. Furthermore, the growth in also represent important applications for imaging systems
fluorescent techniques such as multicolor fluorescence in in cytogenetics.
Benefits security. Partly to address this need for patient record security,
the U.S. Congress passed the Health Insurance Portability and
There are clear advantages to eliminating the need to rely on Accountability Act (HIPAA) in 1996 and subsequently the
photography. Laboratories no longer need to dedicate valuable Health Information Technology for Economic and Clinical
space to creating and maintaining a darkroom, staff no longer Health Act (HITECH) as part of the American Recovery and
need be exposed to the chemicals and fumes involved, and Reinvestment Act of 2009 [3, 4] (see also Chap. 6).
there is no longer a need to arrange and pay for proper disposal While traditional photographic techniques offered some
and reclamation of silver. Nevertheless, a reduction in the time degree of contrast and other image adjustment, automated
it takes to complete an analysis is unquestionably the major imaging systems further offer easy image enhancements,
benefit of an automated imaging system. Laboratories can also visualization techniques, and quantification, potentially pro-
save operator time by automating metaphase scanning, karyo- viding additional information.
typing, and FISH applications, in some cases resulting in a
faster turnaround time and higher throughput of cases.
Reduction in labor also translates to reduced costs. Limitations
Another big advantage of digital images is easy and com-
pact storage. With current compression technologies and Despite image enhancement features, the quality of the
digital storage devices, this is much easier and less space final image is ultimately dependent on the quality of the
consuming than it was with photographs and negatives. In original microscope image (see Chap. 5). An image may
addition, photographs could deteriorate over time, making be improved through background elimination, contrast and
them harder to reexamine if necessary. color enhancement, or even longer exposure times, but all
Automated imaging systems can also provide consistency, these will not make up for a poor image due to poor micro-
especially when performing interphase FISH assays. Whereas scope configuration or suboptimal slide preparation (see
manual spot counting can be highly subjective, an automated Chap. 4).
system will use predefined parameters for spot counting and,
using those parameters, will produce consistent results.
Sharing of data can be important in a clinical lab setting Imaging System Components
and is clearly facilitated by the use of digital images; with the
current use of the Internet and electronic mail, digital images In general, an imaging system for cytogenetics comprises a
are more easily shared for consultation and discussion. microscope with a camera adapter, a camera, and a computer
However, with data sharing via the Internet comes the need for and software (Fig. 7.12). A printer and a method of archiving
compression and, more pressing, the need for patient record images are also required.
Fig. 7.12 Ikaros automated chromosome imaging system (Photo courtesy of MetaSystems Group, Inc.)
102 S.L. Gersen
Microscope with Camera Adapter Printer

A detailed discussion of microscopes and microscopy can be Although the trend seems to be moving toward the paperless
found in Chap. 5. As the name already implies, the camera laboratory, hard copy prints can still be used for diagnostic and/
adapter (known as a C-mount) is the device designed to attach or archival purposes. In addition to the high-resolution black-
a camera to a microscope. This adapter also permits the micro- and-white images of karyograms prepared by cytogenetics lab-
scope image to be projected onto the photosensitive area of the oratories, a printer used for FISH applications must be capable
camera, and ensures that the digitally captured image is scaled of reproducing the range of colors generated by modern FISH
correctly to match the microscope objective. It is important to software. Laser printers are widely used for these purposes.
be certain that the captured image reflects the size and propor-
tion of the image viewed through the microscope. This can be Archival Device
accomplished through the use of an additional lens that will As mentioned earlier, there is a need (often imposed by regu-
enlarge or reduce the size of the image captured. For example, lation) for long-term archiving of patient data. With the use of
a 63× objective would be paired with a 1× camera adapter, automated imaging systems, the data is in digital form and is
which introduces no additional magnification or reduction. easy to store. Currently, DVDs and optical discs can be used
However, a 100× lens would require a .7× camera adapter to as solid media, while many labs choose to store data directly
reduce the size of the image to fit correctly on the camera. on a server, often as part of a network as indicated earlier.
Camera
Though a wide range of different camera options are avail- Applications of Automated Imaging
able (analog, digital, cooled, uncooled, monochrome, color),
the most commonly used camera on automated imaging sys- Preparation of Karyograms
tems for the cytogenetic laboratory is a black-and-white, One main application of automated imaging systems is
uncooled CCD (charge coupled device) camera. karyotyping. This involves separating and classifying chro-
mosomes based on length, position of the centromere, and
Computer banding pattern (See Chap. 3). These automated systems
Though both PC- and Macintosh-based systems have been should provide ease of use, speed, image quality, and
available, the recent trend has been a move toward accuracy.
PC-based imaging systems. The computer(s) can be net- Imaging systems have improved significantly over the
worked, allowing the actual analysis of the images to be years. Early systems required the user to “cut out” the chro-
performed off-line and to facilitate data sharing. Network mosomes using a trackball or mouse, and place them into a
storage of digital images has become more prevalent, karyogram. In semiautomated systems, the system could
allowing laboratories to share data and improve efficiencies, “cut out” the chromosomes, but the user had to arrange them
workflow, and turnaround times. Additionally, network into a karyogram. Today’s fully automated imaging system
storage of images has removed the need to store and main- will capture the image, separate or “cut out” the metaphase
tain data on individual computers. chromosomes, classify them, and arrange them into a karyo-
gram (Fig. 7.13). However, some metaphases contain several
Software
The software for automated imaging systems for cytogenet-
ics consists of at least two parts: acquisition or capture and
the actual analysis. These can be two distinct steps or can be
seamlessly integrated into one application. The acquisition
step drives the camera in order to take a digital picture (cap-
ture an image). It also includes image enhancement features
such as contrast adjustment, background subtraction, shad-
ing correction, and image sharpening. After image capture
and enhancement, the user can analyze the image using the
analysis applications of the software. Though there are many
commercial packages available for image analysis, cytoge-
netics software, especially developed to address the specific
requirements of a cytogenetics laboratory, includes several
important features that are not available in conventional
image analysis packages. Some of these features include the
automatic generation of karyograms and the automated scor- Fig. 7.13 An example of karyotyping software. The original metaphase
ing of interphase FISH slides. is in the upper right (Photo courtesy of MetaSystems Group, Inc.)
overlapping chromosomes, and the user may still need to relocation to a metaphase of interest, speed of scanning, and
intervene and manually separate these using the mouse. Also, sensitivity (the percentage of metaphase cells found by the
abnormal chromosomes are often not identified by imaging system). Newer technologies allow the metaphases finders to
software, requiring user interaction for creation of an accu- automatically select the best metaphases after search, apply
rate karyogram. oil to the slide, and capture these metaphases under high
A fully automated karyotyping system can also be used in magnification.
conjunction with metaphase finding capability. This means Software features important for metaphase finding include:
that the system will automatically scan the slide in search of • Definition of classification parameters to ensure optimum
good metaphase spreads that can be used for analysis and scan results. The user can define the parameters that are
creation of karyograms. utilized by the system.
• The ability to quickly relocate to a metaphase or rare cell
Scanning and Metaphase Finding for review.
Finding metaphases acceptable for analysis is an integral part • A sort function to organize metaphases or cells after scan-
of cytogenetics. In many samples, good quality metaphases ning based on specific parameters and quality preferences.
are abundant. However, in some specimens, such as in cancer A metaphase scanning system can be set up to continu-
cytogenetics, cells are often of poor quality and metaphase ously scan slides for metaphases or rare events while tech-
spreads acceptable for analysis are few and hard to find. nologists are analyzing the detected metaphases or cells on
A system that will automatically scan a slide for metaphase remote review stations. Some laboratories find that this
spreads can greatly reduce the time spent by a technologist on streamlines their workflow, while others find these systems
these samples looking for those metaphases. to be neutral in terms of time gained. Laboratory volume
The microscope in a metaphase finding system is outfitted and workflow will determine whether use of such a system
with a motorized stage and focus drive for automated focus- makes sense.
ing. While automatically scanning one slide saves the user Due to the general nature of the scanning system, it can
time, it does not make much sense to continuously have to also be used in other applications that require scanning for
change slides for scanning. To increase the throughput of the particular cells, such as FISH spot counting for detection of
system, many suppliers add a stage or even slide loader to the tumor cells in body fluids.
system that holds multiple slides (Fig. 7.14). Based on sev-
eral parameters, the system images metaphase spreads at Fluorescent Spot Counting
high power and presents them to the user for review and FISH technology is based on fluorescently labeled probes
analysis (Fig. 7.15). that hybridize to unique DNA sequences along the chromo-
Key factors for a metaphase finding system are the ability somes and can be performed on either metaphase prepara-
to recognize appropriate metaphases or cells, accuracy of tions or interphase cells. One application is fluorescent spot
Fig. 7.14 An automated slide scanning

system, which can be used with both
brightfield and fluorescence microscopy on the
same tray. The tray loader allows up to 81
slides to be scanned without interruption, but
slide trays can also be loaded and unloaded
while the system is operating (Photo courtesy
of Applied Spectral Imaging, Inc.)
104 S.L. Gersen
Fig. 7.15 Software interface of a metaphase finding system, showing thumbnails of the metaphase spreads located by the system (Photo courtesy
of MetaSystems Group, Inc.)
counting and examination used for translocation or copy To visualize the different fluorochromes, the system uses
number analysis performed on interphase cells (Fig. 7.16). different bandpass filters and a single, epi-illuminating light
Generally, an imaging system for FISH needs to be able source (see Chap. 5, Fig. 5.6). An image is acquired for each
to capture low light level images in multiple wavelengths, fluorescent label used in the protocol, and the computer com-
quantify the number of each fluorescent signal, and estimate bines those into a color image. If the system is not equipped
the intensity ratio of the different signals. with an automated microscope with motorized filter block
Since interphase cells are three-dimensional (3-D) struc- changing, a motorized filter wheel that will hold the different
tures, the fluorescent signals in interphase FISH can be pres- filters is highly desirable.
ent in different focal planes. This means that to be able to see The microscope focus, camera, and filter wheel can be auto-
all signals, the user will need to focus on the different planes matically controlled and synchronized by Z-stack software for
(the Z-axis), making the presence of a motorized focus drive multiplane, multicolor fluorescence image capture. Images in
on an automated system imperative. The automated focusing different focal planes are acquired and combined in a focused,
allows for resolution of the multiple signals across a large color image to ensure that faint signals that would otherwise be
focal depth. Images from different focal planes are captured, omitted are incorporated in subsequent analyses.
processed, and compiled into one pseudo 3-D image that FISH analysis is also amenable to automated software. To
shows all signals in focus. This 3-D image capture is often ensure consistent scoring and analysis of interphase FISH,
referred to as Z-stack. such software should include:
Fig. 7.16 Example of a software interface for spot counting of inter- the detection of chromosomal abnormalities associated with the
phase FISH, showing thumbnails of cells and spots located by the sys- recurrence and progression of bladder cancer (Photo courtesy of
tem. This application is using the Abbott Molecular UroVysion® kit for Bioview, Inc.)
• Trainable classifiers to determine which cells to score, so Software that facilitates analysis of multiple (sequential)
users can “teach” the system to work with their own fluorescence assays by relocating previously analyzed cells
results and standards. is also available (Fig. 7.17).
• User-definable parameters to determine the scoring rules.
Such parameters include spot size and spot separation M-FISH
distances (measured three-dimensionally), fusion detec- M-FISH, also referred to as multicolor FISH, multiplex
tion, and the number of cells to score. FISH, or one company’s proprietary name of spectral
• The ability to reprocess the images under different scoring karyotyping (SKY), can be viewed as fluorescent multicolor
rules without having to rescan the slide. karyotyping and is mainly used for the detection and
• The ability to sort the scored cells in user-definable catego- classification of interchromosomal aberrations (see Chap. 17).
ries, allowing for easier analysis of complex signal patterns. In this form of FISH, probes labeled with a combination of
• A reporting function that presents the results for review. different fluors are hybridized with the chromosomes in a
Reports should be customizable to reflect the user’s metaphase spread. Currently, five different fluorochromes are
preferred data layout and should include images of scored used. The five different fluors give 31 (2n−1) color combinations,
cells and different representations of the results, such as bar enough to uniquely identify the 24 different chromosomes in
charts and scatter plots. the human genome. It has been suggested that the resolution
106 S.L. Gersen
Fig. 7.17 ASI MultiStain software for relocation to previously found relocated. This process can be repeated up to three times. Afterward, all
cells after restaining with different markers, enabling cross correlation images of the same, differently stained cells can be viewed side by side
of findings from the same slide after multiple staining. After the slide is in the gallery (Image courtesy of Applied Spectral Imaging, Inc.)
washed following initial staining, the cells can be restained and quickly
of using a 5-fluorochrome set could result in some small • Karyotyping capabilities so that the colored chromosomes
aberrations being missed and that this problem can be elimi- can be arranged in a karyogram (Fig. 7.18; see also Chap.
nated by increasing the number of fluors to seven [5]. 17, Fig. 17.18).
However, this concept has not been introduced into common • Individual pseudo-color display of any single chromosome
laboratory practice. to facilitate visualization of chromosomal aberrations.
From a hardware perspective, the requirements of an
automated system for M-FISH are similar to the require- Comparative Genomic Hybridization (CGH) and
ments of an automated system for interphase FISH: the sys- Chromosome Microarray
tem should include the fluorescent, epi-illuminating light Whereas M-FISH is a useful technique to determine interchro-
source and a filter wheel containing the appropriate filters. In mosomal rearrangements, comparative genomic hybridization
addition, the system could include a metaphase finding capa- (CGH) provides information concerning losses or gains of
bility as well as motorized focusing. DNA within a chromosome (see Chap. 17). In CGH, the
The software for M-FISH incorporates: probes are generated from two different sources: one from
• Sophisticated algorithms that analyze the images to deter- genetically normal cells and the other from the patient sample.
mine the fluor combination a chromosome is labeled with The two different probe sets are labeled with different fluors.
and then assign a pseudo-color to each fluor combination. These two pools of probes are then hybridized to a slide that
These pseudo-colors should be user changeable to improve contains normal metaphases and will compete for hybridiza-
visualization of rearranged chromosomes. tion to the corresponding loci. The ratio of the patient DNA to
Fig. 7.18 M-FISH capture and analysis. The software analyzes the signals produced by the various combinations of fluors, produces a false color
for each chromosome, and arranges the chromosomes into a karyogram. See text for details (Photo courtesy of MetaSystems Group, Inc.)
normal DNA will indicate whether the patient DNA is normal For microarrays, specific DNA targets are “printed” onto
(the ratio is 1:1) or whether there is an addition or deletion of a microscope slide and CGH is performed in situ on the slide
DNA in any given region. When there is an addition, the ratio (see Chap. 18). A scanner reads the slide and sends the data
will increase; when there is a deletion, the ratio will decrease. to a computer for analysis (Fig. 7.19a, b).
Chromosomal CGH requires the use of a high-quality and
quantitative FISH imaging system with dedicated software
that will:
• Accurately measure and average the ratio of the two fluors Multipurpose Instruments
over multiple metaphases. This requires sophisticated
algorithms. Some of the instruments described in this chapter are capable
• Correct the measurements for unequal chromosome length. of performing more than one function in the cytogenetics
• Plot the ratios along the chromosome length for ease of laboratory. Given the direction that instrumentation and elec-
interpretation, highlighting the areas of statistically tronics are going in today’s world, such cross functionality
significant differences (see Chap. 17, Fig. 17.17). will ultimately become more common. Devices designed to
Fig. 7.19 GenePix microarray scanner. (a) This benchtop unit features an 8-position emission filter wheel to allow the user flexibility in choosing
fluorescent dyes. (b) The image on the right shows a schematic of the light path through the scanner (Photos courtesy of Axon Instruments, Inc.)
Fig. 7.20 Multipurpose devices designed to

process chromosome banding, FISH, and
microarrays. (a) SciGene Little Dipper. Wash
time, agitation, buffer temperatures, and drying
are controlled, and the device uses an
integrated centrifuge (Photo courtesy of
SciGene). (b) elja Leonardo® Molecular
Processor. Laptop software allows the user to
modify every aspect of the procedure for
accuracy and reliability (Photo courtesy of
elja, Inc.)
be used for some of the steps involved in chromosome 2. Deng W, Tsao SW, Lucas J, Leung CS, Cheung LM. A new method
banding, FISH analysis, and microarray studies are already for improving metaphase chromosome spreading. Cytometry A.
2003;51A:46–51.
available (Fig. 7.20a, b). 3. Public Law 104-191 August 21, 1996. http://www.gpo.gov/fdsys/
pkg/PLAW-104publ191/pdf/PLAW-104publ191.pdf. Accessed 19
Acknowledgment Special thanks to John Fonte of MetaSystems Group, Oct 2011.
Inc. for his contribution to the section on automated imaging systems. 4. Public Law 111-5 February 17, 2009. http://www.gpo.gov/fdsys/
pkg/PLAW-111publ5/pdf/PLAW-111publ5.pdf. Accessed 19
Oct 2011.
5. Azofeifa J, Fauth C, Kraus J, Maierhofer C, Langer S, Bolzer A,
References Reichman J, Schuffenhauer S, Speicher MR. An optimized
probe set for the detection of small interchromosomal aberra-
1. Spurbeck JL, Zinmeister AR, Meyer KJ, Jalal SM. Dynamics of tions by use of 24-color FISH. Am J Hum Genet. 2000;66:
chromosome spreading. Am J Med Genet. 1996;61:387–93. 1684–8.
Part III
Clinical Cytogenetics
Autosomal Aneuploidy
8
Jin-Chen C. Wang
Introduction all autosomes, with the possible exception of chromosome

21. It has also been reported that the frequency of aneuploidy
The term aneuploidy refers to cytogenetic abnormalities in varies among different individuals and can increase over a
which all or part of one or more chromosomes is duplicated 5-year period in the same individual [13].
or deleted. Autosomal aneuploidy refers to all such abnor- Trisomies for all autosomes have been reported in sponta-
malities that do not involve the sex chromosomes. These can neous abortuses, including trisomy 1, which has been
be either numerical (the topic of this chapter) or structural, reported at least one clinically recognized pregnancy at
the vast majority being trisomies, and may be present only in 8–9 weeks of gestation and in a clinically recognized in vitro
some cells (mosaic aneuploidy) or in all cells (nonmosaic). fertilization (IVF) pregnancy at 6 weeks of gestation [3, 14,
The incidence of autosomal aneuploidy in newborns is esti- 15, 16]. However, no fetal pole ever developed in either case.
mated to be 0.2% [1]. Many autosomal aneuploidies are Unlike in sperm, the observed frequency of each trisomy var-
incompatible with fetal survival and therefore have much ies greatly in spontaneous abortuses or liveborns. For exam-
higher incidences (approximately 27–30%) in spontaneous ple, trisomy 16 accounts for approximately 30% of all
abortuses [2–4]. These are discussed in this chapter and autosomal trisomies in abortuses [3]. In liveborns, the only
covered in detail in Chap. 12. trisomies that have not been reported in either mosaic or nonmo-
Cytogenetic studies of human oöcytes and sperm reveal saic form are those involving chromosomes 1 and 11, although
that the overall frequency of abnormalities is approximately trisomies other than 13, 18, and 21 are rare. Autosomal
15–20 and 10%, respectively [5–7]. More than 90% of the monosomies, on the other hand, are extremely rare in both
abnormalities observed in oöcytes and less than 50% of those liveborns and recognized abortuses.
seen in sperm are numerical. Since structural abnormalities The supposition that, with the probable exception of
are difficult to detect, the observation that the abnormalities trisomy 21, the frequencies of trisomy for each chromosome
identified in oöcytes are mostly numerical could be a result might be similar at the time of conception but differ greatly
of ascertainment bias [8]. Studies using fluorescence in situ among abortuses and liveborns can be explained by the
hybridization (FISH) or primed in situ labeling (PRINS), devastating effect of chromosomal imbalance. Many auto-
which do not require the presence of dividing cells, have somal aneuploidies are so deleterious that they are lethal in
shown that the frequency of autosomal aneuploidy in human the preembryonic stage and thus result in unrecognized and,
sperm is relatively uniform for all chromosomes studied therefore, unstudied in spontaneous abortions. The lethality
(chromosomes 3, 7, 8, 9, 10, 11, 13, 16, 17, 21), with a range of a particular autosomal aneuploidy correlates with the gene
of 0.26–0.34% [9–11]. On the other hand, one study using content of the chromosome involved [14]. Aneuploidies for
FISH reported a higher frequency of aneuploidy for chromo- “gene-rich” chromosomes are less likely to survive. Trisomies
some 21 (0.29%) than for other chromosomes studied (0.08– 13, 18, and 21, which involve chromosomes that are “less
0.19% for chromosomes 1, 2, 4, 9, 12, 15, 16, 18, 20) [12]. It is gene-rich,” are therefore relatively “mild” and fetuses can
therefore possible that meiotic nondisjunction is random for survive to term.
This chapter addresses only those autosomal aneuploidies,
both trisomies and monosomies, that have been observed
in liveborns. Polyploidy, or changes in the number of
J.-C.C. Wang, M.D. (*)
complete sets of chromosomes, are also included, as are
Department of Cytogenetics, Genzyme Genetics,
655 East Huntington Drive, Monrovia, CA 91016, USA aneuploidies that are the result of supernumerary “marker”
e-mail: [email protected] chromosomes.
114 J.-C.C. Wang
nondisjunction of the bivalent chromosomes with both

Mechanism and Etiology homologs going to the same pole (Fig. 8.1d,e). The second
type of error involves premature separation of the sister chro-
Errors in meiosis (nondisjunction) result in gametes that matids of one homolog of a chromosome pair. Subsequent
contain abnormal numbers of chromosomes and, following improper distribution of one of the separated chromatids
fertilization, produce aneuploid conceptuses. Using DNA results in its segregation with the other homolog of the chro-
markers, the parental origin of the additional chromosome in mosome pair [35] (Fig. 8.2d,e). In MII, sister chromatids
autosomal aneuploidies has been studied for trisomies 2, 7, separate. Malsegregation occurs when both chromatids go to
13, 14, 15, 16, 18, 21, and 22 [2, 17–32]. These studies sug- the same pole (Fig. 8.3g,h). It has been shown that error
gest that most trisomies are of maternal origin but that the involving premature sister chromatid separation, especially
proportion varies among different chromosomes and that in the smaller chromosomes, is in fact a more common
(with the exception of chromosomes 7, 13, and 18) nondis- mechanism leading to aneuploidy than malsegregation of the
junction in maternal meiosis stage I accounts for the majority whole chromosome [6, 36, 37].
of cases (Table 8.1). A more recent study on nondisjunction Earlier cytogenetic studies of oöcytes, performed mostly
of chromosome 13 in a large number of cases reported an on unfertilized or uncleaved specimens obtained from in vitro
equal number of maternal meiosis I (MI) and meiosis II fertilization programs, have provided conflicting results
(MII) errors [33]. regarding whether the frequency of gamete aneuploidy actu-
The association between autosomal aneuploidy and ally increases with maternal age [38–41]. A FISH study of
maternal age has long been recognized. In 1933, Penrose human oöcytes using corresponding polar bodies as internal
demonstrated that maternal age was the key factor for the controls demonstrated that nondisjunction of bivalent chro-
birth of Down syndrome children [34]. Why aneuploidy is mosomes (MI error) does in fact increase with maternal age,
maternal age dependent, and what constitutes the mechanism and a study using multiplex FISH on fresh, noninseminated
and etiology of chromosomal nondisjunction have been top- oöcytes also indicated an increase in premature separation of
ics of much research, as summarized later in this chapter. the sister chromatids in MI with increasing maternal age
Nondisjunction can occur during either meiosis I (MI) or [42, 43]. More recent data based on studies of large numbers
meiosis II (MII). In MI, homologous chromosomes pair and of oöcytes further provided evidence for a direct correlation
form bivalents (see Chap. 2). Malsegregation of homologous between advanced maternal age and increased aneuploidy
chromosomes can occur in one of two ways. The first involves frequency [6, 44].
Table 8.1 Parental and meiotic/mitotic origin of autosomal trisomies determined by molecular studies (number of cases)
Maternal Paternal
Trisomy References
MI MII MI or MII Mitotic Totala MI MII MI or MII Mitotic Totala
2 4 1 6 1 13 5 5 [30]
7 2 3 1 6 12 2 2
13 4 17 21 1 1 1 3 [24]
34 33 3 3 5 1 3 [33]
14 3 4 2 9 2 2 [24]
15 21 3 3 27 5 5 [16] (UPD study)
10 10 2 2 [18] (UPD study)
17 2 10 29 4 1 5 [24, 30]
16 56 6 62 0 [25]
18 11 17 56 1 6 [28]
17 5 [21]
16 ³35 3 61 2 2 [26]
10 17 1 28 2 1 3 [31]
21 9 1 22 3 [17]
91 6 [18]
128 38 188 2 7 9 [19]
7 15 8 36 [23] (paternal study only)
174 58 79 311 9 15 8 32 [24]
62 81 10 13 [27]
67 22 97 4 4 10 [29]
22 20 1 15 37 1 1 [24, 30]
a
Total numbers may not add up because not all origins of error can be determined
8 Autosomal Aneuploidy 115
Fig. 8.1 Schematic representation of meiosis I nondisjunction. Fig. 8.2 Schematic representation of meiosis I error resulting from
( a) Prophase I. (b) Metaphase I. (c) Anaphase I. (d) Telophase I, with premature sister chromatid separation. (a) Prophase I. (b) Metaphase I.
both homologs of one chromosome pair segregating together. (e) (c) Anaphase I, with premature separation of centromere of one
Products of meiosis I. (f) Metaphase II. (g) Anaphase II. (h) Meiotic chromosome. (d) Telophase I, with one prematurely separated chroma-
products—two gametes lack one chromosome and two gametes contain tid segregating with its homologous chromosome. (e) Products of
two copies of one chromosome meiosis I. (f) Metaphase II. (g) Anaphase II. (h) Meiotic products—two
gametes with a normal chromosome complement, one gamete
lacking one chromosome, and one gamete containing two copies of
one chromosome
Different mechanisms have been proposed to account for are the first to ovulate in adult life. Another example is the
the observation of the correlation between maternal age and “limited oöcyte pool” model [48]. At the antral stage of each
aneuploidy frequency. One example is the “production line” menstrual cycle, multiple follicles at various stages of devel-
hypothesis [45, 46]. This hypothesis proposes that oöcytes opment are present. When stimulated with high levels of
mature in adult life in the same order as the corresponding plasma follicle stimulating hormone (FSH), only one follicle,
oögonia entered meiosis in fetal life. Oögonia that enter mei- presumably the one at the most optimal stage, will complete
osis later in development may be more defective in the for- MI and eventually achieve ovulation. The number of follicles
mation of chiasmata and thus more likely to undergo in the antral stage decreases with increasing maternal age.
nondisjunction. One direct cytological support for this When the number of these follicles is low, it is more likely
hypothesis was provided by a study that examined the fre- that an oöcyte that is not at the optimal stage will be selected
quency of unpaired homologs in MI pachytene and diplotene for ovulation. If such “less optimal” oöcytes are more likely
in oöcytes obtained from abortuses at 13–24 weeks and to undergo MI nondisjunction, then the ovulated oöcytes
32–41 weeks of gestation [47]. Of the six chromosomes of older women will have higher rates of aneuploidy.
studied (chromosomes X, 7, 13, 16, 18, and 21), the rate of More recent data, however, does not appear to support this
pairing failures in early specimens (0–1.2%) was significantly hypothesis [49, 50].
lower than that in later specimens (1.3–5.5%). No corrobo- One probable factor that predisposes gametes to nondis-
rating data are available. It remains an interesting question junction is aberrant recombination [51] (see Chap. 2).
whether the oöcytes first committed to meiosis in fetal life Data on recombination patterns are available for trisomies
116 J.-C.C. Wang
donors after hormone-induced superovulation demonstrated

that 2.5% of chromosome 16 bivalents had no crossovers and
a high percentage (19.8%) had only a single recombination
[37]. In trisomy 21, there was an overall reduction in recom-
bination with an increase in both zero and one crossover in
maternal MI nondisjunction [52]. Lamb et al. showed that in
maternal MI nondisjunction for chromosome 21, the average
number of recombination events was decreased, with approx-
imately 35–45% of cases having no crossovers [53]. When at
least one crossover was present, it occurred largely at distal
21q. This study, together with one on trisomy 16, suggests
that, at least for trisomies 16 and 21, distal chiasmata are less
efficient in preventing nondisjunction in MI [25]. In contrast,
in maternal MII nondisjunction, the number of recombina-
tion events appeared to be increased, especially in proximal
21q. These proximal recombinations may cause an “entan-
glement” effect. Entanglement of the two homologs can
cause the bivalent to move to the same pole at MI, and then
at MII the two homologs finally separate, resulting in two
disomic gametes each having two chromatids with identical
centromeres. Alternatively, the entanglement may disrupt
sister chromatid cohesion resulting in premature separation
of the sister chromatids at MI. If the two separated sister
chromatids travel to the same pole at MI and again at MII, an
apparent MII nondisjunction would be observed. Thus, these
data suggest that all nondisjunction events may be initiated
during MI. The observation that for chromosome 21, MI
error is associated with distal recombination while MII error
is associated with proximal recombination has been indepen-
Fig. 8.3 Schematic representation of meiosis II nondisjunction. (a)
dently confirmed recently in a study of a population in India
Prophase I. (b) Metaphase I. (c) Anaphase I. (d) Telophase I. (e) [54]. Lamb et al. showed that the alteration in recombination
Products of meiosis I. (f) Metaphase II. (g) Anaphase II, with both sister pattern was not maternal age dependent. They proposed a
chromatids segregating together. (h) Meiotic products—two gametes “two hit” model and hypothesized that certain recombination
with a normal chromosome complement, one gamete lacking one chro-
mosome, and one gamete containing two copies of one chromosome
configurations are less likely to be processed properly in
older women [53, 55]. This could result from, for example,
an age-dependent loss of spindle forming ability, thus
explaining their observation for trisomy 21 that although an
15, 16, 18, and 21. Studies of chromosome 15 nondisjunc- altered recombination pattern is not maternal age dependent,
tion in uniparental disomy (see Chap. 20) revealed that meiotic disturbance is age dependent [56]. The same argu-
there was a mild reduction in recombination in association ment was used by Hassold et al. to explain their findings with
with maternal nondisjunction, with an excess of cases that trisomy 16 [51].
have zero or one crossover and a deficiency of cases that It has been proposed that the cellular mechanism assuring
have multiple crossovers [20]. In contrast, in a study of tri- correct segregation of chromosomes into daughter cells is
somy 18, approximately one-third [5/16] of maternal MI provided by a four-protein complex (SMC1, SMC3, SCC1/
nondisjunctions were associated with a complete absence RAD21, SCC3/SA/STAG) that together form a ring-like
of recombination, whereas the remaining MI and all MII structure known as the cohesin complex [57]. The cohesin
nondisjunctions appeared to have normal rates of recombi- complex acts as “chromosome glue” and thus mediates cohe-
nation [26]. Studies of trisomy 16 and trisomy 21 reported sion of the two sister chromatids during cell division.
similar findings between the two. In trisomy 16, it was Additional proteins are needed for the establishment and
shown that recombination was reduced, but not absent, and maintenance of cohesion. Loss of cohesion of both arms,
that distribution of recombination was altered, with rare telomere, and centromere during the metaphase/anaphase
crossovers in the proximal regions of the chromosome [25]. transition is also tightly controlled by various proteins includ-
A recent study performed on oöcytes from young egg ing a specific protease separin/separase.
It has also been suggested, at least for chromosome 21, for chromosome 21, significantly more were Y-bearing than
that chiasmata and recombination in oöcyte are as efficient X-bearing [65]. This finding was consistent with earlier
as in spermatocyte [58]. The less effective meiotic check- reports showing an excess of males among trisomy 21
point mechanism in oöcytes allowing aneuploid oöcytes to conceptuses that resulted from paternal meiotic errors [23].
progress through meiosis appeared to be the basis for the This preferential segregation of the extra chromosome 21
observation that the majority of trisomy 21 conceptions are with the Y chromosome contributes to a small extent to the
of maternal origin. On the other hand, evidence against a observed sex ratio in trisomy 21 patients. Other mechanisms,
defective spindle assembly checkpoint being the cause of such as in utero selection against female trisomy 21 fetuses,
aneuploidy associated with advanced maternal age has also must also exist.
been reported [59]. Trisomy 21 accounts for approximately 95% of all cases
The possibility of the presence of a genetic predisposition of Down syndrome. Mosaicism and Robertsonian transloca-
to nondisjunction has also been proposed. One study tions (see Chap. 9) comprise the remaining 5%. As described
involving consanguineous families in Kuwait showed that previously, the incidence of trisomy 21 in newborns is closely
the relative risk for the occurrence of Down syndrome was associated with maternal age (Table 8.2).
approximately four times greater for closely related parents
(first cousins, first cousins once removed, second cousins) Phenotype
than for unrelated parents [60]. As consanguinity is usually The clinical phenotype of Down syndrome has been well
perpetuated in certain families or sections of the population, described [68, 69]. Briefly, there is a characteristic craniofacial
these results were taken as evidence for the existence of an appearance with upward-slanting palpebral fissures, epican-
autosomal recessive gene that facilitates meiotic nondisjunc- thal folds, flat nasal bridge, small mouth, thick lips, protruding
tion in homozygous parents. Thus, in a subgroup of trisomy tongue, flat occiput, and small and overfolded ears. Hands and
21 patients, nondisjunction may be genetically determined. feet are small and may demonstrate clinodactyly, hypoplasia
In a study of trisomy recurrence based on North American of the midphalanx of the fifth finger, single palmar crease
data, a significantly increased risk for recurrent of a different (Fig. 8.5), and a wide space between the first and second toes.
trisomy was observed [61]. This supports the hypothesis of Hypotonia and small stature are common, and mental retarda-
possible genetic predisposition to nondisjunction. tion is almost invariable. Cardiac anomalies are present in
While maternal age and altered recombination remain the 40–50% of patients, most commonly endocardial cushion
only well-established risk factors for nondisjunction, our defects, ventricular septal defects (VSD), patent ductus arte-
understanding of the underlying mechanism of this observa- riosus (PDA), and atrial septal defects (ASD). Other observed
tion is still not complete. It is possible that more than one major malformations include duodenal atresia, annular pan-
mechanism, including possibly environmental and hormonal creas, megacolon, cataracts, and choanal atresia. In addition, a
factors, contributes to the observed maternal age effect [62]. 10- to 20-fold increase in the risk for leukemia, most com-
Nondisjunction occurring at mitosis, on the other hand, monly acute megakaryoblastic leukemia, has been observed in
will result in mosaicism, usually with both normal and abnor- Down syndrome patients of all ages, with a bimodal age of
mal cell lines. onset in the newborn period and again at 3–6 years [70].
Discussion of autosomal aneuploidies in this chapter will Moreover, a transient abnormal myelopoiesis (TAM) with
be limited largely to those observed in liveborns only. clinical and morphologic findings indistinguishable from acute
myeloid leukemia occurs in approximately 10% of Down syn-
drome newborns [71]. Spontaneous remission within the first
Autosomal Trisomies 3 months of life is observed in most patients; occasional relapse
and life-threatening disease have also been noted. Of interest
Trisomy 21 is the observation of the presence of a trisomy 21 clone in
association with TAM in 15 phenotypically normal children,
Incidence at least 4 of whom were determined to be constitutional mosa-
Trisomy 21 [47,XX or XY,+21] (Fig. 8.4) was the first chro- ics for Down syndrome [72].
mosome abnormality described in man by Lejeune et al. in Overall, the clinical phenotype is typically milder in
1969 [63]. The phenotype was delineated by John Langdon mosaic Down syndrome patients, but there is no clear corre-
Down (1828–1896) in 1866 and is referred to today as Down lation between the percentage of trisomy 21 cells and the
syndrome [64]. It is the most common single known cause of severity of clinical presentation. This can be as severe in
mental retardation. The frequency in the general population mosaic patients as in nonmosaic trisomy 21 individuals.
is approximately 1 in 700. Down syndrome is more frequent Delineation of the regions of chromosome 21 responsible
in males, with a male-to-female ratio of 1.2:1. A recent study for the Down syndrome phenotype has been attempted using
using multicolor FISH showed that among sperm disomic molecular methods to study patients with partial trisomy 21
1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18
19 20 21 22 X Y
Fig. 8.4 Trisomy 21 Down syndrome male karyogram (47,XY,+21)
Table 8.2 Maternal age-specific risks for trisomy 21 at birth who present clinically with various features of the syndrome
Maternal age Incidence at Maternal age [73–80]. Studies by Korenberg et al. in a panel of cell lines
(years) birth (1 in) (years) Incidence at birth (1 in) derived from 16 partial trisomy 21 individuals suggest that,
15 1,580 33 570 instead of a single critical region, many chromosome 21
16 1,570 34 470 regions are responsible for various Down syndrome features
17 1,565 35 385 [79]. The study was expanded to include a total of 30 sub-
18 1,555 36 307 jects carrying rare segmental trisomies of various regions of
19 1,540 37 242 chromosome 21. By using current genomic technologies
20 1,530 38 189
including high-density isothermal oligonucleotide DNA til-
21 1,510 39 146
ing arrays, a high-resolution genetic map of Down syndrome
22 1,480 40 112
phenotype was constructed corresponding to discrete regions
23 1,450 41 85
of 1.8–16.3 Mb likely to be involved in the development of
24 1,400 42 65
25 1,350 43 49
eight Down syndrome phenotypes: acute megakaryocytic
26 1,290 44 37 leukemia, transient myeloproliferative disorder, Hirschsprung
27 1,210 45 28 disease, duodenal stenosis, imperforate anus, severe mental
28 1,120 46 21 retardation, Down syndrome-Alzheimer disease, and Down
29 1,020 47 15 syndrome-specific congenital heart disease [81]. The map
30 910 48 11 also provided evidence against both the existence of a single
31 800 49 8 Down syndrome consensus region and the previous supposi-
32 680 50 6 tion that a synergistic role of DSCR1, DYRK1A, and/or APP
Modified from Cuckle et al. [66]. Data were based on eight pooled stud- was sufficient for many of the Down syndrome phenotypes.
ies. Restriction of analysis to two studies with the most complete ascer-
tainment yielded higher rates [67]
trisomy pregnancy following an initial trisomy pregnancy

was reported in a study of trisomy recurrence based on
North American data [61].
One study of 13 families with two trisomy 21 children
showed that three had a parent who was mosaic for trisomy
21 (by cytogenetic studies), and two had a parent who was
potentially mosaic (by DNA polymorphism analysis) [85].
In a family with three trisomy 21 children, Harris et al.
reported that the mother was mosaic for trisomy 21 in lym-
phocytes and skin fibroblasts [86]. In another single case
report involving a family with four trisomy 21 children, the
mother was found to have a trisomy 21 cell line in an ovarian
biopsy specimen [87]. In a study compiling data from 80
families with either maternal (61 families) or paternal (19
families) gonadal mosaicism for trisomy 21, a total of 142
Down syndrome offspring were reported [88]. Among these
offspring, mosaicism was observed in 12 families and the
proportion of mosaics among affected female offspring
(14%) was significantly higher compared to that among
affected male offspring (0%). Based on these observations, it
was proposed that female-specific trisomy rescue might be a
mechanism of formation of both gonadal mosaicism and
somatic mosaicism. Gonadal mosaicism in one parent is an
important cause of recurrent trisomy 21 and should be looked
for in families with more than one affected child.
Fig. 8.5 The hand of a Down syndrome child showing small hand, The recurrence risk for mosaic trisomy 21 that results
clinodactyly, only one crease in the fifth finger, and single palmar from mitotic nondisjunction should, in general, not be
crease increased. However, several studies investigating the mecha-
nism and origin of mosaic trisomy 21 have shown that in a
The additional copy of chromosome 21 is proposed to relatively high proportion of cases (probably over 50%), the
result in the increased expression of many of the genes mosaicism results from the loss of one chromosome 21 dur-
encoded by this chromosome. The knowledge of which of ing an early mitotic division in a zygote with trisomy 21 [89,
the genes, when present in three copies, leads to each of the 90]. In such cases, the recurrence risk for nondisjunction will
different Down syndrome-associated phenotype, together be the same as for nonmosaic trisomy 21.
with research using Down syndrome mouse models, may
provide insight into possible pharmacological approach to
improving some of the symptoms [82]. Trisomy 18
Recurrence Incidence
Various estimates of the recurrent risk for trisomy 21 have Trisomy 18 [47,XX or XY,+18] was first described by
been reported. The overall empirical recurrence risk is Edwards et al. in 1960 [91]. The incidence is 1 in 6,000–
about 1% in women under 30 years of age and includes tri- 8,000 births. It is more frequent in females, with a male-to-
somies other than 21. For women over 30, the recurrence female ratio of 1:3–4. The risk for trisomy 18 also increases
risk may not be significantly different from the age-specific with maternal age.
risk [83]. A more recent study reported 5,960 women with
a previous trisomy 13, 18, or 21 pregnancy; 75 of the 3,713 Phenotype
subsequent pregnancies were trisomic [84]. The relative The most common features of trisomy 18 include mental and
risk of a subsequent trisomy 21 compared to the expected growth deficiencies, neonatal hypotonicity followed by
number of trisomies based on maternal age-related risk hypertonicity, craniofacial dysmorphism (prominent occiput,
alone was 2.2. The risk of a different trisomy subsequent to narrow bifrontal diameter, short palpebral fissures, small
trisomy 21 might also be increased (relative risk 1.4). The mouth, narrow palate, low-set malformed ears, micrognathia)
increase in risk was greater for women under age 35 at the (Fig. 8.6), clenched hands with a tendency for the second
first trisomic pregnancy. A similar increase in the rate of finger to overlap the third and the fifth finger to overlap the
120 J.-C.C. Wang
Fig. 8.6 Profile of a trisomy 18 child showing prominent occiput, Fig. 8.7 Trisomy 13 stillborn with midline cleft lip and holoprosencephaly
low-set malformed ear, and micrognathia
fourth, short dorsiflexed hallux, hypoplastic nails, rocker subsequent abortuses (e.g., [108–110]) are recorded. In the
bottom feet, short sternum, hernias, single umbilical artery, same studies referenced for trisomy 21, an increased risk of
small pelvis, cryptorchidism, hirsutism, and cardiac anom- trisomy 18 subsequent to a previous pregnancy with trisomy
alies (mainly ventricular septal defect [VSD], atrial septal 18 was observed [61, 84]. The relative risk was 1.7–3.8.
defect [ASD], and patent ductus arteriosus [PDA]). Studies Again, the increase in risk was greater for women under age
show that median survival averages approximately 5 days, 35 at the first trisomic pregnancy. Given the low baseline
with 1-week survival at 35–45%; one later study indicated age-related risk, the absolute risk of recurrence is nonethe-
a median survival of 14.5 days [92–96]. Fewer than 10% of less quite low.
patients survive beyond the first year of life. A few patients
over 10 years of age, all females with one exception, have
been described; however, the presence of a normal cell line Trisomy 13
in these patients was not always searched for [97–99].
Mosaic trisomy 18 patients have, in general, milder phe- Incidence
notypes. At least six mosaic trisomy 18 patients, again all Trisomy 13 [47,XX or XY,+13] was first described by
females, with normal intelligence and long-term survival Patau et al. in 1960 [111]. The incidence is estimated to be
have been reported [100–105]. 1 in 12,000 births. It is seen slightly more in females than
Two molecular studies, performed on a total of 10 patients in males. Again, the risk for trisomy 13 increases with
with partial trisomy 18, suggest that the region proximal to maternal age.
band 18q12 does not contribute to the syndrome, while two
critical regions, one proximal (18q12.1→q21.2) and one dis- Phenotype
tal (18q22.3→qter), may work in cooperation to produce the The most prominent features of trisomy 13 include the holo-
typical trisomy 18 phenotype [106, 107]. In addition, severe prosencephaly spectrum (Fig. 8.7), scalp defects, microceph-
mental retardation in these patients may be associated with aly with sloping forehead, large fontanels, capillary
trisomy of the region 18q12.3→q21.1. hemangioma (usually on the forehead), microphthalmia,
cleft lip, cleft palate, abnormal helices, flexion of the fingers,
Recurrence polydactyly, hernias, single umbilical artery, cryptorchidism,
Single case reports of trisomy 18 in sibs (e.g., [105]), and of bicornuate uterus, cardiac abnormalities in 80% of patients
trisomy 18 and a different trisomy in sibs or in prior or (mostly VSD, PDA, and ASD), polycystic kidneys, increased
polymorphonuclear projections of neutrophils, and persis-

tence of fetal hemoglobin. Prognosis is extremely poor, with
a median survival of 2.5–7 days and a 6-month survival of
5% [94, 96]. Severe mental deficiencies, failure to thrive and
seizures are seen in those who survive. Mosaic trisomy 13
patients are, again, in general less severely affected; however,
the degree is very variable and can be as severe as in nonmo-
saic trisomy 13 individuals.
Development of a karyotype-phenotype correlation by
studying partial trisomies for different segments of chromo-
some 13 has also been attempted [112, 113]. These studies
were based on cytogenetic methods and suggested that the
proximal segment (13pter→q14) contributes little to the
trisomy 13 phenotype, while the distal segment (all or part
of 13q14→qter) is responsible for the complete trisomy
13 features. A prenatally diagnosed pure partial trisomy 13
involving 13q14→qter with breakpoints delineated by array
comparative genomic hybridization (aCGH; see Chap. 18)
analysis was reported recently. The fetus had agenesis of
the corpus callosum and the diaphragm, severe pulmonary
hypoplasia, and generalized hydrops [114].
Recurrence
Fig. 8.8 An infant with mosaic trisomy 8. Note prominent forehead,
An increased risk of trisomy 13 subsequent to a previous strabismus, broad nasal bridge, upturned nares, long upper lip, and
pregnancy with trisomy 13 was noted in the same references everted lower lip
as recorded for trisomy 18 above [61, 84]. The relative risk
was 3.8–8.6. The relative risk for a subsequent different
trisomy was 1.9 [61]. The absolute risk remains very low. in mosaic trisomy 8 patients [122, 123]. This is of particular
interest because trisomy 8 is a frequently acquired cytoge-
netic abnormality in myeloid neoplasms (see Chap. 15).
Trisomy 8 When studied, the abnormal cells in these patients appeared
to have developed from the trisomic cell population. The
Trisomy 8 [47,XX or XY,+8] was first reported by Grouchy significance of this is not clear, but the possibility remains
et al. in 1971 [115]. It is rare, with an unknown incidence. that constitutional trisomy 8 may predispose individuals to
More than 100 cases have been reported in the literature, most myeloid neoplasia.
of them mosaics [47,+8/46] [116–121]. The male-to-female There is no direct correlation between the proportion
ratio is 2–3:1. of the trisomy 8 cells and the severity of the phenotype.
Growth and the degree of mental deficiency are variable. The percentage of trisomic cells is usually greater in skin
Mild to severe retardation is seen, while a proportion of fibroblasts than in blood lymphocytes. In addition, the propor-
patients have normal IQs. Craniofacial dysmorphism tion in lymphocytes usually decreases with time.
(Fig. 8.8) includes prominent forehead, deep-set eyes, stra- The risk for recurrence is not known.
bismus, broad nasal bridge, upturned nares, long upper lip,
thick and everted lower lip, high arched or cleft palate,
micrognathia and large dysplastic ears with prominent anti- Trisomy 9
helices. Skeletal abnormalities include a long, thin trunk,
hemivertebrae, spina bifida, kyphoscoliosis, hip dysplasia, The first cases of trisomy 9 in either nonmosaic [47,XX or
multiple joint contractures, camptodactyly, dysplastic nails, XY,+9] or mosaic [47,+9/46] form were reported in 1973
and absent or dysplastic patella. The presence of deep pal- [124, 125]. More than 40 cases of liveborns or term stillborns
mar and plantar furrows is characteristic. Renal and ureteral with trisomy 9 have been reported. Most were mosaics
anomalies and congenital heart defects are common. A case [126–130]. The male-to-female ratio is close to 1:1.
with extremely elevated maternal serum alpha-fetoprotein Clinical features include craniofacial anomalies (high nar-
noted prenatally without open defect was recorded [121]. A row forehead, short upward-slanting palpebral fissures, deep-
few cases of hematological malignancy have been reported set eyes, microphthalmia, low-set malformed auricles, bulbous
122 J.-C.C. Wang
nose, prominent upper lip, micrognathia), skeletal malformations cases are not cytogenetically confirmed in postnatal life.
(abnormal position/function of various joints, bone dysplasia, It appears that in conceptuses capable of surviving to the sec-
narrow chest, 13 ribs), overlapping fingers, hypoplastic ond trimester, trisomy 20 cells are largely confined to extra-
external genitalia, and cryptorchidism. Cardiac anomalies are embryonic tissues. Liveborns with documented mosaic
seen in more than 60% of cases, most frequently VSD. Renal trisomy 20 have been reported and most were phenotypically
malformations are present in 40% of patients. A case of mosaic normal at birth [143–150]. In cases with long-term follow-
trisomy 9 with holoprosencephaly and another case with XX up, hypopigmentation, mild psychomotor delay, and facial
sex reversal were reported [131, 132]. The majority of patients dysmorphism have been observed in some cases. The possi-
die in the early postnatal period. With rare exceptions, all sur- bility of a more consistent phenotype associated with mosaic
vivors have severe mental deficiency. Mosaic patients tend to trisomy 20 has been recently suggested, including spinal
survive longer, but the proportion of trisomy 9 cells does not abnormalities (spinal stenosis, vertebral fusion, kyphosis),
predict the severity of the condition or the length of survival. hypotonia, lifelong constipation, sloped shoulders, and
It is possible that a normal cell line could be present in some significant learning disabilities [150]. No case of liveborn
tissues in apparently nonmosaic patients. nonmosaic trisomy 20 has been recorded.
The mean maternal age of women bearing trisomy 9 Phenotypic abnormalities in abortuses with cytogeneti-
offspring was reported to be significantly increased over that cally confirmed mosaic trisomy 20 include microcephaly,
of the general population [127]. This suggests that the occur- facial dysmorphism, cardiac defects, and urinary tract anoma-
rence of trisomy 9 may also be associated with advanced lies (megapelvis, kinky ureters, double fused kidney) [151].
maternal age. The risk for recurrence is not known. Trisomy 20 cells have been found in various fetal tissues
including kidney, lung, esophagus, small bowel, rectum,
thigh, rib, fascia, and skin [142, 151, 152]. Postnatally, they
Trisomy 16 have been detected in cultured foreskin fibroblasts and urine
sediments [143–148]. The detection of trisomy 20 cells in
Trisomy 16 is the most frequently observed autosomal aneu- newborn cord blood has been reported in one case, but sub-
ploidy in spontaneous abortuses (see Chap. 13). Full trisomy sequent study of peripheral blood at 4 months of age pro-
16 is almost always lethal during early embryonic or fetal duced only cytogenetically normal cells [145]. There are no
development, although a single case of a stillborn at 35 weeks other reports of trisomy 20 cells in postnatal blood cultures.
gestation has been recorded [133]. The risk for recurrence is probably negligible.
Mosaic trisomy 16 fetuses, however, may occasionally
survive to term. More than ten such cases have been reported
[134–141]. Intrauterine growth restriction is nearly invari- Trisomy 22
able. An elevated maternal serum hCG or alpha-fetoprotein
level during pregnancy was noted in more than 50% of cases. Trisomy 22 was first reported in 1971 [153]. Since then,
Congenital cardiac defects (mainly VSD or ASD) were pres- more than 20 liveborns have been reported in the literature
ent in 60% of patients. Other clinical findings included post- [154–161]. Although most cases were apparently nonmosaic
natal growth retardation, mild developmental/speech delay, full trisomies, the presence of an undetected, normal cell line
craniofacial asymmetry, ptosis, flat broad nasal bridge, low- confined to certain tissues cannot be excluded, as pointed out
set dysplastic ears, hypoplastic nipples, umbilical hernia, by Robinson and Kalousek [162].
deep sacral dimple, scoliosis, nail hypoplasia, and single The most consistent phenotypic abnormalities include
transverse palmar crease. One patient had normal growth and intrauterine growth restriction, low-set ears (frequently asso-
development at 11 months of age [141]. Approximately 50% ciated with microtia of varying degrees plus tags/pits), and
of the patients died within the first year of life. Long-term midfacial hypoplasia. Other frequently seen abnormalities
follow-up is not available; however, survival to more than are microcephaly, hypertelorism with epicanthal folds, cleft
5 years has been observed (Hajianpour and Wang, personal palate, micrognathia, webbed neck, hypoplastic nails, anal
observation). atresia/stenosis, and hypoplastic genitalia. Cardiac defects,
The risk for recurrence is probably negligible. complex in some cases, are seen in 80% of patients. Renal
hypoplasia/dysplasia is also common. Skin hypopigmenta-
tion (hypomelanosis of Ito) is usually present in mosaic
Trisomy 20 cases. Intestinal malrotation and Hirschsprung disease were
recently reported in a prenatally diagnosed mosaic trisomy
Although mosaic trisomy 20 is one of the most frequent 22 infant with normal development [160]. A 4-year-old girl
autosomal aneuploidies detected prenatally, its occurrence in with confirmed trisomy 22 mosaicism in skin had normal
liveborns is very rare [142]. The majority of prenatally diagnosed cognitive, behavioral, and physical development [161].
Prolonged survival to over 20 years has been observed in One case of postnatally confirmed mosaic trisomy 5 has
mosaic patients. been reported [172]. The trisomic cells were detected in pre-
Most nonmosaic patients die in the first months of life. natal amniocytes and confirmed postnatally in skin fibroblasts,
The longest survival reported is 3 years [163]. That patient but not in blood lymphocytes. The patient had multiple dys-
had severe growth and developmental delay and died a few morphic features and congenital anomalies, including even-
days before his third birthday. tration of the diaphragm and ventricular septal defect.
Trisomy 22 cells can be detected in both blood lympho- At least two cases of mosaic trisomy 6 have recently been
cytes and skin fibroblasts. The risk for recurrence is unknown reported. The first patient was born at 25 weeks of gestation.
and probably negligible. Clinical features included heart defects (ASD and peripheral
pulmonary stenosis), large ears, cleft right hand, cutaneous
syndactyly, overlapping toes of irregular shape and length,
Other Rare Autosomal Trisomies and epidermal nevi. Growth was considerably delayed, but
development was relatively normal at age 2¾. Trisomy 6
As noted in the introduction, mosaic or nonmosaic autosomal cells were detected in skin fibroblasts but not in blood [173].
trisomies for chromosomes other than 1 and 11 have been Mosaic trisomy 6 was prenatally diagnosed in fetal urine in
reported in liveborns. Trisomies are detected much more fre- the second case. The infant was born at term with normal
quently in spontaneous abortuses or in prenatal diagnostic growth parameter, heart defect, and malformations of hands
specimens, following which elective terminations are often and feet [174].
performed. Thus, the occurrence of such trisomies in live- At least seven cases of cytogenetically documented
borns is extremely rare and only isolated case reports are mosaic trisomy 7 in skin fibroblasts have been recorded
available. The risks for recurrence for these rare trisomies [175–178]. All patients were phenotypically abnormal.
are probably negligible. The following discussion will Common features included growth and developmental delay,
include cytogenetically confirmed postnatal cases only. skin pigmentary dysplasia with hypo- and hyperpigmenta-
At least two cases of liveborn mosaic trisomy 2 have been tion, facial or body asymmetry, and facial dysmorphism. One
reported [164, 165]. In one case, the mosaicism was detected mentally retarded male was 18 years old at time of report.
in amniocytes and confirmed postnatally in liver biopsy A few cases of liveborn mosaic trisomy 10 have been reported
fibroblasts (4 of 100 cells) but not in blood, skin fibroblasts, [179–181]. One patient was mosaic for trisomy 10 and
or ascites fluid cells. At 16 months of age, the child had hypo- monosomy X in skin fibroblasts, whereas only monosomy X
tonia, microcephaly, and growth and developmental delay. In cells were present in blood. This infant died at 7 weeks of
the second case, mental retardation, multiple congenital age from heart failure. Another patient was mosaic for tri-
anomalies, and dysmorphic findings similar to Pallister- somy 10 and had maternal uniparental disomy for chromo-
Killian syndrome were observed. Another case of possible some 10 in the diploid cell line [181]. The common clinical
mosaic trisomy 2, detected at amniocentesis and observed in phenotype included growth failure, craniofacial dysmor-
a single cell of a foreskin fibroblast culture following the birth phism (prominent forehead, hypertelorism, upslanted palpe-
of a dysmorphic child, was reported in an abstract [166]. bral fissures, blepharophimosis, dysplastic large ears,
Three cases of mosaic trisomy 3 have been reported; one retrognathia), long slender trunk, deep palmar and plantar
of these, a severely mentally retarded woman, was alive at age fissures, cardiac defects, and short survival.
32 [14, 167, 168]. Clinical features in the three cases vary, At least seven cases of cytogenetically confirmed trisomy
except all had prominent forehead, ear, and eye anomalies. 12 have been reported in liveborns; all were mosaics [110–
At least two cases of postnatally confirmed mosaic tri- 113, 115–187]. The earliest reported case was that of an infer-
somy 4 have been reported [169, 170]. In both cases, the tri- tile man. A more recent case was a girl with pituitary
somic cells were detected in prenatal amniocytes and malformation associated with growth retardation responding
confirmed postnatally in skin fibroblasts, but not in blood to growth hormone therapy. The patient also had a polycystic
lymphocytes. One of the cases also had low-level mosaicism right ovary. Phenotypic presentation was variable among
for trisomy 6 with clinical features of prenatal growth restric- patients and included facial dysmorphism, scoliosis, ASD,
tion, right facial hypoplasia, dysplastic and posteriorly PDA, dysplastic pulmonary and tricuspid valves, short stature,
rotated right ear, high vaulted palate, retrognathia, aplasia of and mental retardation. Trisomy 12 cells have been found in
the right thumb, hypoplasia of the fingernails, deep sacral lymphocytes, skin fibroblasts, urine sediments, and internal
dimple, and patchy skin hypopigmentation of the right leg organs including liver, spleen, adrenal, ovary, and thymus.
[170]. Long-term follow-up was available on the other case More than 20 cases of mosaic trisomy 14 have been
[171]. The patient had right hand and ear anomalies. At age reported in liveborns [188–190]. The most consistent pheno-
14, she had delayed puberty with no menarche, asymmetri- typic abnormalities were growth and mental retardation,
cal breast development, and low normal intelligence. broad nose, low-set dysplastic ears, micrognathia, congenital
124 J.-C.C. Wang
heart defects, and micropenis/cryptorchidism in males. One findings of an electively terminated 20-week female fetus
prenatally diagnosed patient had alobar holoprosencephaly after mosaic monosomy 21 was diagnosed by repeated
and died at 36 days of age [189]. Survival varied from days amniocenteses [207]. The facial abnormalities previously
to more than 29 years. Trisomy 14 cells were detected in described were present in this abortus. In addition, a com-
both lymphocytes and fibroblasts, with a generally higher plex cardiac malformation, malrotation of the bowel, uterus
percentage in lymphocytes. There was no clear correlation didelphys, small dysplastic ovaries, and focal cystic dyspla-
between the proportion of trisomic cells and the severity of sia of the lung were noted.
the phenotype. In patients with body asymmetry, trisomic Approximately ten cases of apparently nonmosaic mono-
cells were usually limited to the atrophic side. somy 21 have been reported in liveborns [208–211]. Some of
At least ten cases of liveborn trisomy 15 have been these cases have subsequently been shown to represent par-
recorded, two of them were reportedly nonmosaics [191– tial monosomy 21 resulting from an undetected subtle trans-
197]. In some cases, the trisomy 15 cell line was present only location [212–214] with part of chromosome 21 material
in skin fibroblasts and not in peripheral blood lymphocytes. attached to a derivative chromosome, explaining the obser-
The concurrent finding of maternal uniparental disomy 15 vation that mosaic monosomy 21 is less commonly observed
(see Chap. 20) in the normal cell line was reported in two of than apparently nonmosaic monosomy 21 and indicating that
the cases [192, 194]. These cases appeared to have the most complete monosomy 21 is almost always incompatible with
severe phenotype. Phenotypic abnormalities include hypoto- life. The phenotypic features were similar to those observed
nia, various craniofacial dysmorphisms, minor skeletal anom- in the mosaics and included intrauterine growth restriction,
alies, congenital heart defects, and short survival. One patient postnatal growth and mental deficiencies, microcephaly,
with longer survival had short stature, mild mental retarda- hypertelorism with downward slanting palpebral fissures,
tion, hemihypotrophy, atrial septal defect, bilateral branchial large low-set ears, prominent nose, cleft lip/palate, microg-
cleft fistulas, and abnormal skin pigmentation [195]. nathia, cardiac anomalies, and abnormal muscle tone. Most
At least four cases of confirmed mosaic trisomy 17 have patients died before 2 years of age. A case of full nonmosaic
been reported [198–200]. The trisomic cells were not seen in monosomy 21 confirmed by fluorescence in situ hybridiza-
lymphocytes but were found in high percentage in skin tion analysis was reported in a liveborn who died shortly
fibroblasts. One patient, age 8½ years at the time of report- after birth [211]. The phenotype of this infant included severe
ing, had mental and growth retardation, microcephaly, minor intrauterine growth restriction, microcephaly, semilobar hol-
dysmorphism, seizures, hearing loss, attention deficit hyper- oprosencephaly, hypotonia, bilateral microphthalmia, facial
activity disorder, and autistic behavior. Peripheral motor and dysmorphism, agenesis of the external auditory meatus,
sensory neuropathy, hypoplastic cerebellar vermis, zonular redundant skin in the neck, narrow chest, cryptorchydism,
cataract, and body asymmetry have also been reported. hypospadias, micropenis, camptodactyly, congenital heart
At least two cases of mosaic trisomy 19 were in the litera- disease, and agenesis of the right kidney.
ture, one of them was a stillborn male and the other died on
day 13. Clinical features were varied and included facial dys-
morphism with no report of major malformation [201, 202]. Monosomy 22
At least four cases of mosaic monosomy 22 in liveborns have

Autosomal Monosomies been reported [215–218]. All four were male. One was a
34-week premature infant with gastroschisis who died from
As noted in the introduction, autosomal monosomies are intracranial hemorrhage shortly after birth. No dysmorphic
extremely rare in either liveborns or abortuses, reflecting the features were noted, and autopsy was not performed [217].
severity of the genetic imbalance resulting from the loss of Two patients had growth and developmental deficiencies,
an entire chromosome. The only monosomies that have been microcephaly, and mild facial dysmorphism. The fourth
reported are monosomy 21 and mosaic monosomy 22. patient was a 30-week premature infant with facial features
of DiGeorge syndrome, hypertonicity, limited extension of
major joints, and flexion contractures of all fingers.
Monosomy 21
Mosaic monosomy 21 was reported in four liveborns in the Polyploidy

early literature [203–206]. The most prominent features
included intrauterine growth restriction, postnatal growth Polyploidies are numerical chromosome abnormalities with
and mental retardation, hypertonia, facial dysmorphism with changes in the number of complete sets of chromosomes.
downward slanting palpebral fissures, large low-set ears, and They are usually incompatible with fetal survival and are
micrognathia. A more recent report described pathological extremely rare in liveborns.
Triploidy somes in such cases is maternal (digyny) [221–224]. Digyny

can result from a failure of the first maternal meiotic divi-
The chromosome number in triploidy is 3n = 69 (Fig. 8.9). It sion, generating a diploid egg, or from retention of the sec-
is estimated to occur in approximately 1% of all human con- ond polar body. While the occurrence of triploidy does not
ceptions and is found in 17–18% of all chromosomally appear to be associated with maternal age, digyny may play
abnormal abortuses [219, 220]. Only very rarely do triploid a major role in the generation of triploidy in the advanced
conceptuses survive to term. Two distinct phenotypes have maternal age group [220]. Early cytogenetic studies indi-
been recognized [221]. One type presents as a relatively cated that the majority of triploid conceptuses were diandric
well-grown fetus with or without microcephaly, and an partial moles [222, 225]. Later studies based on DNA poly-
abnormally large and cystic placenta usually classified as a morphisms have suggested that a maternal contribution to
partial hydatidiform mole. The parental origin of the extra triploidy may occur more frequently than was previously
haploid set of chromosomes in such cases is determined to realized [223, 226]. Yet in a more recent study of 87 informa-
be paternal (diandry) by analysis of cytogenetic heteromor- tive cases of triploid spontaneous abortuses at 5–18 weeks of
phisms or DNA polymorphisms [221, 222, 180]. Diandry gestation, Zaragoza et al. showed that approximately two-
results from the fertilization of a normal ovum with either thirds are androgenetic in origin and that many, but not all,
two sperm (dispermy) or a sperm that has a diploid chromo- androgenetic triploids developed a partial molar phenotype
some complement resulting from a failure of meiotic divi- [227]. The sex chromosome complement in triploidy is either
sion. The other type is characterized by severe intrauterine XXX or XXY, with XYY occurring only rarely. For example,
growth restriction with relative macrocephaly and a small the reported numbers of XXX:XXY:XYY cases in two stud-
and noncystic placenta. The extra haploid set of chromo- ies performed on spontaneous abortuses were 82:92:2 and
Fig. 8.9 Karyogram of a triploid fetus (69,XXX)

126 J.-C.C. Wang
26:36:1, and in one study performed on amniotic fluid cells second polar body into one of the early blastomeres. The trip-
this ratio was 6:8:0 [3, 177, 228]. It has been suggested that loid cell line in this case is digynic [242].
69,XYY triploid conceptuses are incompatible with
significant embryonic development [3].
The observation that the phenotype of triploidy depends Tetraploidy
on the parental origin of the extra set of chromosomes is an
example of genomic imprinting, or the differential expres- The chromosome number in tetraploidy is 4n = 92. It is rarer
sion of paternally and maternally derived genetic material than triploidy in spontaneous abortuses, seen in approxi-
[229, 230]. It correlates well with observations obtained mately 6–7% of such specimens with chromosome abnor-
from mouse embryo studies using nuclear transplantation malities [219, 220]. Tetraploid conceptuses usually abort
techniques, which demonstrated that maternal and paternal spontaneously early in gestation and only rarely do they sur-
genomes function differently and are both required for nor- vive to term. A probable origin of tetraploidy is chromosome
mal development [231–233]. See Chap. 20. duplication in the zygote resulting from a failure of cytoplas-
More than 50 cases of apparently nonmosaic triploidy, mic division during the first division. Other theoretically
either 69,XXX or XXY, have been reported in liveborns. possible mechanisms require the occurrence of two indepen-
Most patients died shortly after birth. Eight patients with sur- dent, rare events and are thus highly unlikely.
vival longer than 2 months have been reported, with the lon- At least nine apparently nonmosaic tetraploid liveborns
gest being 10½ months [234, 235]. The origin of the extra set have been reported [243, 244]. The sex chromosome comple-
of chromosomes was determined by cytogenetic polymor- ment was either XXXX or XXYY. No 92,XYYY or XXXY
phisms or human leukocyte antigen (HLA) to be maternal in conceptuses have been reported. The most frequent abnor-
three cases and paternal in one case [236]. One study based malities were growth and developmental delay, hypotonia,
on DNA polymorphism in an infant who survived for 46 days craniofacial anomalies (short palpebral fissures, low-set
indicated a maternal meiosis II failure as the origin of the malformed ears, high arched/cleft palate, micrognathia), and
triploid [236]. These findings suggest that in general digynic contracture/structural abnormalities of the limbs, hands
triploids survive longer than diandric triploids. The most fre- and feet. Cardiac defects were present in four cases. Urinary
quent phenotypic abnormalities include intrauterine growth tract abnormalities, such as hypoplastic kidneys, have also
restriction, hypotonia, craniofacial anomalies (macro/hydro- been recorded. Most patients died before 1 year of age. One
cephalus, low-set dysplastic ears, broad nasal bridge), syn- girl had survived to 22 months at the time of report [245].
dactyly, malformation of the extremities, adrenal hypoplasia, Mosaic tetraploidy (diploid/tetraploid mixoploidy) has
cardiac defects, and brain anomalies. been reported in at least 12 liveborns [246, 247]. This can
Mosaic triploidy (diploid/triploid mixoploidy) has been occur as a result of postzygotic nondisjunction with failure
reported in approximately 20 patients. Triploid cells were of cytoplasmic division in a diploid conceptus. Tetraploid
found in both lymphocytes and fibroblasts, although in a cells were seen in peripheral blood lymphocytes, skin
number of cases the triploid cell line was limited to fibroblasts fibroblasts, and bone marrow cells. In one severely mal-
[237]. Patients with such mixoploidy are less severely formed patient who died at 2 days of age, tetraploid cells
affected than nonmosaics, and survival beyond 10 years has were found in 95% of bone marrow cells [248]. In two
been observed. Usual clinical features include intrauterine females, aged 11 and 21 years, with severe intellectual handicaps
growth restriction, psychomotor retardation, asymmetric and skin pigmentary dysplasia, tetraploid cells, were found
growth, broad nasal bridge, syndactyly, genital anomalies, only in skin fibroblasts [247]. In lymphocytes, the proportion
and irregular skin pigmentation [238]. Truncal obesity was of tetraploid cells decreases with age [249]. Overall, clinical
seen in some patients [239]. A recent case of a 46,XX/69,XXY features are similar to, but less severe than, those in nonmosaic
diploid/triploid mixoploid 8-year-old girl with normal female tetraploidy patients. In addition to the longer survivals
genital and ovarian development despite normal expression already mentioned, survivals to 6 years at the time of report-
of SRY expression was reported [240]. ing have also been recorded [247, 250].
Mitotic nondisjunction cannot readily explain the occur-
rence of diploid and triploid cell lines in the same individual.
One possible mechanism is double fertilization of an ovum by Partial Autosomal Aneuploidies
two sperm; one sperm nucleus fuses with the ovum nucleus
producing the diploid line, followed by a second sperm fertil- Partial duplication/deletion as a result of structural rearrange-
izing one of the early blastomeres producing the triploid line. ment is discussed in Chap. 9. Only those partial autosomal
Cytogenetic evidence for such a mechanism has been reported aneuploidies that result from the presence of a supernumerary
in at least one case [241]. Another proposed mechanism sup- chromosome and have been detected in postnatal specimens
ported by molecular evidence is delayed incorporation of the will be presented in this chapter.
Tetrasomy 5p of the heterochromatic region of the long arm, or the entire

short arm and part of the long arm extending to the euchro-
Tetrasomy 5p [47,XX or XY,+i(5)(p10)] resulting from the matic region. No consistent phenotypic differences have
presence of a supernumerary isochromosome for the entire been observed among the three types. Both mosaic and
short arm of chromosome 5 is rare and has been reported in apparently nonmosaic patients have been reported. The tetra-
only five liveborns, all of whom were mosaics with both nor- somy 9p cells were seen in both lymphocytes and skin
mal and abnormal cell lines [251, 252]. The abnormal cell line fibroblasts. In contrast to tetrasomy 12p (described later), the
has been found in lymphocytes, skin fibroblasts, and chondro- 9p isochromosomes were present only in lymphocytes in five
cytes. The phenotype appears to be similar to that of trisomy patients and in fibroblasts at a much lower percentage than in
5p. This includes hypotonia, seizures/abnormal electroen- lymphocytes in two others [258, 259, 263, 264, 265, 266].
cephalogram (EEG), psychomotor retardation, macrocephaly, The mechanism for this observed tissue-limited mosaicism
facial dysmorphism, and respiratory difficulties. Skin hyper- for different chromosomes is not clear.
pigmentation was observed in two patients. Survival was vari- Survival is variable, ranging from a few hours to beyond
able; the most recent case reported was a 35-year-old male 10 years. The most frequent phenotypic abnormalities
with a normal phenotype [252]. One patient died at 6 months include low birth weight, growth and developmental delay,
of age, and another was 5 years old at the time of reporting. craniofacial anomalies (microphthalmia, low-set malformed
ears, bulbous tip of the nose, cleft lip/palate, micrognathia),
short neck, skeletal anomalies, joint contracture, nail hypo-
Tetrasomy 8p plasia, and urogenital anomalies. Cardiac defects are present
in more than 50% of patients. Diaphragmatic hernia was
Tetrasomy 8p [47,XX or XY,+i(8)(p10)] usually results from reported in an apparently nonmosaic patient [262].
the presence of a supernumerary isochromosome for the Overall, nonmosaic patients are more severely affected.
entire short arm of chromosome 8. All except one of the One patient, who had the i(9p) present in 75% of lympho-
cases reported were mosaics, with both normal and abnormal cytes but not in skin fibroblasts, had only mild developmen-
cell lines. The abnormal cell line was found in lymphocytes tal delay and minor anomalies [258].
and skin fibroblasts. In some cases, the origin of the abnor-
mal isochromosome was confirmed by molecular cytoge-
netic (FISH) studies [253–255]. At least 12 cases have been Tetrasomy 12p
reported [255–257]. A few patients died before the first year
of life, but survival beyond 5 years was not uncommon. Tetrasomy 12p (Pallister-Killian syndrome) results from the
Weight and head circumference were normal at birth. The presence of a supernumerary isochromosome for the entire
most frequently observed phenotypic features include men- short arm of chromosome 12 [i(12)(p10) or i(12p)] (Fig. 8.10).
tal retardation, speech and motor delay, dilatation of cerebral The syndrome was first described in 1977 by Pallister et al.
ventricles, mild facial dysmorphism (depressed nasal bridge, in two adults, a 37-year-old man and a 19-year-old woman
short nose, upturned nares, low-set and posteriorly rotated [267]. In 1981, Killian and Teschler-Nicola reported a 3-year-
ears), and vertebral abnormalities. Agenesis of the corpus old girl with similar clinical manifestations [268].
callosum was noted in six patients and cardiac defects in Subsequently, many cases have been reported, and many
five. Deep palmar and plantar creases have also been reported. more have been observed but not reported in the literature
The phenotype resembles, to some degree, that of mosaic [269, 270]. All cases were mosaics, with a normal cell line in
trisomy 8. A single apparently nonmosaic case was recorded addition to cells containing i(12p). Maternal age for reported
with isochromosome 8p present in all blood lymphocytes cases has been shown to be significantly higher than that for
while prenatal amniocytes showed a normal karyotype [257]. the general population [271]. This observation has been
The girl had congenital ventricular septal defect, agenesis of taken to suggest that the isochromosome arises from a mei-
corpus callosum, and facial, ear and bone anomalies. otic error and that the normal cell line results from subse-
quent loss of the i(12p) from some cells. In 6 of 7 cases
studied by molecular analysis, the meiotic error was deter-
Tetrasomy 9p mined to be maternal [272, 273]. Tissue specificity and both
the in vivo and in vitro age dependencies of the i(12p) have
Tetrasomy 9p [47,XX or XY,+i(9)(p10)], resulting from the been well demonstrated [274]. The i(12p) is found in a high
presence of a supernumerary isochromosome, has been percentage of skin fibroblasts and amniocytes but is rarely
reported in more than 20 liveborns [258–262]. The isochro- seen in blood lymphocytes. The percentage of cells containing
mosome consists of either the entire short arm of chromo- the isochromosome also decreases with age. The presence of
some 9 as previously described, the entire short arm and part tetrasomy 12p in 100% of bone marrow cells has been
128 J.-C.C. Wang
Fig. 8.10 Tetrasomy 12p female karyogram
reported in at least two newborn infants and in only 6% of result from differential selection against cells containing
marrow cells in a 3½-year-old child [275–277]. In lympho- i(12p) in different tissues and that this selection can occur
cytes it has been found in fetal blood, but has never been seen both in vivo and in vitro.
beyond childhood [274, 278]. In a case reported by Ward Many patients die shortly after birth, but survival to adult-
et al., the i(12p) was present in 10% of lymphocytes initially hood is possible. Clinically, a distinct pattern of anomalies is
but was not seen in these cells when the patient was 2 months observed in these patients. Growth parameters at birth are
old [275]. The isochromosome is more stable in skin usually normal. Profound hypotonia is present in the new-
fibroblasts and can be found in adults, usually at lower per- born period, while contractures develop later in life. Sparse
centage than in younger patients. When fibroblast cultures scalp hair, especially bitemporally, is observed in infancy,
were examined, the percentage of cells containing the iso- with coarsening of facial features over time. Craniofacial
chromosome decreased with increasing numbers of cell pas- dysmorphism includes prominent forehead, large malformed
sages [272, 274–276, 279]. One study using FISH showed ears, hypertelorism, epicanthal folds, broad flat nasal bridge,
that in lymphocytes, the i(12p) was present in a significantly short nose, upturned nares, long philtrum, thin upper lip, and
higher proportion of interphase nuclei than in metaphase high arched palate. Most patients have a generalized pigmen-
cells [280]. With the availability of array CGH (see Chap. tary dysplasia with areas of hyper- and hypopigmentation.
18), gain of 12p has been detected in total genomic DNA Other abnormalities include short neck, macroglossia,
from blood specimens [281]. These indicate that lympho- micrognathia progressing to prognathia, accessory nipples,
cytes containing i(12p) may fail to divide upon phytohemag- umbilical and inguinal hernias, urogenital abnormalities, and
glutinin (PHA) stimulation. These observations suggest that congenital heart defects. Severe mental retardation and sei-
tissue-limited mosaicism in Pallister-Killian syndrome may zure are seen in those who survive.
All cases are sporadic. The recurrence risk is probably Other Partial Autosomal Aneuploidies
negligible.
Supernumerary Marker Chromosomes
In addition to the tetrasomies described previously, partial
Tetrasomy 18p autosomal aneuploidies can result from the presence of small
supernumerary marker chromosomes of cytogenetically
Tetrasomy 18p [47,XX or XY,+i(18)(p10)] results from the indeterminate origin. The frequency of such markers is
presence of a supernumerary isochromosome for the entire approximately 0.7 per 1,000 in newborns and 0.8–1.5 per
short arm of chromosome 18. The syndrome was first 1,000 in prenatal specimens [293–296]. Since their cyto-
described by Froland et al. in 1963, although identification of genetic origins are not initially known, these markers may or
the marker as an i(18p) was not made until after the introduc- may not represent autosomal aneuploidy. Identification of
tion of banding techniques in 1970 [282]. Confirmation of the such markers is now typically achieved using FISH or array
origin of the marker has been possible in recent years by FISH CGH and is covered in Chap. 17.
studies. Of interest is the finding of a loss of approximately These supernumerary markers are often classified as
80% of chromosome 18 alpha-satellite DNA in the i(18p) in satellited or nonsatellited and are frequently present in
one case [283]. mosaic form. They are a heterogeneous group and the clinical
At least 60 cases have been reported [284–287]. Most are significance of a marker depends on its origin and character-
nonmosaics. The i(18p) is usually readily detectable in lym- istics. Markers that contain only heterochromatin and/or the
phocytes. Its presence in amniocytes and cultured chorionic short arms of acrocentric chromosomes are typically of no
villus cells has also been reported [283, 288]. phenotypic consequence. On the other hand, markers that
The most frequent clinical features include low birth contain euchromatin are generally not benign and can result
weight, microcephaly, feeding problems, various degrees of in phenotypic abnormalities. Among these are the dicentric
psychomotor retardation, spasticity, seizures, craniofacial bisatellited markers that contain variable amounts of long
characteristics (oval shaped face, arched eyebrows, strabis- arm euchromatin of an acrocentric chromosome.
mus, low-set dysplastic ears, small pinched nose, small trian- Markers derived from all autosomes have been reported
gular mouth, high arched palate, micrognathia), narrow [297–300]. The most common marker is the so-called
shoulders and thorax, small iliac wings, scoliosis, campto- inverted duplication of chromosome 15, “inv dup(15)”. This
dactyly, and simian creases. Cardiac defects including ASD, is an archaic misnomer that dates from an incorrect assess-
VSD, and PDA have been observed in some cases. Urogenital ment of the mechanism of formation of such chromosomes
anomalies including horseshoe kidneys, double ureter, and and represents a heterogeneous group of small markers con-
cryptorchidism have occasionally been seen. One case with sisting of two copies of the short arm of chromosome 15,
aggressive behavior was reported in a 41-year-old male who with or without variable amounts of long arm material. These
also had dysmorphic features, marked obesity, and profound are correctly identified as isochromosomes or isodicentric
mental retardation [289]. chromosomes and account for approximately 40% of all
It is not clear whether patients with tetrasomy 18p are marker chromosomes [299, 301]. The amount of long arm
born to mothers of increased age. Most of the reported euchromatin present in the marker dictates its phenotypic
cases are sporadic. The presence of i(18p) in maternal significance. A direct correlation has been observed between
lymphocytes has been reported in at least three families. the presence of the Prader-Willi/Angelman syndrome regions
In two families, the mothers had an abnormal chromo- (located at 15q11.2) on the marker and mental retardation or
some 18 with deletion of the short arm and a supernu- developmental delay [302–304]. Of particular interest is the
merary i(18p), and thus were trisomic for 18p. The observation of a few patients with this type of marker who
offspring inherited the normal chromosome 18 and the present clinically with Prader-Willi syndrome or Angelman
i(18p), and were, therefore, tetrasomic for 18p [ 290, syndrome [303, 305–309]. Molecular studies performed on
291 ]. In the third family, the mother had low-level mosa- some of these patients indicate that the abnormal phenotype
icism for a supernumerary i(18p) and was mildly affected results not from the presence of the marker, but from either
clinically. The child apparently had nonmosaic tetrasomy uniparental disomy of the two normal chromosomes 15 or a
18p and had the full clinical presentation of the syn- deletion of 15q11.2-q13 on one of the apparently cytogeneti-
drome [292 ]. In another report, the presence of an i(18p) cally normal 15s [303, 308, 309].
in two maternal half siblings was observed. No i(18p) Another type of marker chromosome that results in a clin-
was found in the mother’s lymphocytes or fi broblasts, ically recognizable multiple congenital anomaly syndrome is
raising the possibility of gonadal mosaicism [287] . The the supernumerary bisatellited dicentric marker derived from
recurrence risk in such families will be high. chromosome 22. This marker contains two copies of a small
130 J.-C.C. Wang
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10. Girardet A, Coignet L, Andreo B, Lefort G, Charlieu JP, Pellestor F.
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Characteristic features include craniofacial anomalies (vertical nique. Am J Med Genet. 1996;64:488–92.
coloboma of the iris, which gives the syndrome its name; 11. Pellestor F, Girardet A, Coignet L, Andreo B, Charlieu JP.
coloboma of the choroid or optic nerve; preauricular skin Assessment of aneuploidy for chromosomes 8, 9, 13, 16, and 21 in
human sperm by using primed in situ labeling technique. Am J
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Structural Chromosome
Rearrangements 9
Kathleen Kaiser-Rogers and Kathleen W. Rao
Introduction somatic and germ cells. These types of exchanges ensure

mixing of the gene pool and appear to be obligatory for nor-
The subject of structural chromosome rearrangements is an mal cell division. It is only when exchanges occur between
immense one, to which entire catalogs have been devoted. nonallelic chromosomal regions that structural rearrange-
Indeed, there are theoretically an almost infinite number of ments result. Since chromosome breakage can theoretically
ways in which chromosomes can reconfigure themselves occur anywhere within the human genome and the involved
from the familiar, normal, 23-pair arrangement. While struc- chromosome(s) can recombine in innumerable ways, the
tural rearrangements are often thought of in terms of chro- number of potential rearrangements that can result is
mosome pathology, some rearrangements are fairly immense. In practice, however, there appear to be particular
innocuous. In fact, a few such benign rearrangements (such areas of the genome that are more susceptible to breakage
as certain pericentric inversions of chromosome 9) are seen and rearrangement than others because of their underlying
frequently enough to be considered polymorphic variants of architecture. The presence of a DNA sequence that is repeated
no clinical significance. elsewhere in the genome, susceptible to the formation of
This chapter will discuss and provide examples of the ways double-stranded breaks, and/or capable of forming a particu-
in which chromosome rearrangements can occur, and will begin lar secondary DNA structure, all appear to influence the like-
with an overview of general concepts that relate to all structural lihood that a particular chromosome region is involved in a
rearrangements and their association with human pathology. structural rearrangement [1–7].
Each category of structural rearrangement will then be dealt Numerous studies have now shown that many recurring
with as a unique entity in the second half of the chapter. and some sporadic rearrangements occur secondary to recom-
bination between nonallelic regions of homology. While these
regions of homology sometimes represent high-copy-number
Mechanism of Formation repeats such as Alu or satellite DNA sequences, the majority
appear to involve low-copy repeats (LCRs). There are now
The exchange of genetic material between sister chromatids many examples in the literature of recurring duplications,
and homologous chromosomes is a normal occurrence in deletions, inversions, translocations, isochromosomes, and
marker chromosomes that form secondary to LCR-mediated
nonallelic homologous recombination (NAHR). The LCRs
K. Kaiser-Rogers, Ph.D. (*) that serve as substrates for these recombination events typi-
UNC Hospitals Cytogenetics Laboratory, Department of Pathology
cally range in size from 10 to 500 kilobase pairs (kb) and
and Laboratory Medicine, Pediatrics, and Genetics,
University of North Carolina Hospitals, share ³95% sequence identity. Although distributed through-
Room 1071, 1st Floor Memorial Hospital, out the genome, LCRs may appear preferentially within peri-
101 Manning Drive, CB# 7525, Chapel Hill, NC 27514, USA centromeric chromosomal regions. The ultimate size and
types of rearrangements that result from these nonallelic
K.W. Rao, Ph.D. homologous recombination events appear to reflect the loca-
Cytogenetics Laboratory, North Carolina Memorial Hospital,
tion, size, and orientation of the involved LCRs, as well as the
Chapel Hill, NC, USA
number of crossover events that occur between them.
Department of Pediatrics, Pathology and Laboratory Medicine,
Direct LCRs (those with the same orientation) located on
and Genetics, The University of North Carolina at Chapel Hill,
Chapel Hill, NC, USA the same chromosome can mediate both duplications and
e-mail: [email protected] deletions, as shown in Fig. 9.1. When a single, nonallelic,
140 K. Kaiser-Rogers and K.W. Rao
Fig. 9.1 Chromosome rearrangements can be produced by nonallelic deletions and acentric ring chromosomes (c). If instead recombination
homologous recombination between shared sequences or repeats of occurs between inverted repeats within a single chromatid, a chromo-
identical (direct repeats) or opposite (inverted repeats) orientation. some inversion is produced (d). Translocations and other more complex
Recombination between direct, nonallelic repeats on homologous chro- rearrangements can occur secondary to recombination events between
mosomes (a) or sister chromatids (b) can produce complementary shared sequences that are located on different chromosomes (e). Shared
duplications and deletions. Recombination between direct repeats sequences or repeats are designated by arrows and lower case letters
located at different sites within a single chromatid can produce both represent unique sequences
homologous recombination event involving homologous result in Mendelian genetic disorders such as Hunter syn-
chromosomes (interchromosomal) or sister chromatids drome, hemophilia A, familial juvenile nephronophthisis,
(intrachromosomal) is mediated by direct LCRs, comple- and others [6, 9]. In contrast to single gene rearrangements,
mentary duplications and deletions occur (Fig. 9.1a, b). Only recombination events that utilize nonallelic homologous
deletions are predicted to occur, however, if nonallelic sequences that are separated by large regions of the genome
homologous recombination involving direct LCRs occurs (typically 1.5–5 Mb) or are located on different chromo-
within a single chromatid (intrachromatid; Fig. 9.1c). As somes altogether can produce cytogenetically visible rear-
shown in Fig. 9.1d, inversions can form secondary to intrac- rangements involving multiple genes. Included within this
hromatid recombination events within a pair of nonallelic group are many of the recognized microdeletion and micro-
homologous inverted LCRs. Nonallelic recombination events duplication syndromes, as well as several recurring rear-
involving LCRs located on completely different chromo- rangements such as the (4;8) translocation with breakpoints
somes would be expected to produce translocations (Fig. 9.1e) at 4p16 and 8p23 and the inverted duplicated chromosomes
as well as other more complex rearrangements [8]. derived from chromosomes 15 and 22 (see respective sec-
The size of the inversions, duplications, and deletions tions later).
produced by the recombination events described earlier is While LCRs appear to serve as the recombination
dependent upon the length and proximity of the LCRs medi- substrates for many chromosomal rearrangements, high-
ating the rearrangement. In general, the larger the rearranged copy-number repeats such as Alu or satellite DNA sequences
region, the larger the LCR that mediates the recombination also play a role. At least 32 cases of single gene disorders
event. Single gene rearrangements occur when the recom- and 16 cases of cancer have been attributed to intrachromo-
bining homologous sequences flank or are within a single somal Alu-mediated recombination events [10]. While much
gene. These rearrangements are submicroscopic, require less common, interchromosomal Alu-Alu recombination
molecular techniques for their identification, and typically events also appear to occur. This is evidenced by a report of
9 Structural Chromosome Rearrangements 141
an XX male who carried an XY translocation mediated by suspected of forming primarily by NHEJ include the 1p36
Alu repeats [11]. Additionally, interchromosomal nonallelic and 11p11.2 deletions associated with 1p36 deletion and
recombination events mediated by high-copy satellite DNA Potocki-Shaffer syndrome, respectively, the 10q24 duplica-
sequences and/or other adjacent repetitive sequences located tions associated with split hand-split foot malformation [20],
within the short arms of the acrocentric chromosomes are and others [7].
hypothesized to be responsible for the formation of at least Recently, a third mechanism, fork stalling and template
some of the Robertsonian translocations (see sec- switching (FoSTeS), has been proposed to explain the forma-
tion “Robertsonian Translocations”, later). Recombination tion of multiple complex nonrecurrent structural chromo-
events mediated by satellite DNA sequences are also thought some rearrangements that do not appear to have formed
to be responsible for the recurring inversions involving the secondary to either NAHR or NHEJ [21, 22]. The complex
heterochromatic region within the proximal long arm of rearrangements associated with FoSTeS contain duplications
chromosome 9 [12]. and/or deletions that are interspersed with nonduplicated or
That DNA architecture may create “hot spots” for chro- nontriplicated segments that are believed to form secondary
mosome rearrangements has been supported by studies to a promiscuous lagging replication strand that may not
addressing the recurring (11;22) translocation (see section only have moved discontinuously within its own replication
“Reciprocal Autosomal Translocations”, later). The break- fork but may also have invaded other replication forks. This
points involved in this translocation are not associated with abnormal replication process is thought to be initiated by
regions of homology but rather with unstable AT-rich palin- fork stalling secondary to particular DNA structures and/or
dromic sequences (DNA sequences that contain two inverted protein-DNA complexes. Once stalled, the lagging DNA
regions complementary to each other) susceptible to double- strand disengages from its original template and, using short
stranded breaks that are repaired by a process referred to as regions of homology, reinitiates replication elsewhere within
nonhomologous end jointing (NHEJ). These palindromic the same chromosome, the homologous chromosome, or a
sequences are predicted to form imperfect hairpin- or nonhomologous chromosome in close proximity. Whether
cruciform-shaped secondary structures susceptible to nucle- the same or a different chromosome has been invaded, the
ases that produce double-stranded breaks, which are then location of strand invasion is upstream or downstream rela-
ligated to form the resulting translocation. Short, typically tive to the original replication fork, and whether the leading
2–5 base pair regions of homology at, or in close proximity or lagging strand of the new replication fork has been invaded
to, the double-stranded breaks are often, but not always, used will dictate whether an interstitial or interchromosomal rear-
to promote ligation. rangement has formed and whether genetic material has been
Despite the fact that the breakpoints of a number of trans- duplicated in a direct or inverted orientation or has been
locations have now been examined, palindromic sequences completely deleted. The number of serial replication fork
have rarely been identified. Among the few that have been disengagements and invasions that occur prior to reestablish-
identified, with one exception, the same AT-rich palindromic ing normal replication on the original strand will dictate the
sequence within the proximal short arm of chromosome 22 complexity of the resulting rearrangement. Many of the
employed in the recurring (11;22) translocation has been uti- PLP1 gene duplications associated with Pelizaeus-
lized [13]. Two reported cases involve independent (17;22) Merzbacher disease, as well as the nonrecurrent 17p11.2
translocations mediated by a palindrome within intron 31 of duplications associated with Potocki-Lupski, are believed to
the neurofibromatosis I (NFI) gene, at least five cases involve occur secondary to FoSTes [23].
a recurring (8;22) translocation with nearly identical break- In contrast to the maternal bias noted for numerical chro-
points within an AT-rich palindrome at 8q24.13, while the mosome abnormalities, approximately 75% of structural
others involved palindromic sequences at 1p21.1 and 4q35.1 chromosome rearrangements appear to be paternally derived
[13–18]. Most rearrangements formed secondary to NHEJ [24, 25]. Exactly why the male bias for de novo structural
therefore do not appear to utilize palindromic sequences. rearrangements exists is currently unknown. It has been sug-
Instead, they tend to occur within other areas of the genome gested, however, that the lifelong mitotic proliferation of
that are predicted to form cleavage-sensitive chromatin struc- spermatogonial cells, compared to the finite number of mitotic
tures vulnerable to the formation of double-stranded breaks divisions responsible for oögonial cell production in the
such as topoisomerase II cleavage sites, DNase I-sensitive female embryo, may promote the accumulation of mutations.
sites, scaffold attachment regions, or expanded trinucleotide Additionally, studies on mouse and drosophila suggest that
repeat regions [1, 19]. Additionally, although the most thor- male gametogenesis may be more sensitive to mutagens than
oughly studied example of a rearrangement formed second- oögenesis [26]. It is interesting to note, however, that although
ary to NHEJ is the recurring (11;22) translocation described structural rearrangements as a group are more commonly
earlier, most rearrangements that utilize this mechanism are paternal in origin, there are some exceptions to this rule. For
nonrecurring. Examples of nonrecurring rearrangements example, approximately 90% of de novo nonhomologous
Robertsonian translocations and 80% of terminal chromo- 3–5 × 106 base pairs. This number will vary, however,
some 1 short arm deletions are maternal in origin [27, 28]. depending on the quality of the chromosome preparations
Several supernumerary isochromosomes and inverted dupli- and the skill of the cytogeneticist examining the karyogram(s).
cated chromosomes also appear to form primarily during The ability to resolve or identify a rearrangement will also be
maternal gametogenesis [29–32]. No parental bias has been influenced by the degree to which the banding pattern, over-
noted for several other types of rearrangements including the all size, and centromere location of an involved chromosome
interstitial microdeletions associated with DiGeorge and is altered. Obviously, the more apparent the change, the more
Williams syndrome [33, 34]. Although the differences noted likely it is to be detected. A number of molecular cytogenetic
in male versus female gametogenesis are thought to affect our techniques such as fluorescence in situ hybridization (FISH),
respective predispositions to producing specific types of 24-color karyotyping, and chromosome microarray analysis
de novo rearrangements, other factors, such as the effect of are currently being used to detect submicroscopic or other-
imprinting on fetal survival, have also been proposed to play wise cryptic rearrangements that cannot be detected using
a role (see Chap. 20). traditional cytogenetics (see Chaps. 17 and 18). Recent stud-
In theory, chromosome breakage, rearrangement, and ies suggest that chromosome imbalances can be found in an
reunion can occur during meiosis or mitosis. Meiotic errors, additional 10–14% of patients using chromosome microar-
since they occur prior to conception, would be expected to be ray analysis [35–37]. Additionally, chromosome microarray
present in every cell of the resulting pregnancy. Postconception analysis has demonstrated that as many as 40% of pheno-
mitotic errors, in contrast, would be predicted to produce a typically abnormal individuals with apparently balanced
mosaic pregnancy containing both normal and abnormal simple chromosome rearrangements detected by traditional
cells. Interestingly, with the exception of mitotically unstable karyotyping actually contain submicroscopic gains and/or
chromosomes such as rings or dicentrics, structural chromo- losses at or near one or more rearrangement breakpoints,
some rearrangements are rarely seen in mosaic form. While while others have been found to have cryptic imbalances
this observation suggests that many structural rearrange- unrelated to their identified balanced rearrangements
ments may be formed during meiosis, ascertainment bias [38–41].
likely plays a role as well. Since mosaic individuals typically
have milder phenotypes than comparable nonmosaics, they
are less likely to be ascertained and karyotyped. This would Associated Risks
be especially true of individuals carrying mosaic balanced
rearrangements. Additionally, mosaicism is difficult to Once a structural chromosome rearrangement is detected,
detect, particularly when it is limited to a specific tissue or regardless of whether it is balanced or unbalanced, the subse-
group of tissues, is present at a low level, and/or involves a quent steps to take depend on the type of specimen that was
subtle structural change. analyzed. For prenatal samples or those obtained from a child,
parental karyotypes or molecular cytogenetic studies should
be obtained whenever possible to assess whether the rearrange-
Differentiating Between Balanced ment has been inherited or represents a de novo mutation. If
and Unbalanced Structural Rearrangements neither parent is found to be a carrier of the rearrangement, the
most likely scenario is that it represents a de novo abnormality
Structural rearrangements are often divided into two general rather than an inherited one. Non-paternity should be consid-
categories, balanced and unbalanced. Balanced rearrange- ered. Since the possibility of gonadal mosaicism can never be
ments contain no net loss or gain of genetic information and excluded, this family would be given a very low risk of having
the individuals who carry them are generally phenotypically another child with the same structural abnormality. Prenatal
normal. In contrast, additional and/or missing genetic mate- testing would also be offered for all future pregnancies.
rial is present in individuals who carry unbalanced rearrange- In contrast to the very low recurrence risk quoted to a
ments. Just as modifications in the amount of the various couple with a child or pregnancy carrying a de novo rear-
ingredients added to any recipe cause changes in the final rangement, the risk of chromosomally abnormal conceptions
product, deviation from the normal disomic genetic comple- for an adult who carries a balanced structural rearrangement
ment results in a clinically affected individual. is much higher. In fact, for some familial rearrangements, the
While it is easy to define balanced and unbalanced rear- risk can approach 50%, and for very rare carriers of a homol-
rangements, distinguishing between a truly balanced and an ogous Robertsonian translocation, the risk for an abnormal
unbalanced rearrangement using traditional cytogenetic conception is 100%. It is therefore imperative that these
techniques is often impossible. The maximum level of reso- families are identified so that they can be given accurate
lution obtained using standard microscopy of G-banded pro- genetic counseling regarding their reproductive risks and
metaphase chromosomes is reported to be 3–5 megabases or options. In situations where a familial rearrangement is
identified, it must be remembered that it is not just the imme- always higher for an individual with an apparently balanced
diate family but distant relatives as well who may be at risk de novo rearrangement than for an individual who has inher-
for having children with unbalanced karyotypes and associ- ited a similar rearrangement from a normal parent. Obviously,
ated mental and/or physical abnormalities. By systematically these individuals also carry a significantly higher risk for
karyotyping the appropriate individuals in each generation, phenotypic abnormalities than their chromosomally normal
all those with elevated reproductive risks can be identified counterparts. Several population studies have shown, for
and appropriately counseled regarding their risks and options. example, that the incidence of de novo apparently balanced
Although there has been some debate regarding the appropri- rearrangements among the mentally retarded is approxi-
ateness of karyotyping the phenotypically normal minors of mately seven times that reported in newborns [42]. Apparently
balanced carriers, 50% of whom would be expected to be balanced de novo rearrangements detected at amniocentesis
balanced carriers themselves, there is a consensus that these have also been associated with a risk for congenital abnor-
children should be referred for appropriate genetic counsel- malities that is two- to threefold that observed within the
ing when they reach reproductive age. general population [1].
The situation becomes a bit more complex when chro- A number of different mechanisms are thought to be
mosome analysis of a bone marrow or tumor specimen responsible for the abnormal phenotypes observed in chil-
results in an apparently balanced rearrangement, not associ- dren with apparently balanced de novo rearrangements. One
ated with any particular neoplasm, in all cells examined. In possibility is that the translocation is not truly balanced. As
these cases, it is imperative to ascertain whether such a rear- discussed earlier, structural rearrangements that appear bal-
rangement represents a patient-specific acquired change anced at the microscopic level may actually contain large
(which can then be monitored during treatment, remission, duplications and/or deletions at the molecular level. Another
relapse, or any change in disease aggression) or a constitu- possibility is that the rearrangement is “balanced,” but a
tional abnormality present from birth. The reasons for this break has occurred within a critical gene or its surrounding
are twofold. First of all, from the point of view of the physi- regulatory sequences such that the gene product or its expres-
cian treating the patient, the presence of any acquired cyto- sion is altered. This scenario has been demonstrated in sev-
genetic change is significant (see Chaps. 15 and 16). Equally eral patients with Duchenne muscular dystrophy, for example
as important in the long term, however, is establishing [43]. A position effect, in which the expression of a specific
whether the chromosome rearrangement is familial and gene or group of genes is altered when the chromosome seg-
therefore has potential reproductive consequences for the ment containing them is moved to a different location, could
extended family. While the potential familial reproductive also result in an abnormal phenotype. Such an effect has
issues are understandably not the primary concern of oncol- been demonstrated in several X;autosome translocation chro-
ogy patients nor their physicians, given the critical nature of mosomes in which inactivation seems to spread from the
their disease and the fact that cancer patients are often well inactive X chromosome into neighboring autosomal seg-
beyond childbearing age themselves, this is an issue that ments. This phenomenon has been documented in drosophila
should not be overlooked. Genetic counseling is covered in and plants as well. Finally, the possibility that an individual’s
detail in Chap. 21. abnormal phenotype may be completely unrelated to his or
her rearrangement must always be examined. Other non-
chromosomal genetic disorders, prenatal exposures, birth
De Novo Rearrangements trauma, non-paternity, etc., must all be considered.
Every chromosome rearrangement was at one time a new or

de novo rearrangement that carried the risks associated with Familial Rearrangements
an undefined entity. Children who carry unbalanced rear-
rangements, regardless of whether they represent new muta- Balanced structural rearrangements may pass through mul-
tions or an unbalanced form of a familial rearrangement, tiple generations of a family without detection. When these
almost inevitably demonstrate an abnormal phenotype. An families are ascertained, it is usually due to the presence of
imbalance is an imbalance regardless of how it arose. infertility, multiple spontaneous pregnancy losses, and/or
In contrast, accurate predictions regarding the phenotype clinically abnormal family members (Fig. 9.2). Meiotic
of a child or fetus that carries an apparently balanced de novo events that result in cytogenetically unbalanced conceptions
chromosome rearrangement are more difficult to make. In can explain the presence of all three occurrences within these
this situation, it is not known what has occurred at the molec- families.
ular level within the rearrangement, and there are no family During normal meiosis, homologous chromosomes pair
members with the rearrangement from whom inferences can utilizing a mechanism of formation thought to depend, at
be made. The risk for an abnormal phenotype is therefore least in part, upon interactions between their shared
One important factor that is considered when assessing

the reproductive risks of a carrier parent is the extent of
imbalance demonstrated by the potential segregants. In gen-
eral, the smaller the imbalance, the less severe the pheno-
type, and the more likely the survival. An additional rule of
thumb is that the presence of excess genetic material is less
deleterious than the absence of genetic material. Another
variable to be considered is the quality of the genetic infor-
mation involved. Some chromosomes, such as 16 and 19, are
infrequently involved in unbalanced structural rearrange-
ments. Presumably, this occurs because of the importance of
maintaining a critical dosage for a gene or group of genes on
these chromosomes. Conversely, imbalances involving other
chromosomes such as 13, 18, 21, X, and Y appear to be more
easily tolerated. In fact, a complete trisomy involving any of
these chromosomes is survivable.
Each family’s reproductive history can also provide
important clues regarding the most likely outcome for an
unbalanced pregnancy. As might be expected, those families
or individuals who have had a liveborn child or children with
Fig. 9.2 A pedigree of a family in which a balanced Robertsonian congenital abnormalities, especially when an unbalanced
(13;14) translocation is segregating. Multiple spontaneous abortions form of the familial rearrangement has been documented, are
(see individuals II-2, III-2, and III-4), abnormal children (III-5), and
infertility are frequently observed in families segregating a balanced at highest risk for having unbalanced offspring. In families
rearrangement or individuals in whom multiple spontaneous abortions and/
or infertility are noted, the risk for liveborn unbalanced off-
sequences. Under normal circumstances, all 23 pairs of spring would be expected to be lower. In these families, it is
homologous chromosomes align themselves to form 23 assumed that the unbalanced conceptions are being lost very
paired linear structures or bivalents that later separate and early as unrecognized pregnancies (infertility) or later during
migrate to independent daughter cells (see Chap. 2). In cells gestation. Interestingly, the sex of the carrier parent, in some
carrying structurally rearranged chromosomes, pairing can- cases, also influences the risk of having unbalanced off-
not occur in a simple linear fashion. Instead, complex pair- spring. In situations where a sex bias does exist, the female
ing configurations are formed in an attempt to maximize carrier invariably possesses the higher risk. Why male carri-
pairing between homologous regions that now differ with ers appear to produce fewer unbalanced offspring than their
regard to their chromosomal location and/or orientation (see female counterparts is not known. Perhaps fewer unbalanced
the later sections “Deletions,” “Duplications,” “Inversions,” segregants form during spermatogenesis relative to oögene-
“Reciprocal Autosomal Translocations,” and “Insertions”). sis, and/or the selective pressure against unbalanced gametes
Chromosome malsegregation and/or particular recombina- is greater in the male, and/or imprinting effects may cause
tion events within these complex configurations can then the unbalanced embryos of male carriers to be less viable
lead to unbalanced conceptions, many of which never implant than those of their female counterparts. Male infertility may
or are spontaneously lost during gestation. also play role [26, 44] (see Chap. 11).
Cytogeneticists are frequently asked to make predictions On rare occasions, an abnormal phenotype is observed in
regarding a balanced carrier’s risk of producing an abnormal an apparently balanced carrier of a familial rearrangement.
liveborn child. While this is a legitimate question, it is in While some of these cases may simply represent coinciden-
practice very difficult to answer accurately. One source of tal events, other possible explanations exist as well. Very
difficulty is the fact that, with very few exceptions, each fam- rarely, abnormal offspring resulting from uniparental dis-
ily’s rearrangement is unique. Therefore, unless a family is omy, or the inheritance of both homologous chromosomes
large and accurate information regarding the reproductive from a single parent, has been documented in the offspring
history and phenotype of each family member is available, of balanced translocation carriers [45] (see Chap. 20).
typically no empiric data are available from which to obtain Incomplete transmission of a partially cryptic rearrangement
risk values. A second source of difficulty one encounters in has also been observed in the abnormal offspring of a pheno-
assessing the reproductive risks associated with a particular typically normal carrier parent. Wagstaff and Herman, for
balanced rearrangement is the breadth and complexity of the example, describe a family in which an apparently balanced
variables involved. (3;9) translocation was thought to be segregating [46]. After
Fig. 9.3 In the example here, the mother (top left) carries a recessive translocation from her father and the normal hatched chromosome
point mutation (asterisk) within a gene (black box) located on one from her mother. Although their second child (bottom right) is also a
chromosome homolog (hatched). The father (top right) carries a balanced translocation carrier, she has inherited two mutated copies
mutation in the same gene secondary to interruption via a transloca- of the gene and therefore manifests the recessive disease. The allele
tion event. Because the second homolog in each parent contains a she inherited from her mother contains a point mutation, while the
normal allele, both parents are phenotypically normal. This is also comparable paternally inherited allele has been interrupted secondary
true for their first child (bottom left) who inherited the balanced to a translocation
the birth of two phenotypically abnormal offspring with functions within a biological pathway identical to or related
apparently balanced karyotypes, molecular analysis demon- to the first hit. The idea is that each parent carries a single
strated that the father’s apparently balanced (3;9) transloca- mutation that is benign or causes only mild clinical manifes-
tion was actually a more complex rearrangement involving a tations of disease. When both mutations are inherited
cryptic insertion of chromosome 9 material into chromo- together, however, the involved pathway(s) is sufficiently
some 8. Abnormal segregation of this complex rearrange- altered to cause disease. Alternatively, one predisposing
ment led to a cryptic deletion of chromosome 9 material in mutation could be inherited, while the other occurs de novo.
one sibling and a duplication of the same material in the The two-hit scenario described earlier has also recently
other. been invoked to provide one possible explanation for the
Phenotypic discrepancies between child and parent may variable expressivity and decreased penetrance observed in
also be explained by the presence of a recessive allele that is association with many microdeletions and microduplica-
inherited from a chromosomally normal parent. While the tions. This is especially true of some of the smaller copy
parent is phenotypically normal due to the presence of a number changes that are currently being identified by chro-
complementary normal allele on the homologous chromo- mosome microarray analysis [47, 48].
some, the abnormal allele can be expressed in the offspring,
who has no normal allele. The affected child inherits two
mutant alleles; one mutant allele is inherited secondary to the Deletions
balanced chromosome rearrangement, while the other is
inherited from the cytogenetically normal parent (Fig. 9.3). Autosomal deletions that can be detected by traditional,
In a slight variation of this theme, the second inherited hit or “high-resolution,” or molecular cytogenetic methods produce
mutation in the affected child is nonallelic but presumably monosomies that are generally associated with significant
Fig. 9.4 A terminal deletion involving the distal short arm of chromo- Fig. 9.5 An interstitial deletion involving the long arm of chromosome
some 5 [del(5)(p15.3)]. Patients with similar deletions are said to have 13 [del(13)(q21.3q33)]
cri du chat or cat cry syndrome because of the characteristic catlike cry
present in many during infancy
pathology. Some exceptions, however, do exist. Loss of the normal banding pattern to its terminus. All stable chromo-
short arm material from acrocentric chromosomes during the somes have telomeres comprised of the human consensus
formation of Robertsonian translocations, for example, has telomere sequence (TTAGGG)n. Chromosomes with appar-
no impact on phenotype. Similarly, the striking size variation ent terminal deletions are no exception and are assumed to
of heterochromatic regions in normal individuals suggests have acquired “new” telomeres following the deletion
that loss of some, if not all, of this material is insignificant. event.
There have even been reports of “benign” deletions in regions Several mechanisms for acquiring or retaining a telom-
that are considered euchromatic. Gardner and Sutherland ere have now been documented among chromosome dele-
catalog deletions of this type in bands 2p11.2-p12 and 2q13- tions. One mechanism referred to as telomere healing
q14.1; 3p25.3-pter; 5p14; 8p23.1-pter and 8q24.13-q24.22; involves the addition of a new (TTAGGG)n sequence at or
9p21.2-p22.1; 11p12; 13q21; 16q21 and 16q13-q22; and near the deletion breakpoint [51–53]. In these cases, a
18p11.2-pter [49]. Close examination of these regions telomerase recognition site in the vicinity of the deletion
reveals that many are relatively gene poor and therefore less breakpoint is bound by the enzyme telomerase, which syn-
likely to contain a dosage-sensitive gene. For example, the thesizes a completely new telomere. These therefore repre-
11 Mb of genomic material within the 5p14 band and the sent true terminal deletions. Other chromosomes with
7 Mb of genomic material within the 11p12 band contain apparent terminal deletions have been shown to actually
only eight and nine genes, respectively. represent derivative chromosomes that have acquired their
Among deletions of pathological significance, classic subtelomeric and telomeric regions from another chromo-
cytogenetic deletions that can be detected by routine meth- some secondary to a translocation event. These transloca-
odology tend to be larger and associated with major malfor- tion or “telomere capture” events are hypothesized to occur
mations. Generally, large deletions have a more significant secondary to homologous recombination mediated by
impact on phenotype and survival than smaller ones. The regions of shared homology that exist within the deleted
nature of the deleted material, however, also plays an impor- chromosome and the subtelomeric region of a separate
tant role in determining whether a specific deletion is viable. chromosome [54, 55]. Still other deletions appear to be ter-
Thus, deletions of large segments of the short arms of chro- minal by traditional cytogenetic analysis but have been
mosomes 4 and 5, and of the entire short arm of chromosome shown by molecular analysic analysis to be interstitial. It is
18, are recurrent abnormalities among infants with major estimated that 7–25% of apparent terminal deletions fall
malformations, while deletions of similar size involving the into this category [56–58]. Because a chromosome with an
short arms of chromosomes 17 and 19 are rarely, if ever, seen interstitial deletion retains its original telomere, there is no
in liveborns [50]. reason to synthesize or acquire a new one.
Classic deletions have traditionally been described as The use of “high-resolution” banding and molecular
either terminal (Fig. 9.4) or interstitial (Fig. 9.5) based on cytogenetic techniques has led to the identification of
chromosome banding patterns. A deletion is considered another class of cytogenetic abnormality variously referred
“terminal” if there is no discernable material beyond the site to as chromosomal microdeletions, contiguous gene syn-
of initial breakage. Conversely, interstitial deletions have a dromes, and segmental aneusomy syndromes (SAS).
proximal breakpoint, missing material, and a more distal These abnormalities are mostly very small interstitial
breakpoint beyond which the chromosome continues with a deletions, often at or below the resolution of microscopic
analysis, that recur with appreciable frequency and are vidual who does not carry it. However, despite their elevated
associated with distinct clinical phenotypes. The term, relative risk, it is important to note their absolute risk
“microdeletion,” is descriptive but fails to include the remains low at approximately 1 in 1,750. Several studies
minority category of “microduplications” (e.g., CMT1A; have now demonstrated that, at least in some cases, these
see also section “Duplications,” later) and the variable eti- inversions increase the probability of a rearrangement by
ologies for some of the disorders. The term “contiguous producing better LCR substrates for recombination. In the
gene syndromes” was introduced in 1986 to describe the case of the recurring 15q13.3, 16p12.1, and 17q21.31
involvement of multiple contiguous genes in the produc- microdeletions, for example, the associated inversion poly-
tion of a clinical phenotype [59]. While this terminology morphism increases the probability of an unequal exchange
remains appropriate for some of the disorders in this new by changing the directional relationship between the medi-
category, others are actually single gene disorders, or the ating LCRs [48, 69, 70]. Without the inversion polymor-
result of imprinting defects or uniparental disomy (see phism, the LCRs are in an inverted orientation and unequal
Chap. 20). In an effort to more accurately characterize the exchange is not promoted. With the inversion, however, the
pathogenesis of these disorders, the term segmental aneu- LCRs are placed in a direct orientation and as such are ide-
somy syndrome was proposed to imply that the phenotype ally suited for the NAHR events between homologous chro-
is the result of “inappropriate dosage for a critical gene(s) mosomes and sister chromatids that are primarily responsible
within a genomic segment” [60]. for these deletions (Fig. 9.1a, b). In other cases, the inver-
Williams syndrome is one example of an SAS that results sion may improve upon the involved LCR substrates by
from a small deletion [61]. These patients typically carry increasing their length. It is also possible that the inversion
a ~1.5 Mb deletion within the proximal long arm of chro- loop that forms to maximize homologous chromosome pair-
mosome 7 that encompasses approximately 30 different ing in the heterozygous parent renders the paired chromo-
known and predicted genes. At least some of these genes somes more susceptible to unequal crossing-over (see
appear to be responsible for the cardiovascular abnormali- section “Inversions” and Fig. 9.12 later).
ties, growth and developmental delays, infantile hypercal- As noted for Williams syndrome, flanking LCR sequences
cemia, and dysmorphic facial features that are associated have also been found at the deletion sites of several other
with Williams syndrome. Deletion of the elastin gene SASs. Recombination events localized to these LCR
(ELN), for example, has been implicated in the cardiovas- sequences appear to account for the size consistency and the
cular abnormalities. This gene is also presumed to play a frequency of the deletions associated with these disorders as
causative role in some of the other features associated with well. A partial listing of classic cytogenetic deletion or SASs
this syndrome including renal artery stenosis, hypertension, can be found in Table 9.1. Given the recent widespread use
hoarse voice, premature sagging of the skin, and perhaps of chromosome microarray technology in many research and
some of the facial features. Similarly, loss of LIM-kinase 1 clinical cytogenetics laboratories, the number of microdele-
(LIMK1), a novel kinase expressed in the brain, is predicted tion and microduplication syndromes being identified and
to explain some of the cognitive abnormalities in these characterized is growing at a rapid pace. For an extensive list
patients. Presumably, some, or all, of the remaining genes of chromosome abnormalities that includes some of the more
identified within the common Williams syndrome deletion recently indentified syndromes and their associated pheno-
also contribute to the physical features associated with this types, see the Websites for Wellcome Trust Sanger Institute
contiguous gene syndrome. “DECIPHER,” the European Cytogeneticists Association
Molecular studies of the Williams syndrome deletions Register of Unbalanced Chromosome Aberrations
have revealed the presence of flanking low-copy repeat (ECARUCA), and Unique – The Rare Chromosome Disorder
(LCR) sequences at the common breakpoint sites. These Support Group [71–73].
LCR sequences appear to provide recombination sites for In contrast to the size consistency and recurrent use of
unequal meiotic and mitotic exchange events that produce specific LCR sequences documented among many of the
the recurring Williams syndrome deletions [62–64]. In some interstitial SAS deletions, other deletions appear to have
cases, these unequal exchange events seem to be promoted multiple independent breakpoints and vary considerably in
by the presence of heterozygosity for a submicroscopic par- size. This size variability has been noted in association with
acentric inversion that spans the same low-copy repeat multiple deletions including those that involve the short arms
sequences that mediate the common 1.5 Mb Williams syn- of chromosomes 1, 4, and 5 [28, 74, 75]. Nonhomologous
drome deletion [65–68]. It is estimated that the risk of hav- end joining (NHEJ) and fork stalling template switching
ing a child with a Williams syndrome deletion is (FoSTeS), rather than nonallelic homologous recombination
approximately fivefold higher for an individual who is (NAHR), are believed to be responsible for these nonrecur-
heterozygous for this inversion when compared to an indi- ring rearrangements [7, 76, 77].
Table 9.1 Some recurring deletion syndromes

Deletion syndrome Deleted region Key clinical features
Monosomy 1p36 1p36a Mental retardation, growth delay, hypotonia, early puberty, deafness, eye problems,
cardiomyopathy, seizures and/or abnormal EEGs, enlarged anterior fontanel, deep-set
eyes, flat nasal bridge, orofacial clefting or palatal abnormalities, pointed chin, ear
abnormalities
Wolf-Hirschhorn 4pa Mental and growth retardation, microcephaly, hypertelorism, broad nasal bridge,
downturned mouth, cleft lip and/or palate, micrognathia, cryptorchidism, hypospadias
Cri du chat 5pa Mental and growth retardation, cat-like cry in infancy, microcephaly, round face,
hypertelorism, down-slanting palpebral fissures
Sotos 5q 35b Cardinal features include intellectual disability, overgrowth, and characteristic long,
thin facies with a broad forehead, sparse frontoparietal hair, and down-slanted
palpebral fissures. Macrocephaly, advanced bone age, behavior problems, hypotonia,
feeding problems, renal anomalies, scoliosis, and seizures are also seen
Williams 7q11.23b Mental retardation, short stature, supravalvular aortic stenosis, hypercalcemia,
friendly disposition, hoarse voice, periorbital fullness, stellate pattern in the iris,
anteverted nares, long philtrum, full lips
Potocki-Shaffer 11p11.2a Mental retardation, biparental foramina, brachycephaly, turricephaly, multiple
(DEFECT 11 exostoses, micropenis, and minor facial dysmorphism including a high forehead,
syndrome for deletion, small upturned nose with broad tip, downturned mouth
enlarged foramina,
exostoses, cranial
dysostosis, retardation)
Jacobsen 11q24.1–11qtera Mental and growth retardation, trigonocephaly, strabismus, cardiac defects, digit
anomalies, thrombocytopenia
Langer-Giedion 8q24.11–8q24.13a Mental and growth retardation, multiple exostoses, cone-shaped epiphyses, fine scalp
(trichorhinophalangeal hair, bulbous nose, prominent ears, simple but prominent philtrum, loose redundant
syndrome type II) skin in infancy
Angelman Maternal 15q11.2–15q13.1 Mental and growth retardation, frequent laughter, ataxia and jerky arm movements,
deletion complementary to seizures, maxillary hypoplasia, deep-set eyes, large mouth with protruding tongue,
the 15q11.2–15q13.1 widely spaced teeth, prognathia
microduplication syndrome
Prader-Willi Paternal 15q11.2–15q13.1 Mental and growth retardation, hypotonia and feeding problems in infancy, later
obesity associated with hyperphagia, narrow bifrontal diameter, almond-shaped eyes,
small hands and feet, hypogonadism, skin picking
15q13.3 Microdeletion 15q13.3b Developmental delay with mild to moderate learning disability, autism spectrum
disorder, schizophrenia, epilepsy, seizures, digit anomalies, and facial features that
include hypertelorism, short philtrum, and a thick, everted upper lip. Extensive
phenotypic variability and incomplete penetrance have been reported
Rubinstein-Taybi 16p13.3b Mental retardation, postnatal growth retardation, hypotonia, broad thumbs and toes,
cryptorchidism, abnormal facies with downward-slanting palpebral fissures; heavy,
highly arched eyebrows; long eyelashes; prominent and/or beaked nose; hypoplastic
maxilla with narrow palate
Miller-Dieker 17p13.3a Mental and growth retardation, lissencephaly, microcephaly, bitemporal depression,
long philtrum, thin upper lip, mild micrognathia, ear dysplasia, anteverted nostrils
Hereditary neuropathy 17p11.2b Asymmetric recurrent palsies precipitated by focal pressure beginning in the second
with liability to Deletion complementary to or third decade of life and electrophysiologic findings of prolonged sensory motor
pressure palsies the CMT1A syndrome nerve conduction
(HNPP) duplication
Smith-Magenis 17p11.2a Mental retardation, behavioral problems, hyperactivity, sleep disturbance, decreased
pain sensitivity, short stature, brachycephaly, midface hypoplasia, prognathism,
fingertip pads, hoarse voice
17q21.3 Microdeletion 17q21.3b Mental retardation/developmental delay, delayed speech, friendly disposition,
Deletion complementary to hypotonia, normal growth, epilepsy, heart anomalies, renal/urologic anomalies,
the 17q21.2 microduplica- abnormal hair color or texture, and typical facies with high broad forehead, ptosis,
tion syndrome blepharophimosis, up-slanting palpebral fissures, epicanthal folds, a tubular- or
pear-shaped nose, prominent ears
Alagille 20p12.2b Cholestasis, peripheral pulmonic stenosis, vertebral arch defects, posterior embryo-
toxon, abnormal facies including deep-set eyes, broad forehead, long straight nose,
prominent chin, small low-set or malformed ears
(continued)

Deletion syndrome Deleted region Key clinical features
DiGeorge/velo-cardio- 22q11.2a Learning disabilities, short stature, overt or submucous cleft palate, velopharyngeal
facial (Shprintzen) Deletion complementary incompetence, prominent nose with squared nasal root and narrow alar base,
to proximal 22q11.2 conotruncal cardiac defects, and psychiatric disorders in some
microduplication syndrome
Phelan-Mcdermid 22q13.3a Moderate to severe developmental delay, severe expressive speech delay, behavior
disturbance, increased tolerance to pain, hypotonia, normal to accelerated growth,
dysplastic toenails, large hands, and minor dysmorphic features including
dolichocephaly, ptosis, abnormal ears, pointed chin
Kallmannc Xp22.3b Hypogonadotropic hypogonadism, eunuchoid habitus, anosmia or hyposmia,
bimanual synkinesia
Ichthyosis (X-linked)c Xp22.3b Hypertrophic ichthyosis, corneal opacities without impairment of vision
a
Deletion is frequently visible or btypically not visible using traditional cytogenetics
c
This has been seen in association with several other X-linked disorders when it occurs as part of a contiguous gene syndrome
Duplications
The term “duplication” as applied to chromosome abnormal-

ities implies the presence of an extra copy of a genomic seg-
ment resulting in a partial trisomy. A duplication can take
many forms. It can be present in an individual as a “pure
duplication,” uncomplicated by other imbalances (Fig. 9.6),
or in combination with a deletion or some other rearrange-
ment. Examples of some types of rearrangements that involve Fig. 9.6 A duplication involving the distal long arm of chromosome
duplications include isochromosomes, dicentrics, deriva- 15 [dup(15)(q24q26.3)]. This duplication was initially observed in the
bone marrow of a patient with mental retardation and leukemia. By
tives, recombinants, rings, and markers. The origins and obtaining a peripheral blood karyotype, it was possible to demonstrate
behavior of these abnormal chromosomes are discussed else- that the duplication was constitutional and apparently unrelated to the
where in this chapter. leukemia
Tandem duplications represent a contiguous doubling of
a chromosomal segment. The extra material can be oriented established microdeletion syndromes. These complementary
in the same direction as the original (a direct duplication) or microduplication/microdeletion syndromes are thought to
in opposition (an inverted duplication). Most cytogeneti- represent the reciprocal products of recurring unequal
cally detectable tandem duplications in humans appear to be exchange events that are mediated by flanking homologous
direct [78]. low-copy repeat (LCR) sequences. The causative unequal
Autosomal duplications produce partial trisomies and exchange events can occur following misalignment of either
associated phenotypic abnormalities. As mentioned in the sister chromatids or homologs as shown in Fig. 9.1.
introduction, the phenotypes associated with duplications One of the first complementary microduplication syn-
are typically less severe than those associated with compa- dromes identified involves the Prader-Willi and Angelman
rable deletions. Relatively few duplications, however, have syndrome (PWS/AS) region within the proximal long arm of
occurred with sufficient frequency or been associated with chromosome 15 (Fig. 9.7). These duplications are mediated
such a strikingly characteristic phenotype that they have been by the same LCRs and encompass the same loci that are
recognized as defined clinical syndromes (Table 9.2). A few deleted in PWS/AS. The clinical significance of these par-
cases of distal 3q duplication have been reported in patients ticular duplications was initially difficult to assess because
with features similar to Cornelia de Lange syndrome. many affected patients had phenotypically normal relatives
However, these patients also have additional abnormalities with the same apparent duplication. Molecular studies have
not usually associated with the syndrome [50]. Paternally now provided us with an explanation for the apparent absence
derived duplications of distal 11p have also been associated, of a genotype-phenotype correlation in these families. With
in some cases, with Beckwith-Wiedemann syndrome [79]. few exceptions, the clinically affected individual(s) within
More intriguing, and perhaps more significant, is the emerg- these families demonstrate mental retardation, decreased
ing recognition of recurring duplications that involve the motor coordination, autism spectrum disorder, and mild to
same genomic segments that are associated with some of the no dysmorphic features and carry a maternally derived
150
Table 9.2 Some recurring duplication/triplication syndromes

Duplication/triplication
syndrome Duplicated/triplicated region Key clinical features
Duplication 3q ?3q26.3 A Cornelia de Lange-like phenotype that includes mental retardation postnatal growth retardation, long philtrum, palate
anomalies, anteverted nares, clinodactyly, talipes, renal and cardiac abnormalities
7q11.23 7q11.23b Cognitive abilities range from normal to moderate mental retardation, but all have speech delay. Other features include
Microduplication Duplication complementary to the autism, hypotonia, heart defects, diaphragmatic hernia, cryptorchidism, and dysmorphic facial features including a short
17q21.2 microdeletion syndrome philtrum, thin lips, and straight eyebrows
Beckwith-Wiedemann 11p15.5b (Paternal) Macrosomia, macroglossia, organomegaly, omphalocele, ear creases, hypoglycemia, tumor susceptibility. Beckwith-
Wiedemann patients with cytogenetic duplications are more likely to have learning difficulties
Pallister-Killian Mosaic tetrasomy 12p usually secondary Mental retardation, streaks of hyper- and hypopigmentation, sparse anterior scalp hair, sparse eyebrows and eyelashes,
to an extra metacentric isochromosome prominent forehead, protruding lower lip, coarsening of face with age
Proximal 15q11.2 15q11.2 15q13.1a Mild to severe intellectual impairment particularly with regard to language, autism spectrum disorders, decreased motor
microduplication Complementary to Prader-Willi/ coordination, hypotonia, reduced deep tendon reflexes, joint laxity, mild or no dysmorphic features. Phenotype typically
Angelman syndrome deletion region associated with maternal (but not paternal) duplication
Pseudodicentric 15 Tetrasomy 15pter–15q13 due to the Mental and growth retardation, autism, behavioral disturbance, seizures, low posterior hairline, epicanthal folds, low-set
(“inverted duplicated presence of an extra pseudodicentric ears, strabismus
15”) chromosome The smaller pseudodicentric 15 chromosomes may not cause phenotypic abnormalities
17p13.3 Duplication 17p13.3 Mental retardation and/or learning difficulties, speech difficulties, autism, hypotonia, subtle hand and foot malformations,
Most are unique nonrecurrent lack of severe congenital anomalies, and normal to increased growth parameters. Facial features include prominent
duplications that overlap with but do not forehead and pointed chin
correspond to the recurring Miller-Dieker
syndrome deletion region
Charcot-Marie-Tooth 17p11.2b Duplication complementary to Abnormal nerve conduction velocities, distal muscle weakness, muscle atrophy, and sensory loss. Symptoms begin
1A (CMT1A) the HNPP syndrome deletion between ages 5 and 25 and progress slowly
Potocki-Lupski 17p11.2a Mild to borderline mental retardation, behavioral problems, hypotonia, failure to thrive, cardiac anomalies, and variable
Duplication complementary to the dysmorphic features that include triangular face, frontal bossing, microcephaly, hypertelorism, wide nasal bridge,
Smith-Magenis syndrome deletion epicanthal folds, and a flat philtrum
17q21.3 Duplication 17q21.3 b Intellectual disability, autism spectrum disorders, and variable dysmorphic features
Duplication complementary to the
17q21.3 microdeletion syndrome
Proximal 22q11.2 22q11.2a Currently no clearly established phenotype recognized. Some patients noted to have features that overlap with DiGeorge
microduplication Duplication complementary to the syndrome including mental retardation and developmental delay, abnormalities of the palate, conotruncal heart defects,
DiGeorge syndrome deletion absent thymus, and corresponding T-cell deficiency. Phenotype variable and ranges from mild to severe
Cat eye Tetrasomy 22q11.2 (occasionally Usually mild mental retardation, coloboma of the iris, down-slanting palpebral fissures, preauricular tags and/or fistulas,
trisomy) usually secondary to an extra anal atresia
pseudodicentric or ring chromosome
a
Duplication is frequently visible or btypically not visible using traditional cytogenetics
K. Kaiser-Rogers and K.W. Rao
Fig. 9.7 A duplication involving the proximal long arm of chromo- Fig. 9.8 This benign inversion of chromosome 9 [inv(9)(p11q13)]
some 15 [dup(15)(q11.2q13.1)] that appears to be complementary to represents a pericentric inversion with breakpoints in both chromosome
the common deletions that are observed in Prader-Willi syndrome and arms. The material between the two breakpoints has been inverted, the
Angelman syndrome patients. Duplication of the Prader-Willi/ block of heterochromatin that normally sits in the long arm has been
Angelman syndrome region was confirmed using fluorescence in situ shifted to the short arm, and the banding pattern has been subtly
hybridization (not shown.) changed. Because the breakpoints have not occurred symmetrically
with respect to the centromere, the short arm-to-long arm ratio of the
inverted chromosome has been altered as well
duplication. In contrast, the duplicated chromosome in the

normal relatives of these patients is typically paternally
inherited. These data suggest that imprinting within the
PWS/AS region is responsible not only for the phenotypic
differences we observe with maternal versus paternal dele-
tions but also for the presence or absence of a clinical pheno-
type in patients with a duplication (see Chap. 20).
In addition to the PWS/AS region, complementary
microdeletion-microduplication syndromes have also been
documented for the Williams, Smith-Magenis, 17q21.31,
and DiGeorge syndrome critical regions, as well as for the
hereditary neuropathy with liability to pressure palsies
(HNLPP)/Charcot-Marie-Tooth type I region (Table 9.2).
Fig. 9.9 Although this recurring pericentric inversion [inv(2)(p11q13)]
More recently, chromosome microarray analysis has resulted is considered to be benign, individuals who carry this inversion might
in the identification of additional previously unrecognized have a slightly increased risk for miscarriages
complementary recurring microdeletions and duplications
involving 1q21.1, 3q29, 15q13.3, 16p11.2, 16p13.11, 17q12,
distal 22q11.2, and multiple other regions [48]. For an exten- both breakpoints on the same side of the centromere, or
sive list of chromosome abnormalities that includes some of within a single chromosome arm (see Chap. 10, Fig. 10.6).
the more recently indentified microduplication and microde- In paracentric inversions, the centromere position does not
letion syndromes and their associated phenotypes, see the change, and the only clue to their presence is an alteration
Websites listed earlier in the section “Deletions” [71–73]. in the chromosome banding pattern. Prior to the develop-
ment of banding techniques, the existence of paracentric
inversions was theorized but could not be proven.
Inversions In those studies in which parents of a proband with a
unique inversion have been karyotyped, the inversion is
Inversions are intrachromosomal rearrangements formed found in a parent as often as 85–90% of the time [80–82].
when a chromosome breaks in two places and the material Most inversions of both types therefore appear to be inher-
between the two breakpoints reverses orientation. ited. Additionally, among the handful of apparent recurring
Inversions can be of two types: pericentric or paracentric. inversions studied thus far, most have not been formed from
In pericentric inversions, the breakpoints lie on either side multiple independent events but instead have been inherited
of the centromere and formation of the inversion often from a single common distant ancestor. Examples include
changes the chromosome arm ratio (centromere position) inv(3)(p25q21), inv(5)(p13q13), inv(8)(p23q22), inv(10)
and alters the banding pattern of the chromosome (Figs. 9.8 (p11.2q21.2), and inv(11)(q21q23). The recurrent variant
and 9.9). Paracentric inversions, on the other hand, have inv(2)(p12q13), however, appears to be one of the few
Fig. 9.10 Several models for meiotic pairing in a pericentric inversion the material that is distal to the inversion breakpoints has been dupli-
heterozygote. (a) An inversion loop containing a single crossover and cated/deleted in each recombinant chromosome. (b, c) Alternate mod-
the resulting parental and recombinant chromosomes. Note that only els for pairing during which only partial pairing or synapsis occurs
exceptions (Fig. 9.9). This inversion has occurred in the Unique inversions are those observed in a single individual
human genome on multiple occasions, and its recurring for- or family. The clinical significance of these inversions must
mation appears to occur secondary to nonallelic homolo- therefore be determined on a case-by-case basis; as described
gous recombination (NAHR) [83, 84]. later, some inversions can impart substantial reproductive
risk, depending on the chromosome segment involved.
Excluding the variant inversions discussed previously, the
Pericentric Inversions frequency of pericentric inversions in the human population
has been estimated at 0.12–0.7% [49].
As discussed earlier, both recurring and unique pericentric
inversions have been reported in man. Some recurring inver- Meiotic Behavior and Risks for Carriers
sions are considered normal variants. In some of these variant of Pericentric Inversions
inversions, a block of heterochromatin normally situated in the In order to understand the reproductive risks of an inversion
proximal long arm of the chromosome is inverted into the carrier (heterozygote), the meiotic behavior of inverted chro-
short arm of the chromosome. Such inversions are found in mosomes must first be considered. In meiosis, homologous
chromosomes 1, 9 (Fig. 9.8), and 16. A second group of appar- chromosomes pair in close association. During this pairing
ently benign recurring inversions, which have breakpoints phase, genetic information is exchanged between homologs
very near the centromere in both the long and short arms, are through a process known as crossing-over or recombination
found in chromosomes 2, 3 and 10, and in the Y chromosome. (see Chap. 2). Crossing-over appears to be a necessary step
These variant forms have been observed in a large number of for orderly chromosome segregation and is the mechanism
families and appear to segregate without deleterious effect. that ensures human genetic individuality. A chromosome
One group of investigators, however, has reported an increased pair that consists of one normal chromosome and one chro-
risk for miscarriage among carriers of a pericentric inversion mosome with an inversion cannot achieve the intimate pair-
of chromosome 2 [inv(2)(p11q13), Fig. 9.9] [85]. Other inver- ing of homologous regions necessary for normal meiosis
sions have been observed in many families but are not without through simple linear alignment. The classic model for pair-
consequence. Of particular note is the inversion of chromo- ing in an inversion heterozygote is the inversion or reverse
some 8 with breakpoints at p23 and q22, which has been seen loop demonstrated in Fig. 9.10a. In this model, the inverted
in families of Mexican-American descent [86]. segment forms a loop that can then pair with homologous
regions on the normal chromosome. The noninverted por- form recombinant chromosomes. The opposite is true of
tions of the chromosome (the chromosome segments distal small inversions with large distal segments, which are usu-
to the inversion breakpoints) pair linearly with homologous ally associated with a very low risk of liveborn abnormal
regions on the normal chromosome. An odd number of offspring.
crossovers between the same two chromatids within the In addition to the size of the inverted segment, other fac-
inversion loop will result in the production of recombinant tors must be considered when determining the reproductive
chromosomes, while an even number of crossovers between risk associated with any given pericentric inversion. Since
the same two chromatids within the inversion loop should monosomies are generally more lethal than trisomies, an
result in the production of normal or balanced inversion that produces a recombinant with a very small
chromosomes. monosomy may be associated with a relatively high risk of
Two types of recombinant chromosomes are formed when abnormal offspring.
crossing-over occurs between the inversion breakpoints. One The nature of the genetic material in the inverted chromo-
recombinant will contain a duplication of the material distal to somes can also be important. For instance, both trisomy and
the breakpoint on the short arm and a deletion of the material partial monosomy of chromosomes 13, 18, and 21 are seen
distal to the breakpoint in the long arm. The second recombi- in liveborn infants with birth defects and mental retardation.
nant is complementary to the first and contains a short arm Once the duplications and deficiencies associated with the
deletion and a long arm duplication (Figs. 9.10 and 9.11). recombinants from a particular inversion are identified,
Both recombinants are known as duplication-deficiency review of the medical literature for evidence that these dupli-
chromosomes. cations and/or deficiencies are compatible with survival can
Alternate models for pairing in an inversion heterozygote aid in predicting the magnitude of the risk associated with
are seen in Fig. 9.10b, c. In inversions with very small that particular inversion.
inverted segments (breakpoints are close to the centromere Another clue to the level of risk associated with a given
and the distal segments are large), the noninverted segments inversion is the manner in which the inversion was ascer-
of both chromosomes may pair in linear fashion, with asyn- tained. If a balanced inversion is ascertained fortuitously, for
apsis or failure to pair in the small inverted segment. In this instance, during a prenatal chromosome study because of
model, crossing-over can only take place in the noninverted advanced maternal age, the risk associated with such an
segments of the chromosomes, and thus abnormal recombi- inversion is probably very low. On the other hand, an inver-
nant chromosomes are not formed. In the opposite situation, sion that is ascertained through the birth of an infant with
where the inverted segment is very large relative to the size anomalies secondary to the presence of a recombinant chro-
of the entire chromosome and the distal segments are small, mosome is associated with a much higher risk since the
pairing may occur only between the inversion breakpoints, important question of whether the recombinant offspring is
and the distal material will remain unpaired. In this situation, viable has already been answered. Careful examination of
a crossover between the inversion breakpoints would pro- the family history in both types of ascertainment can provide
duce recombinant chromosomes in a manner similar to the additional important information in assessing risk.
reverse loop model discussed previously. Crossing-over Gardner and Sutherland reviewed several studies that
could not take place in the segments distal to the inversion contain data about the risks associated with pericentric
breakpoints since those regions do not pair. inversions and estimated the risk for an inversion heterozy-
Careful examination of the recombinant chromosomes gote to have an abnormal child secondary to a recombinant
produced when crossing-over takes place between the chromosome [49]. This risk was estimated to be 5–10% in
breakpoints in a pericentric inversion reveals that the families ascertained through an abnormal child and approx-
genetic imbalance always involves the material distal to the imately 1% for families ascertained for any other reason.
inversion breakpoints. Thus, large inversions have small For families segregating very small inversions, the risk of
distal segments and produce recombinant chromosomes having a liveborn recombinant child may be close to zero.
with small duplications and deficiencies, while small inver- In cases of recurring inversions, additional information
sions have large distal segments and produce recombinant about the risks can be gained from studying the literature.
chromosomes with large duplications and deficiencies. In In the case of the inversion (8)(p23q22) mentioned earlier,
general, then, large inversions are associated with a greater for example, enough recombinant offspring have been
risk of producing abnormal liveborn offspring since the observed to derive an empiric risk of 6% for a heterozygote
recombinant chromosomes associated with them carry to have a liveborn recombinant child [87]. Large inversions
small duplications and deficiencies that have a greater prob- with distal segments that have been seen in liveborn chil-
ability of being compatible with survival. Furthermore, the dren as monosomies or trisomies may be associated with
larger the inversion, the greater the likelihood that a recom- high risk regardless of their mode of ascertainment in a par-
bination event within the inversion loop will occur and ticular family.
Fig. 9.11 Left: a normal chromosome 5 and a chromosome 5 with a that lies distal to the short arm inversion breakpoint and has two copies
large pericentric inversion, with breakpoints at p15.33 and q33.1, of the long arm material positioned distal to the long arm inversion
observed in a parent (* 5p terminal material, * 5q terminal material). breakpoint. Right: a metaphase hybridized with subtelomere probes for
Middle: a normal chromosome 5 and a recombinant chromosome 5 the short arm (green) and long arm (red) of chromosome 5. Note that
[rec(5)dup(5q)inv(5)(p15.33q33.1)] resulting from recombination the recombinant or duplication-deficiency chromosome (arrowed) has
within the inversion loop of the parental inversion carrier. This two red long arm subtelomere probe signals and no green short arm
duplication-deficiency chromosome is missing the short arm material subtelomere probe signal
Paracentric Inversions polymorphism that spans the same low-copy repeat sequences
that mediate the recurring Williams syndrome deletion has
The presence of paracentric inversions in the human popula- been observed in the transmitting parent of some Williams
tion was only appreciated after the advent of chromosome syndrome parents [65–68]. The presence of a submicrosco-
banding, and they are still reported less frequently than peri- pic inversion polymorphism has also been associated with
centric inversions. Their incidence has been estimated at several other recurring microdeletions including Sotos,
0.09–0.49 per thousand [80]. While various cytogenetically Angelman, the 15q13.3, 16p12.1, and 17q21.3 microdeletion
visible recurrent paracentric inversions have been reported in syndromes, as well as several other types of disease-
a variety of chromosomes, recent data suggests that most do associated structural rearrangements. For example, submi-
not represent multiple independent mutation events but are croscopic inversions that span the olfactory receptor gene
instead identical by decent [83]. One of the most frequently clusters at both 4p16 and 8p23, and mediate the recurring
cited is the recurring inv(11)(q21q23), which is believed to (4;8) translocation, have been seen in all nine of the transmit-
have arisen as a single mutation in the Netherlands and is ting parents examined [8, 90]. The same 8p inversion poly-
now observed in a large number of families in the Netherlands morphism associated with this (4;8) translocation has also
as well as in Canadian Hutterites [88, 89]. With the aid of been observed in each of the nine mothers who transmitted a
fluorescence in situ hybridization (FISH) and other molecu- recurring inverted duplicated 8p or supernumerary derivative
lar techniques, a number of recurring submicroscopic inver- 8p chromosome to their offspring [91]. Recombination
sions within the human genome are being identified. between the homologous PRKX and PRKY genes located on
Several studies have now demonstrated that some recur- the short arm of the X and Y chromosome, respectively,
ring submicroscopic inversions confer susceptibility to other results in a translocation involving the material distal to the
rearrangements. For example, a submicroscopic inversion involved breakpoints and the formation of both XX males
Fig. 9.12 The type of recombinant chromosome produced depends on in the formation of an acentric and a dicentric recombinant chromo-
which mechanism of chromosome exchange occurs within the paracen- some (top), whereas a U-type exchange produces only monocentric
tric inversion loop. A classic crossover within the inversion loop results chromosomes (bottom)
and XY females. This particular recombination event also (Fig. 9.12). The dicentric recombinant is genetically unstable
appears to occur preferentially in association with a Yp because each of the two centromeres could potentially orient
inversion polymorphism [92]. toward opposite poles of the dividing cell. The material
The manner in which small paracentric inversion poly- between the two centromeres would remain stretched
morphisms promote the formation of some chromosome between the poles of the two reorganizing daughter nuclei or
rearrangements is not currently known in most cases. It has break. Thus, with each cell division, the dicentric recombi-
been demonstrated, however, that in the case of the recurring nant chromosome has a new opportunity to contribute a dif-
15q13.3, 16p12.1, and 17q21.31 microdeletions the inver- ferent and possibly lethal genetic imbalance to a new
sion produces an improved substrate for nonallelic homolo- generation of cells. The acentric fragment, on the other hand,
gous recombination (NAHR). In both cases, the inversion has no ability to attach to a spindle since it lacks a centrom-
converts a pair of inverted LCRs into a pair of direct LCRs ere. Consequently, at cell division, it can be passively
that are ideally suited to mediate the causative unequal included in the daughter nuclei or be lost. Dicentric and
exchange event [48, 69, 70]. acentric recombinant chromosomes are almost always lethal
and are rarely found in liveborns (see sections “Acentric
Meiotic Behavior and Risk for Carriers Chromosomes” and “Dicentric Chromosomes,” later).
of Paracentric Inversions Although dicentric and acentric recombinants are very
As with pericentric inversions, the classic solution to the rarely seen, there have been several reports of monocentric
problem of homologous pairing in paracentric inversions is recombinants among the children of paracentric inversion
the reverse loop. In this case, however, the centromeres are carriers. Pettenati et al., for example, identified 17 recombi-
found in the segment distal to the inversion loop. On a theo- nant chromosomes among 446 inversions [80]. While two of
retical basis, an odd number of crossovers within the inver- these recombinant chromosomes were dicentric, each of the
sion loop of a paracentric inversion should produce one remaining 15 was monocentric with duplications and/or
dicentric and one acentric recombinant chromosome deletions. A variety of mechanisms have now been proposed
for the formation of these abnormal monocentric chromo-

somes, including breakage of dicentric recombinants,
unequal crossing-over, and abnormal U-loop exchanges sim-
ilar to the one diagrammed in Fig. 9.12. All of these mecha-
nisms involve abnormal processes of one type or another.
There is currently a fair amount of controversy surround-
ing the question of risk for liveborn children with abnormali-
ties secondary to the presence of a familial balanced
paracentric inversion. Much of this controversy may be based
on our inability, in some cases, to distinguish between a par-
Fig. 9.13 A pseudoisodicentric chromosome involving the entire short
acentric inversion and an intrachromosomal insertion using arm and a portion of the long arm of chromosome 9. It appears to have
G-banded chromosomes [93]. Indeed, if the distance between one constricted active centromere (upper dot) and one unconstricted
the original site of the inserted segment and the new point of inactive centromere (lower dot). This chromosome was found in each
of the cells of a phenotypically abnormal infant with the following
insertion is small, the resulting insertion is more likely to be
karyotype: 47,XY,+psu idic(9)(q21.1)
interpreted as an inversion rather than a relatively rare inser-
tion. By inadvertently combining data from intrachromo-
somal insertion carriers, whose risk for a recombinant eres, the chromosome will be simultaneously pulled in two
offspring can approach 50%, with that from true paracentric opposing directions. As a result of this bipolar pulling, the
inversion carriers, some studies may have overestimated the chromosome may continue to straddle both daughter cells in
reproductive risks of paracentric inversion carriers. a state of limbo until it is ultimately excluded from both.
Generation of an accurate empiric risk estimate has been fur- Alternatively, the chromosome may break, allowing some
ther complicated by ascertainment bias. Some express con- portion to migrate to each daughter cell. Regardless of which
cern that not all of the associations of abnormal phenotypes of these takes place, changes in the genetic content of the
with apparently balanced inherited paracentric inversions resulting sister cells will occur and mosaicism can result.
can be explained by the presence of misidentified intrachro- Interestingly, not all dicentric chromosomes demonstrate
mosomal insertions, ascertainment bias, or coincidence [80, mitotic instability. Some of these stable dicentric chromo-
94]. Others believe that familial paracentric inversions are somes appear to have closely spaced centromeres that func-
relatively innocuous and carry a small risk for abnormal off- tion as a single large centromere [97–99]. The presence of
spring; Gardner and Sutherland estimate that the risk “lies in one active and one inactive centromere is also frequently
the range of 0.1–0.5%” [49, 93, 95, 96]. Clearly, many ques- observed among stable dicentric chromosomes. These
tions remain to be answered concerning the clinical “pseudodicentric” chromosomes contain two copies of the
significance of apparently balanced inherited paracentric centromeric heterochromatin, but only the centromere with
inversions. the primary constriction appears to bind the appropriate
centromere proteins required for activity [97, 100]. An
example of a pseudoisodicentric chromosome 9 is shown in
Dicentric Chromosomes Fig. 9.13.
Any chromosome exchange in which the involved donor and

recipient chromosome segments each contain a centromere “Acentric” Chromosomes
will result in the formation of a chromosome with two cen-
tromeres. These chromosomes are referred to as dicentrics. Because the centromere is essential for chromosomal attach-
The most common dicentric chromosomes are those that are ment to the spindle and proper segregation, chromosomes
derived from a Robertsonian translocation event (see sec- lacking this critical component are rapidly lost. Therefore,
tion “Robertsonian Translocations,” later). Recombination although single cells with acentric chromosomes or frag-
within a paracentric inversion loop is also a well-documented ments are occasionally observed, individuals with constitu-
method by which a dicentric chromosome can form (see sec- tional karyotypes that include a true acentric chromosome
tion “Inversions,” earlier). are never seen. More than 90 chromosomes with atypical
The presence of two active centromeres in a single chro- centromeres have, however, been reported in the literature
mosome has the potential to wreak havoc during cell divi- [101, 102]. With the exception of chromosome 19, these
sion. Normal segregation can only occur when the spindle atypical centromeres or neocentromeres have now been
apparatus from a single pole binds both centromeres of the reported in association with each of the 24 human chromo-
dicentric chromosome. If instead, spindles from both poles somes. The majority of these chromosomes are small
independently bind only one of each of the two centrom- supernumerary chromosomes composed of two mirror image
copies of the terminal end of a single chromosome. However, the fetus or child that carries the isochromosome. It is there-
neocentromeres have also been identified in ring chromo- fore not surprising that, with few exceptions, the most fre-
somes, in linear chromosomes with deletions, and very rarely quently reported autosomal isochromosomes tend to involve
in structurally normal chromosomes that appear to also con- chromosomes with small arms. Some of the more common
tain their original intact centromere in an inactive form. chromosome arms involved in isochromosome formation
While typically associated with unbalanced karyotypes, neo- include 5p, 8p, 9p, 12p, 18p, and 18q. The relatively large
centromeres have been observed as part of a balanced rear- isochromosome involving the long arm of the X chromo-
rangement in a handful of cases. Approximately 82% of some shown in Chap. 10, Fig. 10.4, is the most common
cases demonstrate a partial tetrasomy, while trisomies and structural abnormality found in Turner syndrome patients.
more rarely monosomies are also seen. Mosaicism is fre- Over the years, a number of theories have been proposed to
quently observed. explain the mechanism of isochromosome formation [31, 32,
Like traditional centromeres, neocentromeres are denoted 97, 106–109]. One of the more popular proposals has been
by the presence of a primary constriction, and with the excep- that isochromosome formation is the result of centromere mis-
tion of the centromere protein B (CENP-B), they bind the division (Fig. 9.14). Instead of splitting longitudinally to sepa-
same centromeric proteins. Interestingly, however, neocen- rate the two sister chromatids, the centromere was hypothesized
tromeres are located in noncentromeric regions, they do not to undergo a transverse split that separated the two arms from
react to stains specific for centromeric heterochromatin, nor one another. However, molecular studies suggest that the
do they hybridize to centromere-specific fluorescence in situ breakage and reunion events required to form many isochro-
hybridization probes. These staining differences suggested mosomes occur predominantly within the area adjacent to the
early on that the composition of these neocentromeres dif- centromere rather than within the centromere itself [97, 110–
fered from that of a traditional centromere. Subsequent map- 115]. The resulting chromosome, which appears monocentric
ping and sequencing studies have since confirmed this. at the cytogenetic level, would actually have two closely
Current data suggests that neocentromeres do not contain spaced centromeres and would more appropriately be called
DNA sequences that we typically associate with centrom- an isodicentric chromosome. Other theories that invoke
eres, such as alpha satellite DNA. In fact, the DNA sequence exchanges between homologous chromosomes have also been
within such an atypical centromere does not appear to be challenged as common mechanisms of isochromosome for-
altered relative to the homologous region of the parental mation. Molecular evidence indicating that at least some iso-
chromosome from which it was derived [103]. These data, in chromosomes are formed from genetically identical arms,
addition to DNA modeling studies and the observation that rather than homologous arms, suggests that one predominant
duplicons are frequently present at the sites of neocentrom- mechanism of isochromosome formation may rely on sister
ere formation, suggest that it is the conformation or second- chromatid exchange [109, 110, 116–119]. Breakage and
ary structure formed by the DNA, rather than the DNA reunion involving the pericentromeric regions of sister chro-
sequence itself, that enables a chromosomal region to func- matids, an event sometimes referred to as a sister chromatid
tion as a neocentromere. Neocentromere development may U-type exchange, may therefore represent an important mech-
also be promoted by repair-initiated epigenetic changes that anism of isochromosome formation. Nonallelic homologous
occur during formation of the chromosome rearrangements. recombination events mediated by low-copy repeat sequences
Some speculate that neocentromeres may represent ancient appear to be responsible for many of these U-type exchanges.
centromere sequences that have been reactivated as a conse- Additional molecular studies suggest that most isochromo-
quence of chromosome rearrangement [104, 105]. somes are maternal in origin and that nondisjunction occurs
prior to isochromosome formation in the majority of cases
involving supernumerary chromosomes [30–32, 120, 121].
Isochromosomes Most of these nondisjunction events appear to be meiotic
rather than mitotic in origin.
An isochromosome consists of two copies of the same chro- From existing data, it is clear that multiple mechanisms of
mosome arm joined through a single centromere in such a isochromosome formation are likely to exist. Precisely which
way that the arms form mirror images of one another. mechanism is found to predominate may largely depend on
Individuals with 46 chromosomes, one of which is an iso- the chromosomal origin of the isochromosome, whether the
chromosome, are monosomic for the genes within the lost chromosome is present in a disomic karyotype or represents
arm and trisomic for all genes present on the isochromo- an extra or supernumerary chromosome, and whether forma-
some. Tetrasomy for the involved chromosome segment is tion occurs during meiosis or mitosis. Clearly, additional
present when an isochromosome is present as a supernumer- studies are needed to establish a more complete understand-
ary chromosome. In general, the smaller the isochromosome, ing of isochromosome formation.
the smaller the imbalance and the more likely the survival of
Isochromosome
with a single
Centromere
centromere and
Misdivision Transverse Loss of
centromere Chromosome genetically
split segment Identical arms*
U-type Exchange Isodicentric

Involving chromosome
Homologous Breakage and U-type Acentric with
exchange fragments homologous
Chromosomes lost
arms
Isodicentric
U-type Exchange chromosome
Involving Acentric with
Breakage and U-type
Sister Chromatids exchange fragments genetically
lost
identical arms*
Fig. 9.14 Some of the mechanisms proposed for isochromosome formation. *Because recombination occurs during normal meiotic cell division,
the arms of an isochromosome formed during meiosis would be identical only for markers close to the centromere
Ring Chromosomes
Autosomal ring chromosomes are rare and usually arise

de novo (Fig. 9.15). Reported frequencies range from 1 in
27,225 to 1 in 62,279 in consecutive newborn and prenatal
diagnosis studies [81]. Rings have been reported for all chro-
mosome pairs, although those involving chromosomes 13
and 18 are among the most common [122]. When ring chro- Fig. 9.15 A ring chromosome 18 [r(18)(p11.2q23)]. This ring chro-
mosome is the result of fusion between two broken arms. The chromo-
mosomes replace a normal homolog in a karyotype, they some material distal to the breakpoints in each arm has been lost
often represent a partial monosomy for both long and short because it lacks a centromere
arm material. When rings are present as supernumerary chro-
mosomes, partial trisomies result. In rare cases, a ring chro-
mosome is found as part of a balanced chromosome mechanisms of ring formation. Some of these discontinuous
complement [102, 123]. ring chromosomes are believed to represent the “breakdown”
Rings are traditionally thought to form as a result of products of larger rings, while others appear to be derived
breakage in both arms of a chromosome, with subsequent from a structurally abnormal chromosome rather than a nor-
fusion of the ends and loss of the distal segments. Molecular mal one [126–129].
studies, however, have suggested that additional mechanisms An additional class of ring chromosome that lacks a tradi-
exist. In a 1991 study, Callen et al. characterized ten small tional centromere has also been identified [101, 102]. In con-
supernumerary rings using FISH [124]. They found that trast to the traditional ring chromosomes described earlier,
some of the rings were missing specific satellite DNA which are formed following breakage in both arms or within
sequences from one side of the centromere, suggesting that the centromere and one arm, these rings form following
these rings originated from a “transverse misdivision of the fusion of the ends of a chromosome fragment that lacks cen-
centromere” combined with a U-type exchange on one of the tromeric DNA. In the absence of a traditional centromere, a
chromosome arms. In other studies, investigators have dem- new centromere (neocentromere) forms from previously
onstrated that some rings form by telomere fusion, with no noncentromeric DNA within the resulting ring chromosome
detectable loss of genetic material [125]. A number of ring (see section “Acentric Chromosomes,” earlier).
chromosomes that are composed of discontinuous sequences One of the more striking characteristics of ring chromo-
have also been reported in the literature, suggesting still other somes is their instability. This instability is thought to result
from sister chromatid exchanges that occur in the ring chro- imprinted genes on chromosome 1 that influence phenotype,
mosome before cell division. Such exchanges are normal the resulting disomic cell line would be expected to demon-
events that, because of the unique structure of the ring chro- strate normal viability and perhaps a selective growth advan-
mosome, lead to the formation of double-sized dicentric tage compared to the cell line with partial trisomy 1.
rings and interlocking rings. Rings with even larger numbers One recurring phenotype seen in ring chromosome
of centromeres are also occasionally seen. The centromeres heterozygotes is the “ring syndrome,” originally proposed by
of these multicentric and interlocking rings can orient toward Cote et al. in 1981 [132]. These patients have 46 chromo-
opposite poles during cell division. This can lead to breakage somes, one of which is a ring chromosome with no detect-
of the ring at anaphase, with subsequent generation of new able clinically significant deletion. The ring is derived from
ring structures. Alternatively, the entire ring chromosome one of the larger chromosomes in the karyotype, and the
can be lost. This active process of creating new cells with larger the chromosome, the more severe the phenotype.
altered genetic material is termed “dynamic mosaicism” [49, Typically, these patients have severe growth retardation with-
122]. Not all ring chromosomes exhibit instability. Although out major malformations. Minor anomalies and mild to mod-
the relationship between ring size and stability is not entirely erate mental retardation are often part of the picture. The ring
clear, in most cases, smaller rings appear to be more stable syndrome is believed to result from instability of the ring
than large rings [49]. chromosome. The larger chromosomes are thought to be
In addition to mosaicism, the genetic content and break- more unstable than the smaller ones because they present
points of the rings will also have a significant impact on the more opportunities for sister chromatid exchange. The break-
patient’s phenotype. An individual with one normal chromo- age that occurs during cell division generates new ring struc-
some homolog and one partially deleted ring chromosome tures, most of which represent a more serious genetic
will have clinical findings associated with a partial mono- imbalance than the previous forms and are thus less viable.
somy. The specific phenotype of the individual will depend This results in increased cell death and contributes to growth
on both the amount and the nature of the deleted material. failure and the disturbance of developmental pathways [133].
Similarly, for a patient with a supernumerary ring chromo- Kosztolanyi has proposed that this phenomenon may also
some and therefore a partial trisomy, the size of the ring, its contribute to the severity of the phenotype in patients who
genetic content, and the proportion of cells that contain the have ring chromosomes with obvious deletions [133].
ring will all influence phenotype. A 1991 literature review discovered 32 reported cases in
Another phenomenon that has the potential to impact the which a ring chromosome was inherited from a carrier par-
phenotype of individuals with ring chromosomes is unipa- ent. The authors concluded that no more than 1% of ring
rental disomy (UPD, see Chap. 20). Petersen et al., for exam- chromosomes are inherited, and in 90% of these cases, the
ple, described a patient with mosaicism for a normal cell line mother is the carrier parent. Among the 32 patients with
and a cell line in which one normal copy of chromosome 21 inherited rings, half had a phenotype similar to the carrier
was replaced by a ring [130]. Uniparental isodisomy for parent, while approximately one third were more severely
chromosome 21 was present in the normal cell line. The affected [49, 134].
authors suggested the isodisomy developed when the normal In addition to the risks associated with ring instability,
21 was duplicated in a cell that had lost the ring (“monosomy carriers of ring chromosomes may also be at risk for having
rescue”). Rothlisberger et al. have reported a single case of children with other abnormalities involving the chromosome
mosaicism involving a cell line with a supernumerary ring from which their ring is derived. There are at least three
derived from chromosome 1 and a normal cell line with reports of carriers of a ring chromosome 21 who had off-
maternal uniparental heterodisomy for chromosome 1 [131]. spring with trisomy 21 secondary to a translocation or tan-
The presence of uniparental heterodisomy (rather than isodi- dem duplication of chromosome 21 [134].
somy as described earlier) suggests that both of the abnormal
cell lines in this patient may have arisen secondary to a tri-
somy rescue event (see Chap. 20). Presumably, the original Reciprocal Autosomal Translocations
zygote had three copies of chromosome 1: one paternal chro-
mosome 1 and two different maternal chromosomes 1. Reciprocal translocations represent one of the most common
Conversion of the paternal chromosome 1 into a small ring structural rearrangements observed in man. Estimates of the
would then produce a cell line with a survivable partial tri- population frequency range from 1/673 to 1/1,000 [1, 135].
somy 1 rather than a lethal complete trisomy. Subsequent A reciprocal translocation forms when two different chromo-
loss of the ring chromosome would then ultimately produce somes exchange segments. In the example shown in Fig. 9.16,
a disomic cell with the expected two copies of chromosome a balanced translocation involving chromosomes 1 and 9 has
1 and uniparental maternal heterodisomy for chromosome 1. occurred. The distal short arm of chromosome 1 has replaced
Given that current data suggests there are no maternally the distal short arm material on chromosome 9, and vice versa.
Fig. 9.16 A balanced reciprocal translocation involving the short arm of chromosomes 1 and 9 [t(1;9)(p32.3;p21)]. The translocated segments of
each chromosome have been bracketed
1 der(9)
der(1) 9
Meiotic Cell
Division
2:2 Segregants 3:1 Segregants

Daughter Cell 1 Daughter Cell 2 Daughter Cell 1 Daughter Cell 2
Alternate 1 der(9) Tertiary 1 der(9)
aneuploidy
9 der(1) 9 der(1)
Normal Balanced Partial trisomy 1 Partial monosomy 1

Partial trisomy 9 Partial monosomy 9
Adjacent Interchange
1 der(9) 1 der(9)
aneuploidy
der(1) 9 der(1) 9
Partial monosomy 9 Partial monosomy 1 Trisomy 1 Monosomy 1

Partial trisomy 1 Partial trisomy 9
1 der(9)
der(1) 9
Partial monosomy 9 Partial monosomy 1

Partial trisomy 1 Partial trisomy 9
Fig. 9.17 The expected meiotic pairing configuration for the (1;9) translocation described in Fig. 9.16. Each of the 2:2 and 3:1 segregants typi-
cally produced during meiotic cell division are shown
The individual who carries this balanced translocation is clini- paired linear structures or bivalents that later separate and
cally normal. His rearrangement was identified when his wife migrate to independent daughter cells. In a cell with a
had prenatal karyotyping because of advanced maternal age reciprocal translocation, 21 rather than 23 bivalents are
and a fetus with the same (1;9) translocation was found. formed. The remaining two derivative chromosomes
Although individuals who carry truly balanced recipro- involved in the reciprocal translocation and their normal
cal translocations are themselves clinically normal, they do homologs form a single pairing structure called a quadriva-
have an increased risk for having children with unbalanced lent. The expected quadrivalent for the reciprocal (1;9)
karyotypes secondary to meiotic malsegregation of their translocation described earlier is diagrammed in Fig. 9.17.
translocation. As discussed in the introduction to this chap- Notice that the four chromosomes within the quadrivalent
ter and in Chap. 2, during normal meiotic prophase, all 23 have arranged themselves such that pairing between homol-
sets of homologous chromosomes couple to produce 23 ogous regions is maximized.
Fig. 9.18 A balanced reciprocal

translocation involving the long
arm of chromosomes 11 and 22
[t(11;22)(q23.3;q11.2)]. This is
the first recurring constitutional
translocation reported in
multiple, unrelated families
Segregation of the chromosomes within a quadrivalent The (11;22) Translocation

can occur in multiple ways, most of which will result in
chromosomally unbalanced gametes. Only a 2:2 segregation, The (11;22) translocation, with breakpoints within bands
during which the two alternate chromosomes within the qua- 11q23.3 and 22q11.2, is unique because it represents the first
drivalent travel together to the same daughter cell, yields recognized recurring constitutional reciprocal translocation
chromosomally balanced gametes. In theory, 50% of the in man (Fig. 9.18). More recently, evidence for two addi-
resulting gametes would carry a normal chromosome com- tional recurring translocations, a (4;8) translocation with
plement, while the other 50% would be balanced transloca- breakpoints at 4p16 and 8p23.1 and an (8;22) translocation
tion carriers. Each of the remaining segregation patterns with breakpoints at 8q24.1 and 22q11.2, have been reported
for a reciprocal translocation produces unbalanced gametes. (see later).
A 2:2 segregation, during which two chromosomes with More than 100 apparently unrelated families with this
adjacent rather than alternate centromeres migrate to the (11;22) translocation have been reported to date. For many
same daughter cell, produces gametes with partial trisomies years, it was not known whether the ostensible reoccurrence
and monosomies. Both 3:1 and 4:0 segregations also occur, of this translocation was best explained by the efficient trans-
resulting in trisomies and monosomies. Studies examining mission of a single ancient unique translocation through
the sperm obtained from balanced reciprocal translocation multiple generations or by multiple independent transloca-
carriers suggest that approximately equal numbers of alter- tion events between two susceptible regions. However, it is
nate and adjacent segregants are generally formed and that now known that the latter is the case. Mapping studies involv-
these two groups represent the most common types of seg- ing many different unrelated families have demonstrated that
regants. The remaining 3:1 and 4:0 segregants appear to be the translocation breakpoints cluster within long AT-rich pal-
much rarer. Female translocation carriers are capable of pro- indromic sequences and both the size and symmetry of these
ducing the same types of unbalanced 2:2, 3:1, and 4:0 seg- sequences influence the de novo translocation frequency (a
regants that have been documented in male carriers, and like palindrome is a DNA sequence that contains two inverted
their male counterparts, they produce approximately equal regions that are complementary to each other) [139–142]. In
numbers of alternate and adjacent segregants and relatively each case, the breakpoints are localized to the tip of the
few 4:0 segregants. In contrast, however, the 3:1 segregants imperfect hairpin or cruciform structures that are predicted
that are rarely observed in sperm are a relatively common to form. Palindromic sequences have also been implicated in
finding in oöcytes [136–138]. the formation of several other, mostly nonrecurring, translo-
In addition to being inherited, reciprocal translocations cations (see section “Mechanisms of Formation,” earlier).
can also occur as new or de novo mutations. As discussed Although precisely how these structures promote this trans-
in the introduction to this chapter, the risk for an abnormal location is unknown, it has been suggested that they are sub-
outcome associated with a de novo apparently balanced strates for hairpin-specific nucleases. Once nicked, these
rearrangement is always greater than that associated with structures become susceptible to other nucleases that pro-
an equivalent rearrangement that has been inherited from duce double-stranded breaks and further erosion of the palin-
a normal parent. The actual risk associated with a de novo dromic DNA surrounding the initial nick site. Ultimately, the
apparently “balanced” translocation has been reported to two double-stranded ends are joined utilizing a repair pro-
be approximately 6–9% [1]. This is 2–3 times the overall cess referred to as nonhomologous end joining (NHEJ), to
rate of congenital abnormalities observed in the produce the recurring (11;22) translocation. Interestingly, all
population. eight of the de novo (11;22) translocations examined thus far
have been paternal in origin, suggesting the translocation demonstrate mental retardation, poor growth, hypotonia,
mechanism employed may be spermatogenesis specific heart defects, and an abnormal facies including hypertelor-
[141]. ism, prominent forehead, broad nasal bridge, large down-
The presence of multiple families with the same (11;22) turned mouth, cleft lip and/or palate, micrognathia, and
translocation has made it possible to obtain good empiric dysplastic ears.
data concerning viable segregants, expected phenotypes, and In contrast to the frequent reports of chromosomally
the various risks associated with this rearrangement. It is unbalanced children who inherited the abnormal chromo-
known, for example, that a carrier’s empiric risk for having a some 4, only one chromosomally unbalanced child who
liveborn child with an unbalanced karyotype is 2–10% and inherited the abnormal chromosome 8 has been reported.
that the unbalanced, liveborn offspring of (11;22) transloca- This child was reported to be less dysmorphic and have
tion carriers inevitably have 47 chromosomes: 46 normal milder mental retardation than her third cousin and other
chromosomes plus an extra or supernumerary chromosome unrelated individuals reported in the literature who inherited
representing the derivative chromosome 22 [143, 144]. These the derivative chromosome 4 [151].
individuals are therefore trisomic for the distal long arm of It has been demonstrated that this particular (4;8) translo-
chromosome 11 and the proximal long arm of chromosome cation is a recurring one, mediated by nonallelic homologous
22. Severe mental retardation, congenital heart disease, recombination between olfactory receptor gene clusters
microcephaly, malformed ears with preauricular skin tags located on both chromosomes [8]. Among the six individuals
and/or pits, a high-arched or cleft palate, micrognathia, anal whose translocation breakpoints were examined, the 8p23
stenosis or atresia, kidney anomalies, and genital abnormali- breakpoint was confined to a single olfactory receptor cluster,
ties in males are common features shared by these unbal- while the 4p16 breakpoint was distributed between two dif-
anced (11;22) segregants. In 2004, the condition was named ferent clusters. Interestingly, the translocation was of mater-
Emanuel syndrome, to honor the work of Dr. Beverly nal origin in all five of the de novo cases examined, and each
Emanuel. transmitting mother was heterozygous for a submicroscopic
Balanced carriers of the (11;22) translocation are pheno- inversion at both 4p16 and 8p23. The 4p16 inversion seen in
typically normal, with one possible exception. There is a each of these mothers spanned the two olfactory receptor
single, unconfirmed report in the literature indicating that clusters that serve as breakpoint sites for the (4;8) transloca-
female carriers may have a predisposition to breast cancer tion. Similarly, the 8p23 inversion spanned the distal olfac-
[145]. While cytogenetically the breakpoints involved in this tory receptor cluster involved in the (4;8) translocation and a
translocation appear to be identical to those identified in the more proximally placed cluster. Presumably, both of these
acquired chromosome rearrangements seen in Ewing sar- submicroscopic inversions, like the (4;8) translocation, were
coma, peripheral neuroepithelioma, and Askin tumor, molec- also mediated by these clusters. Of note, heterozygosity for
ular studies have shown that they differ [146–148] (see also the same 8p23 inversion polymorphism was also found in the
Chap. 16). The gene(s) and mechanisms responsible for the transmitting parents of patients who carry several other types
development of these neoplasms therefore have provided no of recurring chromosome 8 rearrangements that are mediated
clues regarding the etiology of breast cancer development in by the same 8p23 olfactory receptor gene clusters implicated
these patients. in the (4;8) translocation [91]. Heterozygosity for a submicro-
scopic inversion has also been reported in association with
several other chromosome rearrangements (see section
The (4;8) Translocation “Paracentric Inversions,” earlier).
At least 18 unrelated families with similar (4;8) transloca-

tions, or a chromosome derived from this translocation, have The (8;22) Translocation
been reported in the literature [8, 149, 150]. In each case,
the breakpoints involved appear to correspond to bands At least 12 families with a recurring (8;22) translocation
4p16 and 8p23. Most of these families have been ascertained involving breakpoints at 8q24.1 and 22q11.21 have been
secondary to the birth of a clinically abnormal child with the identified [13]. Molecular characterization of the transloca-
derivative chromosome 4 but not the complementary abnor- tion breakpoints involved in the five families studied thus far
mal chromosome 8. These children are monosomic for dis- indicates this rearrangement is mediated by AT-rich palin-
tal chromosome 4 short arm material and trisomic for a dromic sequences. Interestingly, this translocation utilizes
small amount of distal chromosome 8 short arm material. the same palindrome at 22q11.2 that has been associated
Despite the presence of 8p trisomy, these patients are clini- with the recurring (11;22) translocation as well as several
cally indistinguishable from Wolf-Hirschhorn patients with other nonrecurring rearrangements (see sections “Mechanism
pure 4p deletions (Table 9.1) [149]. Both groups of patients of Formation” and “The (11;22) Translocation,” earlier).
Also, as with the recurring (11;22) translocation, all via-

ble cytogenetically unbalanced offspring of t(8;22) are the
result of a 3:1 meiotic segregation event. These individuals
carry 46 normal chromosomes in addition to a supernumer-
ary derivative chromosome 22 and are therefore trisomic for
proximal chromosome 22 and distal chromosome 8 long arm
material. The phenotype associated with this imbalance is
variable but is most often associated with a normal birth
weight and normal subsequent growth, mild developmental
delays and/or mild mental retardation, prominent ears with
preauricular pits, and clinodactyly. While the balanced carri-
ers are typically clinically normal, two of the six individuals Fig. 9.19 This (13;14) translocation is the most common Robertsonian
translocation observed in man [der(13;14)(q10;q10), sometimes
reported have autoimmune disorders.
described as der(13;14)(p11.2;p11.2); see Chap. 3]
Robertsonian Translocations
A Robertsonian translocation occurs when the long arms of

any two acrocentric chromosomes join to produce a single
metacentric or submetacentric chromosome [49, 50, 56, 62,
63] (Figs. 9.19 and 9.20). Although these translocations may
in fact be reciprocal, the small complementary chromosome
composed of short arm material is only occasionally seen,
presumably because it is typically acentric and therefore Fig. 9.20 Although less common than the (13;14) translocation, the
lacks the stability conferred by a centromere [152]. Balanced Robertsonian (14;21) translocation is more clinically significant because
carriers of Robertsonian translocations therefore typically the affected offspring of such a carrier are more likely to survive to
have 45 chromosomes rather than the usual 46. The only birth. Their unbalanced offspring will inevitably have three copies of
chromosome 21 long arm material or Down syndrome, a chromosome
notable genetic material within the short arm region of each abnormality that is more compatible with survival than trisomy 13
of these chromosomes is a nucleolar organizer region com- [der(14;21)(q10;q10)]
posed of multiple copies of the ribosomal RNA genes.
Because this is redundant information, loss of this material
from the two chromosomes involved in the translocation is chromosomes. Among this group, the (13;14) and (14;21)
therefore not clinically significant. It has been suggested that translocations are the most common and constitute
the close association of these nucleolar organizer regions approximately 75 and 10% of all nonhomologous
within the cell nucleus may promote the formation of Robertsonian translocations, respectively [49] (Figs. 9.19
Robertsonian translocations. and 9.20). Molecular studies performed to explore the ori-
Since Robertsonian translocations were first described gins of these rearrangements suggest that they occur pre-
by W. R. B. Robertson in 1916, it has come to be recog- dominantly during oögenesis [27, 157].
nized that they are among the most common balanced Despite the monocentric appearance of many of these
structural rearrangements in the human population [153]. chromosomes, most are in fact dicentric [158–160]. The
Numerous studies examining both spontaneous abortions majority of these chromosomes therefore appear to form as a
and liveborn individuals indicate a frequency of approxi- result of short arm fusion rather than centromere fusion or a
mately 1/1,000 [154–156]. Although pairwise association combination of both. A single pair of short arm breakpoint
of the five human acrocentric chromosomes can form 15 regions has been observed in most (13;14) and (14;21) trans-
different Robertsonian translocations, these rearrangements locations, while multiple short arm breakpoint regions are
do not occur with equal frequency, and their mechanisms of utilized during formation of each of the remaining types of
formation appear to differ. Robertsonian translocations [158, 161–163]. Precisely where
the breakpoint occurs within the short arm therefore seems to
be dependent upon the type of Robertsonian translocation
Nonhomologous Robertsonian Translocations being formed and perhaps the mechanism responsible for the
rearrangement. While the mechanisms responsible for
Approximately 95% of all Robertsonian translocations Robertsonian translocation formation are not currently
are formed between two nonhomologous or different known, recombination involving repetitive satellite III DNA
sequences and/or other repetitive DNA sequences located offspring. Trisomy 22 occurring secondary to a Robertsonian
within the short arms of the acrocentric chromosomes has translocation may also represent a rare possibility. Because
been proposed. Nonrandom suppression of one centromere their risk for aneuploidy is greater than that of the general
appears to provide mitotic stability to some of the dicentric population, it is recommended that all Robertsonian translo-
Robertsonian chromosomes [100, 164]. In other cases, both cation carriers be offered prenatal testing (see Chap. 12).
centromeres appear to be active. It is believed that because of Occasionally, abnormal offspring with uniparental disomy
their close proximity, both centromeres are able to function (UPD) have also been observed among the children of bal-
as one in these dicentric chromosomes [165]. It should be anced Robertsonian translocation carriers [45]. UPD has been
noted that current nomenclature (see Chap. 3) still calls for reported in association with both de novo and familial, homol-
the description of all Robertsonian translocations as mono- ogous and nonhomologous, translocations. Currently, the risk
centric and that this is used in most laboratories. for UPD in a fetus with a balanced nonhomologous
Robertsonian translocation is estimated to be 0.6%, while that
for a fetus with a balanced homologous Robertsonian translo-
Homologous Robertsonian Translocations cation is predicted to be approximately 66% [169]. Among
liveborn offspring with congenital anomalies who carry a
In contrast to nonhomologous Robertsonian translocations, balanced nonhomologous or homologous Robertsonian trans-
de novo whole arm exchanges involving homologous or like location, the risk for UPD has been reported to be 4 and 100%
chromosome pairs are very rare. They appear to be predomi- (2/2 homologous Robertsonian cases studied), respectively
nantly monocentric and several of them have been shown to [170]. The higher incidence of UPD noted in association with
form postmeiotically [110, 160, 119, 166, 167]. While his- the balanced homologous Robertsonian translocations paral-
torically all such rearrangements were collectively called lels the observation that most of these translocations actually
homologous Robertsonian translocations, recent molecular represent true isochromosomes. Because both arms of a true
studies have shown that approximately 90% of the chromo- isochromosome are derived from a single chromosome, by
somes within this category may actually be isochromosomes definition, uniparental isodisomy should be present in these
composed of identical rather than unique homologous arms balanced Robertsonian translocation carriers. Whether the
[110, 111, 118, 168]. Molecular studies exploring the paren- risk for UPD varies depending on whether the translocation is
tal origin of de novo homologous Robertsonian transloca- familial or de novo is not currently known.
tions suggest that no parental bias exists. Equal numbers of Postzygotic correction of a trisomy through chromosome
maternally and paternally derived isochromosomes have loss (trisomy rescue) is thought to represent the most likely
been reported, and true homologous Robertsonian transloca- mechanism for UPD, although monosomy correction and
tions in balanced carriers appear to be composed of both a gamete complementation may occur as well [169–171].
maternal and a paternal homolog. Current data indicate that uniparental disomy is most concern-
ing when Robertsonian translocations containing chromo-
somes 14 or 15 are involved since both chromosomes appear
Reproductive Risks for Carriers of Robertsonian to have imprinted regions. Maternal and paternal UPD for
Translocations chromosome 15 result in Prader-Willi syndrome and Angelman
syndrome, respectively [172, 173]. Clinically abnormal off-
Carriers of Robertsonian translocations are at risk for mis- spring have also been documented in association with paternal
carriages and for offspring with mental retardation and birth and maternal UPD for chromosome 14 [174–178]. A single
defects associated with aneuploidy and, rarely, uniparental reported case of maternal UPD in a normal individual has cre-
disomy or the inheritance of both copies of a chromosome ated uncertainty regarding the association between maternal
pair from a single parent (see Chap. 20). The relative risk for UPD 14 and phenotype [175]. Because UPD involving chro-
each of these outcomes is a function of the sex of the mosomes 14 and 15 is associated with an adverse outcome, it
heterozygous parent and/or the particular acrocentric chro- has been proposed by the American College of Medical
mosomes involved. In theory, all chromosome segregations Genetics and Genomics that prenatal UPD testing be offered
within the carrier parent of a homologous Robertsonian when a fetus carrying a balanced Robertsonian translocation
translocation and all malsegregations within nonhomologous involving one or both of these chromosomes is ascertained.
Robertsonian carriers produce monosomic or trisomic con- While an abnormal phenotype is not likely to be directly
ceptions. Since all potential monosomies and most of the associated with UPD for chromosomes 13, 21, and 22,
potential trisomies are lethal during the first trimester, mis- residual disomy/trisomy mosaicism and recessive disease
carriage is not uncommon. Only those Robertsonian translo- resulting from reduction to homozygosity through isodis-
cation chromosomes containing chromosomes 21 or 13 are omy may influence the phenotype of all uniparental disomy
associated with an increased risk for having liveborn trisomic offspring [171]. These etiologies for disease should be
remembered when dealing with any fetus that carries a these repetitive regions and the suspicion that evolutionary
balanced Robertsonian translocation involving these chro- chromosome rearrangements have distributed inactive forms
mosomes, especially if the fetus is clinically abnormal (see of these sequences throughout the genome suggest that
Chap. 20). recombination between homologous sequences may play a
As discussed in the introduction, for some types of rear- role. Evidence also exists for the involvement of fragile sites
rangements, the risk for unbalanced offspring appears to be and viral integration sites in the formation of some jumping
significantly higher for a female carrier than a male carrier. translocations [182]. For now, however, the mechanism by
This appears to be the case for nonhomologous Robertsonian which jumping translocations occur is unknown.
translocations involving chromosome 21. In female carriers
of these translocations, an unbalanced karyotype is detected
in 13–17% of second trimester pregnancies [49, 179]. For Insertions
male carriers, the same risk appears to be less than 2%.
Precisely why male carriers appear to produce fewer unbal- Insertions are complex three-break rearrangements that
anced offspring than their female counterparts is not known. involve the excision of a portion of a chromosome from one
Current data, however, suggests that female Robertsonian site (two breaks) and its insertion into another site (one
translocation carriers produce greater numbers of unbalanced break). The orientation of the chromosomal material that
gametes than their male counterparts [180, 181]. has been moved can remain the same in relation to the cen-
tromere (a direct insertion) or be reversed (an inverted inser-
tion). When the material is inserted into a different
Jumping Translocations chromosome, the insertion is considered interchromosomal,
while with intrachromosomal insertions, material excised
The term “jumping translocation” refers to dynamic or from one portion of a chromosome is reinserted into another
changing translocations that are rarely observed in constitu- portion of the same chromosome. An example of an inter-
tional karyotypes. It is used most often to describe a type of chromosomal insertion involving chromosomes 5 and 11 is
mosaicism in which a specific donor chromosome segment is shown in Fig. 9.21.
translocated to two or more different recipient sites over the While the incidence of insertions detectable by traditional
course of multiple mitotic cell divisions [182–184]. To date, karyotyping is estimated to be approximately 1 in 80,000 to
at least 50 cases have been described, with the majority 1 in 10,000, not surprisingly, more recent studies using
involving at least one acrocentric chromosome [182, 183]. microarray analysis in conjunction with fluorescence in situ
Jewett et al., for example, have described an individual with hybridization (FISH) suggest the incidence is likely to be
four different cell lines in which long arm material of chro- higher; Kang et al. report an incidence of 1 in 500, while Neil
mosome 15 was translocated to five different sites [184]. et al. report an incidence of 1 in 3,400 to 5,200 [189–192].
Within the child’s main cell line, the chromosome 15 long While the large cytogenetically detectable insertions typi-
arm segment was transferred to the distal long arm of chro- cally result in abnormal phenotypes when inherited in an
mosome 8 and the distal short arm of chromosome 7. In
additional cell lines, this same segment was transferred to the
long arm of chromosome 12, the short arm of chromosome
6, or the short arm of chromosome 8.
In other rare situations, families are described in which
translocations involving a common donor chromosome seg-
ment but a different recipient chromosome are observed in
parent and child [185, 186]. Tomkins et al., for example,
describe a mother and daughter with different, apparently
balanced translocations involving the same short arm seg-
ment of chromosome 11 [185]. The mother carried an (11;22)
translocation, while the daughter carried a similar (11;15)
translocation. In families like this, chromosome “jumping”
Fig. 9.21 Insertion. A portion of chromosome 11 short arm material
appears to occur during gametogenesis rather than during has been inserted into the proximal long arm of chromosome 5 to pro-
mitosis as described earlier. duce an apparently balanced, inverted, interchromosomal insertion
The breakpoints observed in jumping translocations fre- [ins(5;11)(q13.1;p15.3p13)]. The individual who carries this insertion
was ascertained following the birth of a cytogenetically unbalanced
quently involve regions known to contain repetitive DNA
child who inherited the derivative 5 but not the complementary deriva-
sequences such as telomeres, centromeres, and nucleolar tive 11 (Courtesy of Dr. Frank S. Grass, Department of Pediatrics,
organizers [184, 186–188]. The location of breaks within Carolinas Medical Center)
unbalanced form, many of the submicroscopic insertions double-loop structures during meiosis. Crossing-over or
appear to cause few, if any, phenotypic consequences. In one recombination in these fully synapsed chromosomes can
study by Kang et al., a parent was found to carry the same result in the generation of chromosomes with duplications,
chromosomal imbalance as the proband in 69% of the cases deletions, or both. Madan and Menko, in their review of 27
involving inherited submicroscopic insertions [189]. cases, observed an overall 15% risk for each pregnancy that
Regardless of the size of an insertion, however, because the a carrier of an intrachromosomal insertion will have a live-
risk for an abnormal pregnancy outcome can approach 50% born child with an unbalanced karyotype [195]. This risk
for a carrier, it is important to identify these individuals. may differ greatly for individual insertions depending on the
size of the inserted segment and the viability of the partial
trisomies and monosomies produced by the abnormal recom-
Intrachromosomal Insertions binant chromosomes.
Intrachromosomal insertions can occur within a single chro-

mosome arm or between chromosome arms. Direct within- Interchromosomal Insertions
arm insertions have occasionally been mistaken for
paracentric inversions [93, 193, 194]. Interchromosomal insertions involve the movement of mate-
During meiotic pairing, the inserted segment and its com- rial from one chromosome to another. As discussed earlier,
plementary region on the normal chromosome may loop out the inserted segment can be either direct or inverted relative
allowing synapsis, or pairing, of the rest of the chromosome to its original position in the chromosome. Approximately
(Fig. 9.22). A single crossover in the paired interstitial seg- 85% are inherited, usually from a carrier mother, and no fer-
ments of such a bivalent would result in the formation of tility differences were noted between the two sexes [189,
recombinant chromosomes that are either duplicated or 192].
deleted for the inserted segment. The theoretical risk for the For relatively small inserted segments, it seems most
formation of such recombinant chromosomes could approach likely that the homologs involved in the rearrangement will
50% for each meiosis, depending on the size of the intersti- pair independently [196]. The inserted segment and its
tial segment. The risk for having a liveborn child with an homologous region on the normal chromosome can loop out,
unbalanced karyotype will depend, to some extent, on the allowing full pairing of the uninvolved segments of the biva-
viability of the duplications and deletions produced. lents (Fig. 9.22). Independent 2:2 segregation of the homologs
Alternatively, in the case of large inserted segments, com- in these two bivalents can result in the formation of four
plete pairing between the homolog with the insertion and its gamete types, two of which have a normal or balanced chro-
normal counterpart can be achieved through the formation of mosome complement and two of which have an unbalanced
Intrachromosomal insertion:
a a a a
b d d
c b b b
c c
d c d
e e e e
Meiotic pairing
Interchromosomal insertion:
a a l l a a
l l
b b
m m b b
c c m m
n c c
Fig. 9.22 Models for meiotic n n n
d d d
pairing during which partial e o d
pairing is observed between the e e e o o
o
insertion chromosome and its
homolog Meiotic pairing
complement, one duplicated and one deleted for the inserted prevalence may also reflect the presence of male sterility
segment. The theoretical risk, in this situation, would be 50% associated with some CCRs.
for producing a conceptus with an unbalanced karyotype. As one might suspect, meiotic pairing and segregation
The risk for having a liveborn abnormal child would depend can become quite complex in a CCR carrier. In theory, the
on the viability of the partial trisomy or partial monosomy of more complex the rearrangement, the more elaborate the
the inserted segment involved. chromosome contortions required to optimize pairing
In the case of very long inserted segments, a quadrivalent between the rearranged chromosomes and their homologs.
containing an insertion loop may be formed, allowing com- Similarly, the greater the number of involved chromosomes,
plete pairing of the chromosomes involved in the rearrange- the greater the potential number of unbalanced gametes. It
ment [197]. If no crossover occurs within the insertion loop, is therefore somewhat surprising that a balanced CCR car-
the consequences are the same as described earlier for non- rier’s empiric risk for an unbalanced liveborn child does not
paired bivalents. If a crossover occurs within the insertion appear to differ significantly from that of a comparable
loop, however, recombinant chromosomes that would lead to simple balanced reciprocal translocation carrier. The risk
the production of gametes with duplications and deletions for miscarriage among these carriers does, however, appear
may be formed. Once again, the risk for having a liveborn to be somewhat higher, suggesting that early loss of unbal-
abnormal child will depend on the viability of the partial tri- anced pregnancies may partially explain this observation
somies and monosomies produced. [179, 199–202]. Selection against grossly unbalanced gam-
Regardless of whether complete pairing is achieved etes at fertilization could also play a role. As discussed in
between the chromosomes involved in an interchromosomal the introduction to this chapter, the actual reproductive
insertion or whether recombination takes place, compared to risks for any CCR carrier will vary depending upon the pre-
carriers of other chromosome rearrangements, an insertion cise rearrangement involved as well as many other vari-
carrier’s risk of having an abnormal liveborn child is among ables. However, the empiric risk for a liveborn child with
the highest. VanHemel and Eussen reviewed the data from a an unbalanced chromosome complement and phenotypic
number of individual case reports and found the average risk abnormalities is estimated to be approximately 18% for a
for having an abnormal child to be approximately 32% for a CCR carrier [202].
male carrier and 36% for a female [192]. The theoretical
risk, as mentioned earlier, approaches 50%.
Variant Chromosomes
Complex Chromosome Rearrangements There are a number of structural chromosome rearrange-

ments that have no apparent clinical consequences for the
Although the definition of what constitutes a complex chro- patients that carry them [203–208]. The chromosomes that
mosome rearrangement (CCR) appears to vary somewhat, a carry these rearrangements are referred to as normal hetero-
rearrangement involving two or more chromosomes and at morphic or polymorphic variants.
least three breakpoints is generally considered to be complex Changes in the C-band-positive heterochromatic DNA
[198]. The more complex the rearrangement, the greater the found in the distal long arm of the Y chromosome and within
number of chromosome breaks and the higher the probabil- the pericentromeric region of every chromosome are respon-
ity that an essential gene has been interrupted or that genetic sible for some of the most common chromosome variants
material has been lost or gained during its formation. It is that we see. Because C-band-positive heterochromatin repre-
therefore not surprising that CCRs are only rarely seen in sents DNA that has been permanently inactivated, it is not
constitutional karyotypes. surprising that alterations in the size, position, and/or orien-
The majority of reported constitutional CCRs represent tation of this material would be benign. Among the most
de novo events that appear to have occurred during sper- common chromosomal variants observed in humans is a
matogenesis. Because many de novo, apparently balanced pericentric inversion of chromosome 9 (Fig. 9.8 and sec-
CCRs have been shown to include cryptic imbalances, a tion “Pericentric Inversions,” earlier). While the heterochro-
high-resolution genome-wide chromosome microarray matic C-band-positive material typically sits within the
analysis may be warranted for such patients, especially if proximal long arm of chromosome 9, when inverted, it
they are phenotypically abnormal [38]. In contrast to the becomes situated within the proximal short arm. Inverted or
male preponderance of de novo CCRs, the less frequently not, the size of this heterochromatic material is also quite
reported familial CCRs appear to be transmitted predomi- variable. Some chromosomes 9 have little to no pericentro-
nately through females, in keeping with the observation that meric heterochromatic material, while in others this region
chromosome rearrangements are more readily tolerated in can be quite large; the largest ones are comparable in length
female meiosis than male meiosis. This female transmission to the long arm of chromosome 17.
The short arms of the acrocentric chromosomes (13, 14, variants have recently been identified by chromosome
15, 21, and 22) represent another major region of variability microarray testing. While many of the identified copy
within the human genome. Both the proximal short arm and number variants are recognized as benign changes in the
distal satellite region of these short arms are composed of genome, the clinical significance of others remains unknown.
repetitive satellite DNA that is devoid of coding sequences. The variants that fall into this latter category are referred to
As described earlier with respect to C-band-positive hetero- as variants of unclear significance (VUS) and constitute a
chromatin, because these sequences do not contain DNA that significant portion of the copy number changes detected by
is expressed, changes in the size, orientation, and position of chromosome microarray analysis. As described earlier for
this acrocentric short arm material is clinically benign. In cytogenetically visible variants, parental studies to establish
contrast to the proximal and distal regions of the acrocentric whether the copy number change in question was inherited
short arms, the stalk region sandwiched between encodes from a clinically normal parent or instead represents a
ribosomal RNA. Typically, many copies of these ribosomal de novo mutation can be helpful for establishing clinical
RNA genes are located within the stalk region of each of the significance. However, because variable expressivity and
five pairs of acrocentric chromosomes. This region of the incomplete penetrance have been reported in association
genome is therefore highly redundant, and the presence of with multiple copy number changes identified by microarray
missing or extra copies of this sequence is of no phenotypic testing, the presence of the same mutation in a parent is not
consequence. Translocation of this region to another chro- considered definitive proof of clinical irrelevance [48]. These
mosome, provided that critical genes have not been deleted copy number changes typically remain VUS until large
or interrupted secondary to the rearrangement, also appears studies are completed to ascertain the incidence of the
to have no clinical consequences. Multiple examples of copy number change in question within a well-vetted normal
de novo and familial normal variant chromosomes with ter- population and the extent of clinical variability within the
minal translocations and interstitial insertions of these ribo- affected population. See also Chap. 18.
somal RNA sequences have been documented in the
literature.
In addition to the common C-band heterochromatic and
acrocentric short arm variants described earlier, numerous
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Sex Chromosomes, Sex Chromosome
Disorders, and Disorders of Sex 10
Development
Cynthia M. Powell
Introduction some disorders may not correspond to the genetic or

gonadal sex (see section “Disorders of Sex Development
It can be argued that the sex chromosomes are the most with “Normal” Sex Chromosomes”).
important pair of chromosomes given their role in deter-
mining gender and, therefore, allowing for reproduction and
procreation. Considered together, sex chromosome aneu- Pseudoautosomal Regions
ploidies are the most common chromosome abnormalities
seen in live-born infants, children, and adults. Physicians in The distal region of the short arms of the X and Y chromo-
many specialties including pediatrics, obstetrics and gyne- somes contains highly similar DNA sequences. During normal
cology, endocrinology, internal medicine, and surgery com- meiosis in the male, crossing-over occurs between these
monly encounter individuals with sex chromosome disorders. regions. Because this resembles the crossing-over that occurs
There has been a great deal of misinformation in the past between autosomes, these regions have been termed pseudo-
regarding outcomes and developmental profiles of these autosomal or PAR1 (Fig. 10.1). There is also a region of
patients, leading to bias and discrimination. This chapter homology (PAR2) at the distal ends of Xq and Yq, which has
attempts to provide a summary of information regarding the been observed to associate during male meiosis, with proven
sex chromosomes, sex chromosome disorders, and disorders recombination events [2]. PAR1 is 2.6 Mb and contains at least
of sex development. 24 genes, whereas PAR2 is only 320 kb and has 4 genes [3].
All genes within PAR1 escape X inactivation in women.
One of the genes in this region, SHOX, requires two func-
The X and Y Chromosomes tional copies, without which there will be short stature with
or without features of Leri-Weill dyschondrosteosis. If no
Role in Sexual Differentiation functional copy of SHOX is present, a more severe skeletal
dysplasia, Langer mesomelic dysplasia, results. Families
Genetic sex is established at the time of fertilization and is segregating deletions or mutations of SHOX can have unusual
dependent on whether an X- or Y-bearing sperm fertilizes the inheritance patterns due to the very high recombination
X-bearing egg. The type of gonads that develop (gonadal sex) frequency in PAR1. Males may inherit the deleted or mutated
is determined by the sex chromosome complement (XX or gene from their mother but pass it to their sons due to recom-
XY) and sex-determining genes. Before the seventh week of bination. This has important implications for genetic coun-
embryonic life, the gonads of both sexes appear identical [1]. seling [4].
Normally, under the influence of the Y chromosome, the
immature gonad becomes a testis. In the absence of the Y
chromosome and with two normal X chromosomes, the X-Chromosome Inactivation
gonad differentiates into an ovary. The term phenotypic
sex refers to the appearance of the external genitalia and in There are thousands of genes on the X chromosome but rela-
tively few on the Y chromosome. The explanation for the
fact that males survive quite nicely with only one X chromo-
C.M. Powell, M.D., M.S. (*) some while females have two involves a concept called
Department of Pediatrics, The University of North Carolina
at Chapel Hill, Chapel Hill, NC 27599-7487, USA
“dosage compensation,” and is termed the Lyon hypothesis
e-mail: [email protected] after its proponent, Dr. Mary Lyon [5].
176 C.M. Powell
Fig. 10.1 Idiograms of the X

and Y chromosomes showing the
pseudoautosomal regions (PAR1
and PAR2), the locations of the
X-inactive-specific transcript
(XIST) gene, the critical region
on Xq, the sex-determining
region Y (SRY), the azoospermia
factor region (AZF), the
heterochromatic region of the Y,
and the male-specific region
located between PAR1 and PAR2
In somatic cells in females, only one X chromosome is mechanism protecting a single X-chromosome inactivation
active. X inactivation occurs early in embryonic life, begin- for every two autosome sets [6]. Genes retaining Y homology
ning about 3 days after fertilization, and is completed by the tend to be expressed on the inactive X [9]. Although all genes
end of the first week of development. The inactivation is in PAR1 escape X-chromosome inactivation (XCI), some in
random between the two X chromosomes. Either the maternal PAR2 do not escape XCI but achieve dosage compensation
or paternal X can be inactivated, and after one X has become through inactivation on both the inactive X and the Y [10].
inactive, all the daughter cells from that original cell have the Early evidence for the existence of the inactive X was the
same inactive X. In female germ cells, the inactive X chro- observation of the Barr body, named for the Canadian cyto-
mosome is reactivated as the cells enter meiosis, and in male logist Murray Barr [11]. This is a dark-staining chromatin
germ cells, the single X chromosome becomes inactive. body, present in one copy in normal females, which is the
The inactive X has properties characteristic of hetero- condensed, inactive X chromosome. Normal males have no
chromatin, with late DNA replication in the S phase of the Barr body. Initially, a buccal smear was obtained from
cell cycle and remaining condensed during interphase. patients to look for Barr bodies. Because of improved methods
Histone proteins associated with the inactive X are under- for looking at sex chromosomes and the inaccuracy of the
acetylated, and the cytosines in the CpG islands are methy- buccal smear technique, it is now considered an obsolete test.
lated [6]. A gene that controls X inactivation is XIST The sex chromatin body in polymorphonuclear leukocytes
(X-inactive-specific transcript) and is located at the takes the form of the “drumstick,” seen attached to the
X-inactivation center (XIC) at band Xq13 (Fig. 10.1). Only nucleus in approximately 2% of these cells in XX women but
the inactive X expresses this gene. XIST codes for Xist RNA, not in XY men [12].
which appears to coat or paint the inactive X chromosome Techniques for detecting the inactive X have been based
and is responsible for inactivation [7]. About 15% of genes on the fact that it is late replicating. The most commonly
on the X-chromosome escape inactivation and remain active used cytogenetic method involves the use of bromodeoxyuri-
to some degree on both X chromosomes in females [8]. An dine (BrdU) [13]. Newer methods for detecting the inactive
additional 10% of genes show variable patterns of inactiva- X involve molecular techniques often using differential
tion and are expressed to different extents from some inac- methylation analysis [14–18].
tive X chromosomes [9]. Many more genes on Xp escape Despite the fact that XIST was identified in 1990, there
inactivation as compared to Xq [6]. These genes are clus- remains an incomplete understanding of the cis- and trans-
tered and primarily map to the distal portion of the short arm acting elements that control X-chromosome inactivation and
[9]. In individuals with extra or missing X chromosomes, a determine how some genes escape it as well as how XIST is
single X remains active. However, in triploids, there are only able to associate with the chromosome from which it is
usually two active X chromosomes, suggesting a counting transcribed [19].
10 Sex Chromosomes, Sex Chromosome Disorders, and Disorders of Sex Development 177
Skewing of X-chromosome inactivation (defined as dispersed on the Y chromosome [26, 27]. One of the most
greater than 80% of cells with one of the two chromosomes likely candidate genes in this region is the testis-specific
inactivated) may occur through XIST promoter mutations or protein Y-encoded (TSPY) gene. This gene functions normally
mutation of other regulatory elements within the XIC, defined in male germ cell proliferation and differentiation but is
as primary skewing. Secondary skewing is post-X-chromosome ectopically expressed in early and late stages of gonadoblas-
inactivation selection due to slower proliferation of cells toma [28, 29].
expressing a mutant allele. Evidence supporting this is a
study by Amos-Landgraf in which 4.9% of cord blood sam-
ples but 14.2% of adult peripheral blood samples showed Numerical Abnormalities
skewing of XCI [20]. of the Sex Chromosomes
Introduction
The Y Chromosome
Numerical abnormalities of the sex chromosomes are one of
The Y chromosome is made up of several different regions. the most common types of chromosomal aneuploidy, with a
These include the pseudoautosomal regions at the distal frequency of 1 in 500 live births. This might be the result of
short and long arm, PAR1 and PAR2, the heterochromatic the fact that abnormalities of sex chromosomes have less
region on the long arm, and the recently sequenced male- severe clinical abnormalities and are more compatible with
specific region of the Y (MSY) located between PAR1 and life as compared to autosomal disorders. Reasons for this
PAR2, with 78 protein-coding genes that encode 27 dis- include inactivation of all additional X chromosomes and the
tinct proteins (Fig. 10.1). Twelve of the MSY genes are small number of genes on the Y chromosome.
ubiquitously expressed in many organs throughout the Sex chromosome disorders are more commonly diagnosed
body, while 11 MSY genes are expressed predominantly in prenatally than autosomal aneuploidies, and genetic counsel-
the testes [21]. ing for these conditions is often more complex and challeng-
The testis-determining factor (TDF) that leads to differen- ing than that for an autosomal abnormality. In the past, many
tiation of the indifferent gonads into testes is located on the individuals with sex chromosome disorders would have gone
short arm of the Y chromosome. TDF was mapped by molec- through life undetected, as they do not have physical or
ular analysis of sex-reversed patients (chromosomally female developmental problems that would have warranted a
but phenotypically male and vice versa), and the gene SRY chromosome study [30]. Women undergoing amniocentesis
(sex-determining region Y) was identified in 1990 [22]. It is and chorionic villus sampling (see Chap. 12) should be
located on the short arm of the Y at band p11.3 in the MSY informed about the possibility of detecting a sex chromosome
region (Fig. 10.1). Deletions and mutations in this gene have disorder, and when a fetal sex chromosome abnormality is
been found in some 46,XY females (see Disorders of Gonadal detected prenatally, information should be provided to the
(Testicular) Development). patient by a clinical geneticist or pediatric endocrinologist
The Y chromosome has a highly variable heterochromatic when possible [30]. Cytogenetics labs reporting results to
region on its long arm. The length of this region is usually physicians and genetic counselors discussing results with
constant from one generation to the next. A gene controlling patients should provide up-to-date and accurate information
spermatogenesis, termed the azoospermia factor (AZF), was about these conditions. It is important for couples faced with
first proposed by Tiepolo and Zuffardi in 1976 and mapped having a child with a sex chromosome disorder to see the
to the distal part of the euchromatic Yq11 region (Yq11.23) potential problems for developmental delay in the context of
[23] (Fig. 10.1). In studies of men with azoospermia or severe any chromosomally normal child having a risk of develop-
oligospermia, deletions in different intervals of Yq11 have mental delay (by definition, a 5% chance) [30].
been found, and three nonoverlapping regions or azoo- There appears to have been a trend toward a higher rate of
spermia factors (AZFa, AZFb, and AZFc, from proximal to pregnancy continuation in more recent years [31]. This might
distal Yq) have been defined as spermatogenesis loci [24] be at least in part from results of long-term studies of indi-
(see also Chap. 11). viduals with sex chromosome disorders revealing a better
A locus for susceptibility to gonadoblastoma (GBY) has prognosis than previously reported. [32]. Average percent-
been proposed on the Y chromosome based on the high ages of pregnancy terminations for sex chromosomal aneu-
incidence of gonadoblastoma in females with 45,X/46,XY ploidies reported range from 10 to 88% depending on the
mosaicism or XY gonadal dysgenesis [25]. Deletion mapping population, type of aneuploidy, maternal age, presence of
has localized this putative gene to a region near the centro- fetal abnormalities on ultrasound, and the medical profes-
mere but has raised the possibility of multiple GBY loci sional providing information [33–39].
178 C.M. Powell
Turner Syndrome Origin of the X Chromosome in Turner Syndrome

In approximately 75% of patients with 45,X, the X chromo-
45,X (and its variants) occurs in approximately 1 in 2,500 some is maternal in origin [37, 45, 46]. There is no parental
live-born females but is one of the most common chromo- age effect [46]. Although phenotypic differences have not
some abnormalities in spontaneous abortions and is esti- been found between Turner patients with a maternal or paternal
mated to occur in 1–2% of all conceptuses. The syndrome X chromosome, there may be some cognitive differences
was first described in 1938, and a report that it is caused by a particularly in memory function [47]. This has been theo-
single X chromosome appeared in 1959 [40, 41]. The older rized to be on the basis of an imprinted X-linked locus; how-
medical literature sometimes referred to the Turner syndrome ever, no human imprinted X-linked genes have yet been
karyotype as 45,XO. This terminology is incorrect and identified [19, 48].
should not be used; there is no O chromosome.
Ninety-nine percent of 45,X conceptuses result in sponta- Phenotype
neous loss, usually by 28 weeks (Fig. 10.2). These fetuses Clinical features of Turner syndrome in newborns may
usually have ultrasound abnormalities including a cystic include decreased mean birth weight (average weight
hygroma and hydrops. Although 45,X is quite lethal in the 2,800 g), posteriorly rotated ears, neck webbing (Fig. 10.3a),
fetus, those that survive to term have relatively minor prob- and edema of hands and feet (Fig. 10.3c, d), although more
lems. The reasons for this are not known, although it has than half are phenotypically normal [49]. Congenital heart
been speculated that all conceptions that survive have some defects, especially coarctation of the aorta, and structural
degree of undetected mosaicism for a normal cell line [42]. renal anomalies are common and should be checked for.
Prenatally diagnosed cases with mosaicism for a 45,X cell Most older children and adults with Turner syndrome have
line and a cell line with a second structurally normal sex short stature and ovarian failure, and variable dysmorphic
chromosome usually result in the birth of a child with a features including down-slanting eyes, posteriorly rotated
normal phenotype [43, 44]. ears, low posterior hairline, webbed neck (Fig. 10.3a), a
broad chest, short fourth metacarpals (Fig. 10.3b), and
cubitus valgus [49]. Adults with Turner syndrome have a
four- to fivefold increased rate of premature mortality,
mainly due to complications of congenital heart disease
[50]. There is a risk of aortic dilatation leading to dissection.
Regular follow-up throughout life with a cardiologist and
cardiac magnetic resonance imaging is recommended [49].
Without hormonal supplementation, there is usually lack of
secondary sex characteristics. The gonads are generally
streaks of fibrous tissue. Although germ cells normally form
in the 45,X embryo, there is accelerated oöcyte loss by
15 weeks of gestation [51]. Standard treatment includes use
of growth hormone and estrogen. It is recommended that
these patients be followed by endocrinologists familiar with
Turner syndrome. There are several published guidelines
for health supervision for children and adults with Turner
syndrome [49, 52–55].
The SHOX gene, located in the distal part of the pseudo-
autosomal region on Xp, escapes X inactivation. Haplo-
insufficiency for SHOX causes short stature and Turner
skeletal features [56–58]. A gene determining lymphedema
has been proposed at Xp11.4 [59].
Some degree of spontaneous puberty occurs in 10–30%
of girls with Turner syndrome, but fewer reach complete
puberty with menarche, and most require estrogen treat-
ment beginning at age 12 for normal secondary sex charac-
teristic development, and throughout adulthood to prevent
osteoporosis [49]. Spontaneous pregnancy may occur in
2–5% [49, 54].
Fig. 10.2 A 45,X fetus with large cystic hygroma and hydrops
Fig. 10.3 Child with Turner syndrome and low posterior hairline and webbed neck (a) and short fourth metacarpals (b). Infant with Turner
syndrome and lymphedema of the hand (c) and feet (d)
Development Mosaicism
Intelligence in individuals with Turner syndrome is average Mosaicism for 45,X and another cell line is found in 15–20%
to above average, although there is an increased risk of a of patients with Turner syndrome with lymphocyte chromo-
nonverbal learning disorder and behavioral problems. Infants some analysis. A 46,XX cell line may modify the phenotypic
can have feeding problems and developmental delay. features of the syndrome. As mentioned earlier, in order to
Problems with visual-spatial skills, working memory, execu- explain why 99% of 45,X conceptions terminate in miscar-
tive functions, and social skills can occur. Verbal IQ scores riage, it has been proposed that most surviving 45,X fetuses
are typically higher than performance IQ [55]. With appro- have some degree of mosaicism. In a study of both lympho-
priate therapeutic and educational intervention, women with cytes and fibroblast cultures in 87 patients with Turner
Turner syndrome can do well academically and socially. syndrome, mosaicism was found in 66.7% [61]. In a patient
with several typical features of Turner syndrome but normal
Turner Syndrome Variants lymphocyte chromosome analysis, analysis of another tissue
Approximately half of all individuals with Turner syndrome such as skin for fibroblast culture or buccal cell analysis
have a 45,X karyotype. The remainder exhibit mosaicism using an X-chromosome probe and fluorescence in situ
and/or structural abnormalities of the X chromosome. In a hybridization (FISH; see also Chap. 17) should be consid-
study of cytogenetic and cryptic mosaicism in 211 patients ered [61, 62].
with Turner syndrome, Jacobs et al. reported pure 45,X in In prenatal diagnosis, when multiple cells from a single
46%, 47% had a structurally abnormal sex chromosome culture are identified with a 45,X karyotype, a moderate
(41% with an abnormal X and 6% with an abnormal Y), and work-up is warranted with examination of an additional 20
7% had a 46,XX or 47,XXX cell line [60]. Two patients were cells from cultures other than the one with the initial finding
found to have cryptic X mosaicism, and none had cryptic Y or 12 colonies from coverslips other than the one with the
mosaicism. abnormality [62, 63]. Markers identified prenatally should
180 C.M. Powell
have their origin determined using X and Y centromere The frequency of occult Y mosaicism in Turner syndrome
FISH. When a small ring or marker chromosome is deter- has varied widely depending on the study and type of analysis
mined to be derived from the X chromosome, FISH with a used. One study using polymerase chain reaction (PCR)
probe for XIST should be performed. Lack of the XIST locus and Southern blot analysis found that 40% of 45,X patients
in a ring X chromosome may be associated with a more had SRY sequences [67]. Most patient samples produced
severe phenotype that includes developmental disability only a faint signal, indicating a low percentage of cells with
(see section “Ring X”). Y-chromosome material (or contamination). A more recent
study using PCR and Southern blot analysis found evidence
Mosaicism with a Y Chromosome of Y mosaicism in only 3.4% of patients with Turner syn-
Patients with 45,X/46,XY mosaicism may have external drome who were cytogenetically nonmosaic [68]. In a popu-
genitalia ranging from normal male to ambiguous to female lation study in Denmark of 114 females with phenotypic
with features of Turner syndrome. The Y chromosome is Turner syndrome, 12.2% of patients had Y chromosome
often structurally abnormal. One study of 92 prenatally diag- material by PCR analysis. Fifty percent of these patients did
nosed cases found that 95% had normal male genitalia. not have evidence of Y detected by karyotype analysis [69].
Abnormal genitalia included hypospadias, micropenis, and Nishi et al. studied 122 patients with Turner syndrome and
abnormal scrotum. In those fetuses for which pathologic compared results of nested PCR in these patients with those
studies were possible, 27% had abnormal gonadal histology, of 100 women with no known chromosome abnormality [70].
classified as dysgenetic gonads. The percentage of mosa- First-round PCR identified Y sequences in four patients
icism found in amniotic fluid samples was a poor predictor (3%); all were also found to have a marker chromosome with
of the phenotype [44]. Another study of 42 cases of cytogenetic analysis. Fourteen percent of DNA samples from
45,X/46,XY mosaicism diagnosed prenatally found pheno- 100 “normal” women showed positive amplification after
typically normal male offspring in 90%, with 10% having nested PCR. The authors hypothesized contamination with
questionably abnormal phenotypes, including three cases PCR amplification products. The possibility of microchimer-
with mixed gonadal dysgenesis [64]. In another study, three ism in these women secondary to having had a male concep-
of 27 patients with mosaic 45,X/46,XY diagnosed postna- tion or a male twin has also been raised [71]. Jacobs et al.
tally had mixed gonadal dysgenesis (a streak gonad on one found no occult mosaicism for Y in 178 patients with Turner
side and testis on the other) and normal plasma testosterone syndrome using Y-specific PCR primers [60]. Thirteen
levels. The streak gonads were removed in these patients but patients had Y-chromosome material detected by routine
the testes were not, and all three had normal puberty. Mild cytogenetic analysis of 100 cells. Only two of the thirteen
intellectual disability (mental retardation) and autism were patients had fewer than 10% of cells with a Y chromosome:
seen in four and two patients, respectively, in this series, one had 7% and one had 8%. It is likely that these would
although there might have been biased ascertainment [65]. have been detected with standard analysis of 30 cells [60].
Abnormal gonadal development including gonadal dysgen- These studies suggest that the presence of cryptic mosa-
esis, infertility, low testosterone level, and azoospermia can icism involving the Y chromosome is rare and might involve
occur in patients with 45,X/46,XY and an apparently normal less than 1% of patients. Nested PCR appears to overesti-
external male phenotype [65]. Fertility cannot be evaluated mate the frequency of Y sequences in patients with Turner
until puberty, but infertility is common. syndrome and should be avoided to prevent false-positive
Using fluorescence in situ hybridization (FISH) analysis, results, which lead to unnecessary surgical treatment in
Robinson et al. looked at the structure of the Y chromosomes these patients [70]. At this time, PCR analysis for Y sequences
present in 14 cases of Turner syndrome with at least one cell in patients with Turner syndrome does not appear to be
line with an abnormal Y chromosome [66]. Ten patients had warranted unless a marker chromosome is found. In such
a pseudodicentric Yp chromosome, two had an isodicentric instances, identification of the marker with FISH or molecular
Yq, one a pseudodicentric Yq, and one had a derivative Y analysis is critical due to the risk of gonadoblastoma. Most
chromosome (see Chap. 9). Results suggested that the marker Y chromosomes in patients with Turner syndrome
majority of Turner syndrome patients with structurally should be detected with standard G-band analysis of 30 cells.
abnormal Y chromosomes contain two copies of most of the The use of FISH analysis to look for Y-chromosome mosa-
euchromatic Y material (see the section “Structural icism in 45,X patients has been recommended [52, 62]. If a
Abnormalities if the Y Chromosome,” later). In a study of 30-cell analysis reveals an apparently nonmosaic 45,X kary-
211 patients with Turner syndrome, Jacobs et al. found a otype in patients without virilizing features, FISH analysis
clinically significant structural abnormality of the Y chromo- using X and Y probes should be performed to identify low-
some in 13 patients (6%) [60]. One hundred cells were exam- level sex chromosome mosaicism to prevent a potential
ined from each patient. No patients were found to have life-threatening cancer [62, 72]. Identification of mosaicism
45,X/46,XY mosaicism with a structurally normal Y. for a marker, ring, or isochromosome derived from the X
chromosome eliminates the need to pursue additional

investigation for Y-chromosome material. All ring and
marker chromosomes should be further analyzed with FISH
to determine their origin.
If a patient with Turner syndrome has evidence of viriliza-
tion with clitoromegaly or other masculinizing features,
mosaicism for a Y-containing cell line is likely. FISH with X
and Y centromere probes should be performed on a mini-
mum of 200 cells to detect low-level mosaicism. If lympho-
cyte analysis is normal, study of a second tissue should be
considered [62].
The risk of gonadoblastoma is increased in phenotypic
females with 45,X/46,XY mosaicism [73]. Some but not all
patients with Turner syndrome and a Y cell line will have
masculinization. In one study, gonadoblastoma was found in
one of ten patients with Y-chromosome material who had
ovariectomy performed [69]. This patient had a 45,X/46,XY Fig. 10.4 Normal X chromosome and isochromosome Xq: 46,X,i(X)
(q10) in a patient with Turner syndrome. Brackets indicate region of
karyotype. None of the patients with only PCR-detected duplication on Xq
Y-chromosome material developed gonadoblastoma. In
another study, 10 of 171 patients with Turner syndrome with-
out clitoromegaly or other evidence of masculinization had female gametogenesis. When the error occurs postzygoti-
Y-chromosome mosaicism with conventional cytogenetic cally, it is likely to be at the first postzygotic division because
analysis of at least 50 metaphase cells. Four additional 45,X/46,X,i(X)(q10)/46,XX mosaics are extremely rare [60].
patients were identified using PCR for SRY and the DYZ3 These patients are phenotypically indistinguishable from
region, two of whom subsequently had marker chromosomes those with a pure 45,X karyotype although there have been
identified cytogenetically. Of the 12 who underwent gonad- reports of an increased risk of autoimmune problems in
ectomy, gonadoblastoma was identified in 4 (33.3%), with patients with an isochromosome X [76]. In a study of 145
the youngest patient aged 2.8 years. The percentage of women with Turner syndrome, 83% with an i(Xq) had posi-
Y-bearing cells in the blood or gonadal tissue did not corre- tive thyroid autoantibodies compared with 33% of women
late with gonadoblastoma incidence. Combining their data with other karyotypes. The women with an i(Xq) chromosome
with previous studies, there was a 26.9% risk of gonadoblas- were also more likely to become hypothyroid (37.5%) and
toma in patients with 45,X and Y-chromosome material. In require thyroxine compared to the 45,X women (14%) [77].
those with gonadoblastoma, the risk of malignancy was 14%
[72]. As the risk of malignant degeneration of gonadoblas- Ring X
toma cannot be predicted, surgical removal of gonadal tissue A subset of patients with Turner syndrome, estimated at
is generally indicated for patients with Turner syndrome and approximately 15%, have one normal X chromosome and a
Y-chromosome material. The appropriate timing for this ring X chromosome, most often as a mosaic cell line [60]. In
surgery should be discussed with a pediatric endocrinologist. a study of patients with a 45,X/46,X,r(X) karyotype in whom
For those patients with a male phenotype and external testes, the parental origin of the ring could be determined, the ring
the risk of neoplasm is not as high, but frequent physical and was derived from the opposite parent from the normal X. In
ultrasound examinations are recommended [74]. one patient, there was uniparental disomy (UPD) for the X
chromosome (see Chap. 20). In two-thirds of patients, the
Isochromosome X normal X was maternal in origin, and in one-third of patients,
An isochromosome X [i(X)(q10)] (see Fig. 10.4), consisting it was paternal, as it is in 45,X patients [78]. The size of the
of two copies of the long arm (missing all or most of the ring varies from minute to nearly the size of a normal X. An
short arm), is seen in 18% of patients, either as a single cell important first step in characterizing a ring is to perform
line or, more commonly, in mosaicism with a 45,X cell line. FISH (see Chap. 17) with a painting probe for the X to
Although most i(Xq) chromosomes look monocentric, confirm that it is derived from the X and not Y [79] (see the
molecular evidence suggests that many are actually dicentric section “Mosaicism with a Y Chromosome”).
and result from breakage within the proximal short arm of a In general, ring X patients lack many of the classic
single X chromosome with subsequent reunion of the sister somatic features of Turner syndrome [80]. Those with ring
chromatids [60, 75]. The i(Xq)s are equally likely to be X often do not have lymphedema and, therefore, are less
maternal or paternal [60]. The error occurs during male or often ascertained at birth compared to 45,X individuals [81].
182 C.M. Powell
Although some have typical features of Turner syndrome, severe phenotypic abnormalities, demonstration of an inac-
others have a severe phenotype with developmental dis- tive ring X is not necessarily reassuring [90]. The etiology of
ability, facial dysmorphism, and congenital malformations. the abnormal phenotype in ring X is complex and cannot be
Some patients with a ring X and severe phenotype have based solely on the inactivation status of the ring. Size and
features similar to those of patients with Kabuki syndrome, gene content, extent of X inactivation, parental origin, and
a multiple malformation syndrome due to mutations in the timing of ring formation and of cell selection likely play a
MLL2 gene on chromosome 12 [80–83]. El Abd et al. role in the broad phenotypic variability [79].
reported a characteristic behavioral profile in five patients
with a small active ring X that included autistic-like fea- 45,X/47,XXX Mosaicism
tures, obsessive compulsive behavior, and social and com- Approximately 2% of patients with Turner syndrome have a
munication problems [84]. 45,X/47,XXX mosaic karyotype [60, 91]. A study of seven
Studies have suggested that the more severely affected girls with this type of mosaicism aged 6.1–20.4 years found
patients have smaller rings that are deleted for XIST. It has that three of seven did not require growth hormone, five of
been hypothesized that the lack of XIST causes the ring to six girls older than 10 years had spontaneous puberty, and
fail to inactivate, thus causing functional disomy for genes four of five girls older than 12 years had spontaneous menarche
present on the ring, resulting in phenotypic abnormalities with regular menstrual cycles without medication. No renal
[85–87]. Larger rings have XIST present and are preferen- or cardiac anomalies and no cognitive or behavioral prob-
tially inactivated. However, Turner et al. reported that in lems were found in this small group of patients. In general,
seven females with 45,X/46,r(X) and a XIST-negative ring, patients with Turner syndrome caused by 45,X/47,XXX
only one had a severe phenotype and this was explained by mosaicism are more mildly affected clinically with regard to
the absence of XIST expression, a large amount of Xp mate- phenotype and ovarian function [91].
rial in the ring, and, possibly, the concomitant maternal
uniparental isodisomy [88] (see Chap. 20). The remaining Marker Chromosomes in Patients with Turner
six patients had physical phenotypes consistent with Turner Syndrome
syndrome. The size of the ring X chromosome lacking XIST It is important to identify the origin of a marker chromosome
correlates with the degree of clinical severity [81, 87–89]. in a patient with Turner syndrome, because of the risk of
Those with extremely small rings have been found to have gonadoblastoma if it is made up of Y material (see the
cognitive functioning similar to those with 45,X. There could section “Mosaicism with a Y chromosome” earlier) or the
be particular gene sequences that, when functionally disomic, increased risk of phenotypic and developmental abnormali-
result in the severe physical phenotype. ties if the marker is of autosomal origin. This can be done
Other factors that could contribute to the phenotype in either with FISH or molecular techniques.
patients with small ring X chromosomes are the tissue-
limited distribution of the ring X cell line or ring formation
from an inactive X after the establishment of X inactivation. 47,XXX
In patients with an inactivated ring X chromosome, having a
larger proportion of cells with the ring was associated with Trisomy X or triple X is the most frequent sex chromosome
lower verbal and nonverbal IQ scores [88, 89]. Migeon et al. disorder present at birth in females, occurring in 1 in approx-
reported two patients with inactive ring X chromosomes, imately 1,000 live female births [92]. It was first described in
mental retardation, and a severe phenotype [90]. Cultured 1959 by Jacobs et al. [93]. Unfortunately, the term originally
fibroblasts from these patients showed a second ring in a used for this cytogenetic abnormality was “superfemale,”
small percentage of cells. The authors hypothesized that the which gives a misconception of the syndrome and is no long-
severe phenotype with an inactive X chromosome is the er in use. Due to variation in phenotype, with some individu-
result of the presence of a second ring X that was active in als very mildly affected, only approximately 10% of cases
some tissues during embryogenesis. are ascertained clinically [94].
The prognosis for patients with small ring X chromosomes
might be better than previously proposed [87]. However, a Origin
ring X chromosome appears to be associated with a substan- Most 47,XXX conceptions result from maternal nondisjunc-
tially increased risk of significant learning difficulties, requir- tion at meiosis I, and so there is an association with increased
ing special educational provision, compared to 45,X [89]. It maternal age. Two of the X chromosomes are inactivated,
might not be possible to accurately predict, prenatally, the and abnormalities could result from three active X chromo-
phenotype that will be associated with the ring X chromosomes early in embryonic development, prior to X inactiva-
some after birth. Although a relatively large, active ring X tion and/or from genes on the X chromosome that escape
(XIST not expressed) is more likely to be associated with inactivation.
Phenotype orientation. Compared to individuals with other types of sex

In contrast to the result of a 45,X karyotype, there is not a chromosome disorders, 47,XXX females seemed to have
recognizable syndrome in females with an additional X chro- the most psychological problems, including depression and,
mosome. The majority are physically normal, although there occasionally, psychoses. However, one woman attended
is a slight increase in the frequency of minor anomalies. The college, and many were able to overcome psychological
mean birth weight is at the 40th percentile, the mean birth problems and become independent, hold jobs, and marry.
length is at the 70th percentile, and the mean birth head Stability of the home environment was somewhat related to
circumference is at the 30th percentile [95]. In general, as outcome but not to such an extent as is seen in other sex
adults, these women have moderately tall stature, with an chromosome disorders [102]. In adulthood most of these
average height of 172 cm (5 ft and 7.7 in.). Pubertal develop- women were employed full time (unskilled or semiskilled
ment is normal, and most have normal fertility, although a jobs), had married, and had children [103]. In another long-
small number have ovarian dysfunction and premature term study, 47,XXX women were found to have more work,
ovarian insufficiency sometimes associated with autoim- leisure, and relationship problems as compared to a control
mune thyroid disease [96, 101, 97]. Renal and ovarian abnor- group [104]. They were found to have poorer psychosocial
malities and congenital heart defects have been reported in adaptation and more psychiatric impairment as compared to
small numbers of patients. There appears to be an increased their female siblings. However, most were self-sufficient and
risk of seizures [94]. Most have good health, although one functioned reasonably well as adults. Severe psychopathology
study found that 25% had recurrent nonorganic abdominal and antisocial behavior were rare, and there was a larger
pain as teenagers [98]. variability in behavioral phenotype than originally appreci-
There is a small but slightly increased risk of chromoso- ated [104]. In another longitudinal study of 16 girls with
mally abnormal offspring of 47,XXX women [99, 100]. 47,XXX ascertained at birth through a cytogenetic survey of
Although they do not, remarkably, appear to be at significantly consecutive newborns, 50% had speech delay and IQ scores
increased risk of having XXX or XXY children, prenatal averaged 85.3 verbal (range 67–109) and 88.3 performance
diagnosis should be offered for all pregnancies. (range 67–110). Higher IQ scores were positively correlated
with the level of parental education. All attended regular
Development schools, but most required extra help in math and reading.
Usually, 47,XXX females have normal intelligence, but Behavior problems required psychiatric referral in 25% of
most have lower IQs than their siblings. There is a great the girls [105].
deal of variability and many women have normal intelli- In a study of five girls ranging in age from 7 to 14 years
gence and are well adjusted. Precise predictions regarding with 47,XXX diagnosed prenatally, only one had motor
an individual child’s prognosis are not possible, but there and language delays and learning problems; the others had
does appear to be a risk for mild to moderate developmental normal IQs (range 90–128) and were doing well in school
problems in the areas of motor, speech and language, and [106]. Another longitudinal study by Linden and Bender of
learning [95, 101 102]. Verbal IQ tends to be lower than 17 47,XXX females, ages 7–18, initially diagnosed prena-
performance IQ, with many having problems with expres- tally, found that eleven needed academic assistance and
sive language. seven required speech therapy. Many were shy in younger
Many studies of 47,XXX females have ascertainment years but became more outgoing as teenagers, and three had
bias; however, in a group of 11 females with 47,XXX ascer- no developmental problems. Their IQs ranged from 73 to
tained at birth by unbiased screening of all newborns who 128, with a mean of 103. The girl with a 73 IQ was from a
were then followed into adulthood, most had serious patterns family with a history of learning problems [107]. Reasons
of dysfunction [102]. Most showed early delays in motor, for the difference between the two groups could be the higher
language, and cognitive development and were described as socioeconomic status and greater stability of the prenatally
shy, withdrawn, and immature, with poor coordination [95]. diagnosed group.
The full-scale IQ was 26 points lower than in normal sibling Experts in this field advocate for anticipatory guid-
controls; average IQ was in the 85–90 range, and subjects ance for these patients, emphasizing the child’s normal
were at the 24th percentile in academic achievement scores, development but remaining prepared to identify and pro-
but mental retardation was rare. Nine of the eleven needed vide early intervention when developmental problems
special education in high school either full time or part occur. Appropriate speech, occupational and physical
time, and less than half completed high school, but two therapy, educational assistance, and psychiatric treatment
achieved “A”s and “B”s, and one excelled in math. Most should be instituted as soon as a need is identified [104].
did not participate in extracurricular activities. They were There have been several recent reviews of 47,XXX [94,
described as socially immature. All had heterosexual 105, 106].
184 C.M. Powell
Variants with Additional X Chromosomes association with increased maternal age in those with mater-
nal meiosis I errors [127]. A recent study found the addi-
48,XXXX tional X chromosome to be paternal in approximately 25%
As compared to 47,XXX, there is almost always mild to of cases, and these were associated with increased paternal
moderate intellectual disability (mental retardation) in indi- age, although other studies have not supported this finding
viduals with 48,XXXX or tetrasomy X syndrome, with an [128, 129].
average IQ of 60, ranging from less than 30 to 75 [110]. One Maternal nondisjunction during meiosis I leads to unipa-
individual was reported to have a normal IQ [111]. Phenotypic rental heterodisomy for the X chromosome, while an error
features include mild hypertelorism, epicanthal folds, during meiosis II results in uniparental isodisomy (see Chap.
micrognathia, and midface hypoplasia [112]. Tall stature is 20). If the father contributes the X, there are two different
common, with an average height of 169 cm (approx. 5 ft X chromosomes with different parental origins. So far, stud-
and 6 in.) [110]. Skeletal anomalies include radioulnar ies have found contradicting results regarding differences in
synostosis and fifth finger clinodactyly. Incomplete develop- phenotype and development between KS males with a pater-
ment of secondary sex characteristics could occur with scant nal versus a maternal additional X [130]. No phenotypic dif-
axillary and pubic hair and small breasts, and some patients ferences between heterodisomy and isodisomy for the X in
have gonadal dysgenesis [113]. Speech and behavioral prob- KS have yet been found. The repeat length and inactivation
lems are common. Fertility is reduced, and primary ovarian status of the androgen receptor at Xq11.2-q12 has also been
failure has been reported, although some have had chromo- studied as to its correlation with the KS phenotype, but so far
somally normal offspring [114]. there have been no definitive findings [130].
49,XXXXX Phenotype
Nondisjunction in successive meiotic divisions is the probable 47,XXY males have taller than average stature, with mean
mechanism for pentasomy X syndrome, and molecular height at the 75th percentile, and might have a eunuchoid
studies have shown that, at least in some cases, the extra X build with long limbs and pear-shaped hips, although there is
chromosomes are all maternally derived [115, 116]. a great deal of phenotypic variability [98]. Head circumfer-
Phenotypic features seen in penta-X females include intra- ence during infancy is usually average but, beginning at age
uterine growth restriction, short stature, microcephaly, up- 4 years, tends to be below the mean for age, although gener-
slanting palpebral fissures, low hairline, and coarse, Down ally within normal limits [131]. Testicular and penile size is
syndrome-like facial features. Congenital heart defects, renal usually small during childhood, although prepubertal pheno-
anomalies, hip dysplasia, and joint subluxation have been type is often unremarkable. Gynecomastia occurs in up to
reported [117–120]. One individual with penta-X and hyper- 50% of 47,XXY males during adolescence. Most enter
IgE syndrome has been reported [121]. An infant with laryn- puberty normally, although there is usually inadequate testos-
gomalacia and extreme neonatal hypotonia has been reported terone production and most require testosterone supplemen-
with mosaicism for tetrasomy X and pentasomy X [122]. tation. Measurement of serum testosterone level in male
Most have moderate developmental disability (IQ range infants with Klinefelter syndrome at 6 weeks of age can help
20–75, average IQ 50) and are described as shy and coopera- predict the amount of natural testosterone production these
tive [110, 120]. There have been no reports of pregnancy in patients will have.
women with this chromosomal aneuploidy [110]. Testes are small in adulthood, and there is hypergonado-
tropic hypogonadism. Almost all have infertility with absent
spermatogenesis, tubular hyalinization, and Leydig cell
47,XXY (Klinefelter Syndrome) hyperplasia. Most are born with spermatogonia, but during
early puberty, the spermatogonia are thought to undergo
Klinefelter syndrome (KS) was the first sex chromosome massive apoptosis along with damage to the germinal epithe-
disorder to be described and its cytogenetic cause identified, lium [132]. Many men with KS are diagnosed in adulthood,
and is the most common cause of hypogonadism and infer- with a chief complaint of infertility, but based on a popula-
tility in males [120, 123–125]. It is found in approximately 1 tion study, as many as 74% might never be diagnosed [30].
in 575–1,000 newborn males, although only 25% are ever There have been more than 200 reports of pregnancies
diagnosed [30, 92, 126]. resulting from intracytoplasmic sperm injection (ICSI) from
nonmosaic 47,XXY patients [130]. In one study of 38 preg-
Origin nancies, there were 34 karyotypically normal neonates, two
In one study, the extra chromosome arose at paternal meiosis karyotypically normal pregnancy losses, one healthy unkary-
I in 53% of patients, 34% at maternal meiosis I, 9% at mater- otyped neonate, and one 47,XXY prenatally diagnosed fetus
nal meiosis II, and 3% from postzygotic errors. There is an [133] (see also Chap. 12).
Development Klinefelter and XYY syndrome (see the section 47,XYY).

Boys with Klinefelter syndrome have been reported to have The incidence is estimated at 1 in 50,000 male births [137].
decreased muscle tone during infancy, delayed speech and Men are tall statured, with adult height above 6 ft, a eunu-
language skills, and an increased incidence of reading choid body habitus, and long thin legs. There is hypergo-
disability and dyslexia [134]. IQs are lower than in controls nadotropic hypogonadism, small testes, and sparse body hair.
and compared to siblings, with the average between 85 and Gynecomastia occurs frequently [110].
90, although there is a wide range [98]. Verbal IQ is usually Most 48,XXYY patients have mild intellectual disability,
lower than performance IQ. The majority require special although IQs ranging from 60 to 111 have been reported.
help in school, especially in the areas of reading and spell- Speech and motor delays and hypotonia are seen in 75%,
ing. They are often described as awkward with mild neuro- with average age at ambulation of 18 months [138].
motor deficits, shy, immature, restrained, reserved, and Developmental outcomes described range from milder
lacking confidence [98]. A group of 13 males with Klinefelter language-based learning disability (reading disability, dys-
syndrome, diagnosed as newborns and followed into adult- lexia) to mild mental retardation in 30% of patients. Overall
hood, were said to have struggled through adolescence with adaptive functioning is often more impaired than would be
limited academic success but were able to function indepen- expected based on IQ scores [139]. Psychosocial and behavior
dently in adulthood [102]. Most needed at least some special problems are generally more severe than in 47,XXY indi-
education help in school; nine completed high school, and viduals, although patients without significant behavior prob-
four went to college. All were heterosexual. Some had lems have been reported [110, 140]. Four patients with
psychological problems, including conduct disorder and 48,XXYY observed over a 10-year period had psychiatric
depression, and difficulties with psychosocial adjustment. disorders, including aggressiveness, self-injury, and mental
A stable and supportive family environment was found to retardation [141].
correlate with better outcome [102]. Another long-term study 48,XXYY is not associated with advanced parental age.
of this group found that as adults, ten of eleven were employed Nondisjunction in both the first and second male meiotic divi-
full time and eight had married [103]. sions leading to an XYY sperm has been hypothesized [142].
In a group of five boys ranging in age from 7 to14 years
who had been prenatally diagnosed with 47,XXY karyo- 48,XXXY
types, there were fewer language and motor deficits in child- This is a rare condition, with more abnormal features and
hood as compared to the postnatally diagnosed group, and all developmental disability as compared to 47,XXY. It was first
were doing well in school. IQs ranged from 90 to 131. The described by Barr in 1959 [143]. Stature is normal to tall, and
reason for the better outcome might be the result of environ- dysmorphic features include epicanthal folds, hypertelorism,
mental and other genetic factors [108]. In a long-term study protruding lips, prominent mandible, radioulnar synostosis,
of 14 prenatally diagnosed boys with Klinefelter syndrome and fifth finger clinodactyly. There is hypergonadotropic
followed to 7–18 years, ten had average school performance, hypogonadism and hypoplastic penis in 25% of patients, and
eight required educational assistance, three had attention gynecomastia is common. Testosterone therapy has been
deficit disorder, and four had speech problems. In general, shown to be beneficial. Affected individuals are infertile.
they were healthy and had pleasant personalities [109 ] . Males with this condition have mild to moderate intellec-
A study of 50 boys with KS diagnosed prenatally and through tual disability, with most in the 40–60 IQ range, although an
clinical ascertainment found problems with complex language individual with an IQ of 79 has been reported [110]. Most
processing, impaired attention, and motor function [134]. have speech delay, slow motor development, and poor
An excellent review of the developmental phenotype in KS is coordination. Behavior is immature, with personality traits
by Boada et al. [135]. described as passive, pleasant, placid, and cooperative [110].
In summary, individuals with Klinefelter syndrome can A 10–15 point decrease in IQ for each additional X chromo-
have productive and fulfilling lives but often require special some has been reported, although there is still wide variability
assistance in school and could have social and behavioral in outcomes [144].
problems. Early evaluation and intervention are strongly
recommended because the prognosis can be improved signi- 49,XXXXY
ficantly with appropriate therapeutic intervention [136]. 49,XXXXY has an approximate incidence of 1 in 85,000
male births, and more than 100 cases have been described in
the literature since this karyotype was first reported by
Variants with Additional X or Y Chromosomes Fraccaro et al. in 1960 [145]. It results from nondisjunction
of the X in maternal meiosis I and II [146].
48,XXYY Common features include low birth weight, short stature
This is the most common variant of Klinefelter syndrome in some patients, craniofacial features consisting of round
[110]. There is overlap between this condition and both face in infancy, and coarsening of features in older age, with
186 C.M. Powell
hypertelorism, epicanthal folds and prognathism, and a short, polymorphic microsatellite DNA markers have shown a
broad neck [110, 142]. Congenital heart defects are found in maternal origin of extra X chromosomes in 30 cases of
15–20%, with patent ductus arteriosus the most common 49,XXXXY and 49,XXXXX [115, 116, 157, 159–162,
defect described, but atrial ventricular septal defects and 165–168 ]. There does not appear to be a maternal age effect as
tetralogy of Fallot are also reported [110, 147, 148]. Skeletal is seen for 47,XXX and 47,XXY. Two cases of 48,XXYY have
anomalies include radioulnar synostosis, genu valgus, pes been shown to arise from paternal nondisjunction [116, 162].
cavus, and fifth finger clinodactyly. Muscular hypotonia and For cases of 48,XXXY studied with Xg blood groups or
hyperextensible joints are often present. There is hypergo- other polymorphic markers, two are maternal and five are
nadotropic hypogonadism [110]. Genitalia are hypoplastic paternal in origin [45, 116, 146, 159–164]. Nondisjunction at
with short penile length, small testicular volume, and cryp- first and second meiotic divisions is proposed versus fertil-
torchidism [110, 139]. Because of the distinctive phenotype, ization of an ovum by an XY sperm followed by postzygotic
some authors have suggested classifying this condition sepa- nondisjunction, because mosaicism has not been detected in
rately from Klinefelter syndrome [139, 148]. these patients [116].
Mild to moderate intellectual disability (mental retarda-
tion) is seen in most patients, although reported IQs range
from 20 to 78. Language development is most severely 47,XYY
impaired with some patients having speech aphasia [148].
Behavior has been described as timid, shy, pleasant, anxious, Origin
and irritable [110]. Testosterone replacement therapy has One in 800–1,000 males has an extra Y chromosome [92].
been found to be beneficial in some patients, with improve- This arises through nondisjunction at paternal meiosis II.
ment in attention and behavior [149]. Recent studies have
suggested that there may be less significant cognitive Phenotype
impairment in these individuals than previously thought Males with 47,XYY tend to have normal birth length and
[150]. In 13 children aged 2–7 years, the mean nonverbal IQ weight, but when older, most are above the 75th percentile in
was 87.1 [139]. height. Minor anomalies are found in 20% of patients, but
the rate of major malformations is not increased. Most infants
49,XXXYY are normal in appearance [152]. Pubertal development is
Only five cases of live-born males with this sex chromosome usually normal, although onset of puberty in one group of
disorder have been described. Physical features include tall patients studied was approximately 6 months later than aver-
stature, dysmorphic facial features, gynecomastia, and hypog- age for males with no sex chromosome disorder. Patients
onadism. All have had moderate to severe mental retardation, often have severe facial acne, are described as tall and thin,
with passive but occasionally aggressive behavior and temper and have good general health. Sexual orientation is typically
tantrums. One patient had autistic-like behavior [110]. heterosexual. Individuals are described as somewhat awkward
and have minor neuromotor deficits [98]. Most have normal
47,XY,i(X)(q10) fertility and are able to father children. It has been estimated
There have been reports in the literature of 19 individuals with that only 12% of men with XYY are ever diagnosed. Half of
Klinefelter syndrome with an isochromosome Xq or i(Xq) those identified were karyotyped because of developmental
[151]. The phenotype is very similar to typical Klinefelter syn- delay and/or behavior problems [30].
drome with azoospermia, hypogonadism, and gynecomastia,
but height is in the normal range, presumably due to a normal Development
copy number of the pseudoautosomal region on Xp/Yp, Intelligence is normal, although there is an increased inci-
including the SHOX gene. Using microdissection, Hockner dence of learning disabilities. There have been two groups of
et al. demonstrated the i(Xq) chromosome observed in their patients with 47,XYY studied long term: one diagnosed in a
patient was an isodicentric, maternally derived, true isochro- newborn screening program and the second diagnosed pre-
mosome and not the result of an Xq;Xq translocation [151]. natally. The latter group of patients comes from families with
These findings are consistent with the proposed mechanism of an above-average socioeconomic status. The first group had
origin for most of the i(Xq) chromosomes observed in Turner an IQ range of 93–109, and all required part-time special
syndrome females (see section “Isochromosome X” above). education. The second group had an IQ range of 109–147,
and all were reported to be getting “A”s and “B”s in school.
Origin of Extra Chromosomes IQ is usually somewhat lower than in siblings [98, 108].
The extra chromosomes in polysomy X syndromes most Most older boys attend college or have jobs after high school.
likely arise from sequential nondisjunction events during Longer follow-up of the boys in the second group found that
either maternal or paternal gametogenesis. Studies using five of 14 required extra assistance in school for academic
difficulties and two were in special education programs for aggressive behavior, were reported [110, 158–162]. In a patient
the learning disabled [109]. Overall, school performance in followed from age 4 months to 26 years, adult height was at
this group was above average. IQ scores were available for the 80th percentile, walking began at 2 years, and IQ at age
six of the boys and ranged from 100 to 147. Most were 7 years was 75, but he was described as having mild to moder-
involved with sports, although five were described as poorly ate mental retardation as an adult with impulsivity, behavioral
coordinated. Two had serious emotional problems. Five had disturbance, and difficulties with social interaction [162].
no academic or behavior problems. In general, there was
wide variability of development and adaptation [109].
Delays in speech and language development occur in Sex Chromosome Aneuploidy and Age
some boys. An increased rate of autism spectrum disorder in
boys with XYY has been reported [153]. In a systematic Increased aneuploidy with advancing age was first reported
literature review, Leggett et al. found that although males by Jacobs et al. in 1961 [169]. This was subsequently found
with XYY had lower IQ scores than expected for their social to be the result of the loss of the X chromosome in females
background, they were not impaired in relation to general and of the Y in males [170]. Premature centromere division
population norms. Although some have problems with reading, in the X chromosome and loss through anaphase lag and
others report reading as a relative strength, and some are formation of a micronucleus are one proposed mechanism in
noted to have proficiency in science and mathematics [105]. females [171, 172]. This is supported by the finding that
Hyperactive behavior, distractibility, temper tantrums, hyperdiploidy for the X is much less common than mono-
and a low frustration tolerance are reported in some boys in somy, which would not be expected if nondisjunction was
late childhood and early adolescence. Aggressiveness is not the mechanism [173]. It is usually the inactive X chromo-
common in older boys. Although early studies raised the some that is missing in monosomy X cells [174]. Another
possibility of an increase in criminal behavior in these indi- proposed mechanism is telomere shortening, as telom-
viduals, recent studies have shown that although there is a eres play a key role in chromosome segregation [175].
higher percentage of males with 47,XYY in prisons than in X-chromosome aneuploidy is not observed in bone marrow
the general population, there was not an increase in violent preparations from older women but is seen in phytohemag-
behavior in these individuals [154]. Differences in rates of glutinin (PHA)-stimulated peripheral blood lymphocytes.
incarceration may be due to the lower intelligence of some Although some early studies suggested an increase in sex
XYY males. A study of a newborn cohort of XYY males chromosome aneuploidy in women with a history of repro-
followed into adulthood reported a higher proportion with ductive loss, recent studies have shown that this is probably
antisocial behavior than their peers, but there was no statistical not true and that it is purely a phenomenon of aging [173, 176].
difference when diagnostic criteria for antisocial behavior In a prospective study of 11 women from 83 to 100 years of
disorder was used and compared with a group of unaffected age, Jarvik et al. found a fourfold increase in X-chromosome
males [105, 155]. loss after 5 years, compared with the initial level [177].
The condition is clearly variable. Most blend into the Galloway and Buckton found a tenfold increase in
population as normal individuals. Better outcomes seem to X-chromosome aneuploidy in women aged 25–35 years com-
be associated with a supportive, stable environment. pared with those between 65 and 75 years [170]. Between 30
and 55 years, the rate of hypodiploid cells was 3–5% in
females, increasing to 8% at age 70 years. This holds true for
Variants with Additional Y Chromosomes the loss of any chromosome, but the most common was loss
of an X chromosome. It should be noted that there is variabil-
(See also the section “48,XXYY” presented earlier). ity of sex chromosome loss between individuals of the same
age, so that what is “normal” aneuploidy at a specific age is
48,XYYY impossible to predict. This makes it difficult to interpret the
There is no consistent phenotype for males with two addi- clinical implications of X-chromosome loss seen in an older
tional Y chromosomes. Mild mental retardation to low normal woman who can also have features of Turner syndrome such
intelligence and sterility has been described [156, 157]. as ovarian failure and/or infertility. Because the age-related
loss is limited to peripheral blood lymphocytes, analysis of
49,XYYYY other tissues such as skin fibroblasts or buccal cells can be
Five nonmosaic cases of 49,XYYYY have been reported. helpful in clarifying these situations. Russell et al. studied
Facial features include hypertelorism, low-set ears, and metaphase cells from cultured peripheral blood lymphocytes
micrognathia. Skeletal abnormalities include clinodactyly, in 655 females ranging in age from birth to 80 years and
radioulnar synostosis, scoliosis, and spina bifida occulta. found that below age 16 years there is 0.07% X-chromosome
Mental retardation and speech delay, along with impulsive and loss and above age 65 years there is 7.3% X-chromosome
188 C.M. Powell
loss. The authors provide their data as a reference graph to following paragraphs. “High-resolution” chromosome anal-
illustrate normal percent X-chromosome loss based on num- ysis should be performed on females manifesting X-linked
ber of cells counted (ranging from 30 to 60 cells) [178]. disorders to look for a structural X abnormality. Chromosome
Age-associated loss of the Y chromosome in men is found microarray can also be helpful to look for X-chromosome
more often in bone marrow than in peripheral blood, and it imbalance (see Chap. 18).
approaches the rate generally seen in peripheral blood in The molecular X-inactivation pattern seems to correlate
women [173]. Studies of bone marrow preparations have with phenotype in women with structural abnormalities of
shown that Y-chromosome loss was restricted to males over the X chromosome. Completely nonrandom X inactivation
age 40–50 years, with a frequency of 8–10% of cells [179, of the abnormal X is generally associated with a normal
180]. For men of varying ages with no hematological malig- phenotype, whereas those with skewed or random inactiva-
nancies, 7.7% had Y-chromosome loss; it was rare below age tion patterns usually have nonspecific intellectual disability
50 years but was found in 20% of men above 80 years of age. (mental retardation) and/or congenital abnormalities. The
The percentage of Y-negative cells ranged from 10 to 100% X-inactivation status of women with structurally abnormal
[180]. X chromosomes and an abnormal phenotype should be
Most studies comparing age-related sex chromosome assayed as part of a routine clinical workup. The phenotype
aneuploidy were done on metaphase preparations and are, could be correlated with differences in X-inactivation ratios
therefore, at risk for preparation aneuploidy. Guttenbach [183]. There have been very few reports on the use of prena-
et al. performed an in situ study of lymphocyte interphase tal X-inactivation studies in amniotic fluid or CVS [184]
nuclei to look at sex chromosome loss and aging [181]. In (see also Chap. 12). Studies comparing prenatal and post-
males, the rate was 0.05% up to age 15 years, 0.24% in 16- to natal analysis of X inactivation and their correlation with
20-year-olds, and then steadily increased to 1.34% at age phenotypic and developmental outcomes are needed before
76–80 years. The mean value of monosomy X cells in these could be used to give prognostic information in female
females was 1.58% in 0- to 5-year-olds and increased to fetuses with X-chromosome abnormalities.
4–5% in women over 65 years. Only women over 51 years of
age showed a distinct age correlation. This study also found
no difference in aneuploidy rates between cultured and non- X;Autosome Translocations
cultured cells [181]. Bukvic et al. performed analysis of sex
chromosome aneuploidy in interphase cells of 16 centenar- Balanced Translocations
ians and found loss of Y signal in 10% of cells in males com- In females, balanced X;autosome translocations can be
pared to 1.6% of cells in younger control men and loss of X divided into four phenottypic categories: normal phenotype
signal in 22% of females compared to 1.7% of cells in young with or without history of recurrent miscarriage, gonadal
women [182]. dysfunction with primary amenorrhea or premature ovarian
These findings should be considered when analyzing failure (POF), a known X-linked disorder, or congenital
peripheral blood chromosomes in older females and bone abnormalities and/or developmental delay [185]. The reasons
marrow from older men, in order to avoid misinterpretation for the variable phenotypes are complex and not fully under-
of normal age-related aneuploidy as clinically significant stood, making genetic counseling in cases of prenatal detec-
mosaicism or acquired changes. tion of these translocations very difficult.
Translocations involving the X chromosome and an auto-
some often lead to primary or secondary ovarian failure and
Structural Abnormalities of the X Chromosome sometimes Turner syndrome-like features if the translocation
occurs within the critical region of Xq13-q26 [186–188].
In addition to the isochromosome Xq commonly found in There are several different hypotheses concerning the cause
patients with Turner syndrome, the X chromosome can be of gonadal dysfunction in these cases, including disruption
involved in translocations, both balanced and unbalanced, of POF-related genes, a position effect resulting from local
and can also have deletions and duplications (see Chap. 9). alteration of chromatin caused by the translocation, and
Structural abnormalities of the X in males are generally incomplete pairing of X chromosomes at pachytene [186–190].
associated with more severe phenotypic manifestations than In cases in which the derivative X chromosome is inactive,
in females. This is partly explained by preferential inactiva- inactivation will spread from the translocated X segment to
tion of the structurally abnormal X in cases of duplications the attached autosomal material, where it will inactivate
or deletions or in unbalanced X;autosome translocations in genes. The other X-chromosome segment will remain active.
females. In cases of balanced X;autosome translocations, There is incorrect dosage of both autosomal and X-linked
there is usually preferential inactivation of the normal X genes in these cells, with functional autosomal monosomy
chromosome. Theories explaining this are discussed in the for the derived (X)t(X;autosome) chromosome that contained
the X-inactivation center and functional X-chromosome X;autosome translocation carriers had a significantly higher
disomy for the portion of the X chromosome translocated number of abnormalities, including developmental delay
onto the active (autosomal) reciprocal translocation product and learning problems, than would be expected from litera-
[191]. There is strong selection against such cells. In general, ture review [185]. Those with congenital anomalies and/or
the normal X is preferentially inactivated in approximately developmental delay showed random X-chromosome break-
75% of such patients [191, 192]. When the translocation dis- point distribution. De novo translocations were significantly
rupts a gene located on the X chromosome, a female with more likely to be associated with an abnormal outcome
such a translocation could manifest a disease condition (18 of 19 cases), suggesting that de novo status versus break-
[193, 194]. Any mutated genes on the derivative X chromo- point location is the most important risk factor in predict-
some will be fully expressed, as they would be in a male [5]. ing phenotypic outcome [185].
Several X-linked genes have been mapped in this way. Some studies have indicated that in those patients with
A “critical region” determining normal ovarian function phenotypic and/or developmental abnormalities, the translo-
has been hypothesized at Xq13-Xq26 [188, 195]. The majority cated X was late replicating (inactive) [200]. However,
of females with balanced translocations with breakpoints in Waters et al. did not find an association with aberrant late
this region usually have premature ovarian failure (secondary replication and abnormal phenotype. Eight of their patients
amenorrhea associated with elevated gonadotropin levels showed a deviation from the expected pattern of consistent
before the age of 40 years). Although there have been several early replication of the derived X chromosome and late
candidate genes for POF identified in this region, molecular replication of the normal X chromosome. Five of these
characterization of the translocation breakpoints of women patients were phenotypically normal [185].
with balanced translocations involving the critical region has Because of variability in X inactivation from one person
often shown no gene disruption [196, 197]. This supports the to another with the same X;autosome translocation, it is
hypothesis that a position effect secondary to chromatin possible for a phenotypically normal mother to have a
alteration or pairing abnormalities at meiosis causes ovarian daughter with phenotypic abnormalities and intellectual
dysfunction. disability (mental retardation) even though both carry the
It has been thought that the majority of females with same such rearrangement. This can be because of skewed
balanced X;autosome translocations with breakpoints above (nonrandom) X inactivation in the former and random X
the X-inactivation center at Xq13 are phenotypically normal inactivation in the latter, leading to functional X disomy and
[198, 199]. Schmidt and DuSart found that most X;autosome functional autosomal monosomy in some cells. This is esti-
translocation patients with phenotypic abnormalities or mated to occur in approximately 25% of females with
developmental delay had breakpoints clustered in the subte- X;autosome translocations [193].
lomeric bands Xp22 and Xq28 [191]. This was thought to be A fertile woman with a balanced X;autosome transloca-
the result of persistence of cells with inactivation of the tion is at risk for having offspring with an unbalanced rear-
translocated X in these patients. However, in a study by rangement (Fig. 10.5). There is also the risk that even
Waters et al. that reported 104 cases ascertained from balanced offspring could be abnormal as a result of random
cytogenetics laboratories in the United Kingdom, female or skewed inactivation of the abnormal X in a female child
Fig. 10.5 Balanced reciprocal translocation between the short arms of daughter with an unbalanced translocation consisting of a normal X, the
chromosomes X and 3: 46,X,t(X;3)(p11.3;p21.2). Brackets indicate derivative X, and two normal chromosomes 3 (partial monosomy Xp
regions involved in translocation on the derivative chromosomes. The and partial trisomy 3p)
patient was a 30-year-old clinically normal, fertile female who had a
190 C.M. Powell
or by disruption of a functional gene on the X in a male. spread of gene silencing [209]. The spreading of late replication
The risk for a female with a balanced X;autosome transloca- is often incomplete and might skip some autosomal bands
tion to have a live-born child with a structural and/or func- and affect others [210]. This suggests that autosomal chro-
tional aneuploidy has been estimated at 20–40% [192]. matin does not transmit or maintain the inactivation signal as
Phenotypic abnormalities can range from mild effects to efficiently as the X chromosome [209].
severe intellectual disability (mental retardation) and birth Sharp et al. reported a family with both a balanced and
defects. unbalanced (X;10) translocation segregation [209]. A female
Males with balanced X;autosome translocations are with the unbalanced form was phenotypically normal except
usually phenotypically normal but almost all are infertile for secondary amenorrhea. Although the derivative X chro-
[201] (see also Chap. 11). There have been reports of mosome was late replicating, the late-replicating region
severe genital abnormalities in males with such transloca- extended only to the X;autosome boundary and did not
tions and of multiple congenital anomalies in a man with an appear to spread into the translocated segment of 10q.
apparently balanced (X;6) translocation inherited from his However, transcriptional analysis showed that the translo-
mother [202, 203]. As noted earlier, there is also a risk of an cated segment of 10q was mostly inactive, consistent with
X-linked recessive disorder because of disruption of a gene the phenotype of the patient. There have been several other
by the translocation. reports of patients with X;autosome translocations with mild
Further characterization of breakpoint regions in phenotypes in which no spreading of late replication into the
X-chromosome rearrangements has revealed that although attached autosome was observed [211, 212]. This suggests
some interrupt specific genes, others are in gene-poor that silencing of autosomal genes by X inactivation can occur
regions. X-linked gene interruption is not a common finding without apparent delay in the replication timing of the
in patients with POF and X;autosome translocations. Two surrounding chromatin. The use of replication-timing studies
critical regions on Xq associated with POF have been pro- to evaluate the extent of spread of inactivation in X;autosome
posed. Critical region I at Xq21 has a low meiotic recombi- translocations can be misleading and should not be used to
nation frequency, is gene poor, is high in repetitive sequences, make predictions of phenotype [209]. In a study of five cases
and is close to the X-inactivation center [204]. Translocations with X;autosome translocations, there appears to be some
in this region could cause POF as a result of a position effect correlation between the pattern of gene silencing and clinical
of autosomal genes important in ovarian function or as a phenotype [15]. However, use of such techniques for prog-
result of meiotic pairing abnormalities [205, 206]. Deletions nostic purposes on a clinical basis awaits further studies.
in this region rarely cause POF. Critical region II, at Cytogenetic features such as depletion of histone acetylation
Xq23-q28, has different properties. Translocations involving and H3 lysine 4 dimethylation provide more reliable indica-
this region could cause POF because of a position effect of tors of the extent of spread of X inactivation than replication-
X-linked genes. Since deletions in this region are frequently timing studies [15].
associated with POF, haploinsufficiency for missing or inter- Prenatal detection of an unbalanced X;autosome translo-
rupted genes in this region could be the cause. cation presents a difficult genetic counseling problem (see
Chap. 21). Although there have been reports of affected
Unbalanced Translocations females having only secondary amenorrhea or mild develop-
In females with unbalanced X;autosome translocations, the mental delay, many have had a more severe phenotype with
abnormal X is generally inactive if the X-inactivation center mental retardation and birth defects [15, 200, 209, 213].
is present, probably secondary to selection against cells with In males with unbalanced X;autosome translocations,
an autosomal imbalance and functional X disomy. If the there is in utero lethality or, if they survive, multiple con-
X-inactivation center is not present in the translocated genital anomalies and intellectual disability (mental retarda-
segment, phenotypic abnormalities usually result from such tion) [214].
imbalances and can include intellectual disability (mental Functional disomy for the distal long arm of the X caused
retardation) and multiple congenital anomalies [207]. There by unbalanced X;autosome translocations involving Xq28
have also been patients described who have unbalanced appears to cause a distinctive phenotype [215] (see also the
X;autosome translocations but no phenotypic abnormalities section on “Xq Duplications” later).
and only mild behavioral problems [208].
Earlier studies relied on replication timing to investigate
inactivation in X;autosome translocations. The translocated Deletions of Xp
autosomal material can become delayed in its replication
timing, and this has been used to examine the extent of the Males with deletions of the short arm of the X show con-
spread of X inactivation in such cases. It has recently been tiguous gene syndromes characterized by different combina-
demonstrated that late replication is a poor correlate of the tions of phenotypes, depending upon the location and length
of the deletion [216]. X-linked ichthyosis, Kallmann syndrome Xq13 to Xq25 had both average and short stature, suggesting
(anosmia and hypogonadism), intellectual disability (mental variable inactivation of growth genes in Xp or proximal Xq
retardation), and chondrodysplasia punctata (skeletal dys- [226]. Deletions in various regions of the long arm are some-
plasia) are seen in males with deletions involving distal Xp. times associated with gonadal dysgenesis or POF. Females
Deletions in Xp21 cause a contiguous gene syndrome of with terminal deletions originating at Xq13 are more likely
Duchenne muscular dystrophy, retinitis pigmentosa, adrenal to have complete ovarian failure, whereas those with dele-
hypoplasia, mental retardation, and glycerol kinase deficiency tions at Xq24 might have POF [221]. In a series of women
[217]. Larger Xp deletions in males are lethal. with Xq deletions ranging from Xq13.3 to Xq27 reported by
In females, there is usually preferential inactivation of the Maraschio et al., seven of eight patients had secondary amen-
structurally abnormal X when the deletion is in or proximal orrhea. One woman with deletion at Xq27 had fertility and
to Xp22.1. In those with breakpoints in Xp22.3, the normal menopause at 43 years [227]. Clinical features of Turner
and abnormal X can be active in various proportions of cells syndrome are less common in Xq as compared to Xp dele-
[218]. Females with Xp deletions do not usually manifest tions but more common in patients with deletions proximal
any of the recessive disorders due to presence of a normal X to Xq25 [227, 228]. The likelihood of an abnormal pheno-
chromosome, although almost all have short stature and type in a female with an Xq deletion is low, although primary
some have phenotypic features of Turner syndrome. Short or secondary ovarian failure is likely. Recent studies using
stature in these patients is likely the result of haploinsufficiency molecular techniques to characterize breakpoints in Xq dele-
for the SHOX gene, within the pseudoautosomal region on tion cases have reported that normal fertility is more likely if
Xp [56, 219]. Turner syndrome features could include vari- the deletion is in critical region I (Xq21) but most women
able skeletal anomalies associated with SHOX deletion and with deletions in critical region II (Xq23-Xq28) have ovarian
soft tissue anomalies such as nuchal webbing and low poste- failure [205]. Women with Xq deletions and fertility should
rior hairline reported in some patients with Xp11.1 terminal be advised about their 50% risk of passing the abnormal X to
deletions possibly related to a proposed lymphedema critical male offspring, with likely miscarriage or severe phenotype
region in Xp11.4 [43, 59]. depending on the size and location of the deletion.
Females with terminal deletions at Xp11.1 usually have
complete ovarian failure, although in a series reported by
Ogata et al., almost 50% of those with deletions in this region Xp Duplications
had spontaneous puberty and one had fertility [220]. Females
with terminal deletions originating at Xp21 are more likely Duplications of Xp involving bands p21.2–21.3, plus a Y
to show premature rather than complete ovarian failure, chromosome, have been reported in patients who were
although they may have normal fertility [220, 221]. The phe- phenotypic females, suggesting a sex-determining gene
notypes associated with Xp deletions can vary, even within locus on Xp [229, 230]. These patients also had mental retar-
the same family [222]. This is most likely due to variable X dation and multiple anomalies. This area of the X has been
inactivation and modifying genes. termed the dosage-sensitive sex reversal (DSS) region (see
Studies have shown that most de novo Xp deletions origi- the section “46,XY Disorders of Sex Development” later).
nate on the paternal chromosome [218]. Uniparental disomy Dosage-sensitive sex reversal is the result of duplication of
(UPD; see also Chap. 20) for the deleted and nondeleted X the DAX1 gene, which, when deleted or mutated, leads to
chromosomes was not found in a study of 25 females with congenital adrenal hypoplasia [231]. Males with duplications
Xp deletions [218]. involving more distal Xp have been reported with mental
Interstitial deletions of Xp in females can have intrafa- retardation and autism but without sex reversal [232].
milial phenotypic variability, even when the involved genes Both normal and abnormal phenotypes, and normal fertility
escape X inactivation [223]. The cause of this variability is as well as amenorrhea, have been reported in females with
not known. Xp duplications and one normal X chromosome [229, 230,
233]. The abnormal phenotype—including Turner syndrome
features, short stature, seizures, and amenorrhea, but normal
Deletions of Xq intelligence—was seen in a female with complete inactiva-
tion of the duplicated X, suggesting that random inactivation
Large Xq deletions in males are not compatible with survival. was not the cause [233]. An interstitial duplication at
Smaller deletions are associated with severe phenotypes [224]. Xp11.1-p21.2 was reported in a female with macrocephaly,
Deletions of the long arm of the X lead to variable pheno- cleft lip, hypertelorism, and other dysmorphic features who
typic outcomes in females. Forty-three percent of women died at age 2 months. There was random X-chromosome
with Xq deletions have short stature [225]. In a study by replication pattern in this patient [234]. In a review of 52
Geerkens et al., it was found that women with breakpoints in females with partial X duplications involving Xp or Xq,
192 C.M. Powell
Matsuo et al. found that random or skewed but not completely were intellectual disability, hypotonia, failure to thrive/feeding
selective X inactivation occurred in nine of 45 patients difficulties, and seizures. Eight phenotypically normal
examined for X-inactivation pattern, independent of the size females tested had random or preferential inactivation of
or location of the duplicated segments [235]. For Xp duplica- the duplicated X as compared to those with an abnormal
tions, four of six patients with random or skewed X inactiva- phenotype in whom the duplicated X was preferentially
tion had an apparently normal phenotype, and three of 12 active in two of seven, preferentially inactive in one, and
patients with selective inactivation of the duplicated chromo- there was random X inactivation in four [242]. Whether
some had clinical abnormalities [235]. microduplication of Xp22.31 is clinically significant and
A dicentric inverted duplication of most of the short arm related to intellectual disability or is a benign copy number
of the X [dic dup(X)(qter→p22.3::p22.3→cen)] has been variant remains unclear.
reported in a mother and daughter with short stature, mental
retardation, and dysmorphic features. The mother had the
duplicated X as the inactive X in all cells, but the daughter Xq Duplications
had the duplicated X active in 11% of lymphocytes [236].
Females with duplications of Xp including the SHOX Males with duplications of the long arm of the X usually
gene region have been reported with tall and normal stature have significant intellectual disability (mental retardation)
[237, 238]. and birth defects due to functional disomy of the duplicated
With the increasing use of chromosomal microarray regions. Most females with Xq duplications have normal
technology (see Chap. 18), recurrent microduplications phenotypes and are ascertained after the birth of an abnormal
associated with variable phenotypes are being delineated male child. However, there have been females with pheno-
and characterized. A recurrent microduplication of typic abnormalities including short stature, microcephaly,
Xp11.22-p11.23 has been reported in association with intel- developmental delay/mental retardation, and gonadal dys-
lectual disability, speech delay, and electroencephalogram genesis reported. Reasons for this variability may be the size
(EEG) abnormalities in both males and females [239]. The or location of the duplicated segment, random or nonskewed
size of the duplication ranges from 0.8 to 9.2 Mb. Other X inactivation, duplication of dosage-sensitive genes and
clinical features include a hoarse or nasal voice, early puberty, genes that normally escape inactivation, incomplete inactiva-
and obesity. X-inactivation analysis of affected females tion of a portion of the duplicated segment, or an imprinting
showed selective inactivation of the normal X chromosome effect [243, 244] (see also Chap. 20).
in six of nine subjects and random inactivation in the other In a review of Xq duplications, phenotypically normal
three. The degree of X inactivation did not correlate with females had smaller and more proximal duplicated Xq
clinical phenotypic severity within families. A female with a segments compared to the Xq duplications in females with
de novo duplication of Xp11.22-p11.4 has been reported clinical abnormalities [243]. In a review of Xq duplications,
with intellectual disability and structural brain anomalies. Zhang et al. also reported that the duplicated segments in
X-inactivation studies in peripheral blood lymphocytes individuals with abnormal phenotypes were more frequently
showed that the duplicated X was active in the majority of located in proximal Xq [244]. A review by Matsuo et al.
cells [240]. This is a gene-rich region, with numerous genes showed that normal phenotypes are more commonly associ-
associated with X-linked intellectual disability, and all genes ated with smaller and more proximal duplications of Xq, and
in this region typically undergo X inactivation, which may abnormal phenotypes tend to have larger and more distal
account for the abnormal phenotypes of both sexes even with duplications, but that there is a great deal of overlap [235].
relatively small duplications [9]. Goodman et al. reported three families with duplication of
Duplication of the region from Xp21-p22 may also be of Xq27-qter on the short arm of the X [245]. Affected males
clinical significance and related to developmental delay and had mental retardation and minor anomalies. The abnormal
intellectual disability in both males and females along with chromosomes were inherited from the mothers, who were
variable dysmorphic features, including genital anomalies phenotypically normal. Replication studies in two of the
and seizures in a male [241]. mothers showed the abnormal X to be late replicating.
An interstitial microduplication of Xp22.31, ranging in However, most phenotypically abnormal females have also
size from 149 kb to 1.74 Mb, and including the steroid been reported to preferentially inactivate the abnormal X
sulfatase gene has been found with a frequency of 0.15% in chromosome [246]. Therefore, both normal and abnormal
a healthy control population and 0.37% in a cohort of indi- phenotypes can be seen even when there is preferential inac-
viduals with abnormal phenotypes that included develop- tivation of the abnormal X. One of three patients with random
mental delay, intellectual disability, autism, dysmorphic or skewed X inactivation had an abnormal phenotype, and 9
features, and/or multiple congenital anomalies. The most of 22 cases with selective inactivation of the duplicated X
frequent clinical features observed in the abnormal group had an abnormal phenotype [235]. The reason for the vari-
able phenotypes but similar inactivation patterns could be the with normal intelligence, even in the same family [256].
result of differential patterns of inactivation along the Males can be phenotypically normal or have mental retarda-
chromosome. The activation status of the material present in tion [255, 257]. Fertility in males is also variable [258,
excess copy number might be what differentiates females 259].
with normal phenotypes from those with abnormal pheno-
types. The functional disomy of genes might affect the Pericentric Inversions
phenotype [246]. Most females with pericentric inversions of the X have
Replication studies cannot distinguish phenotypically normal phenotypes and fertility [260–264]. However, peri-
normal and abnormal females with Xq duplications [233, 243]. centric inversions of the X have been reported in females
Correlations of X-inactivation pattern and phenotype in with gonadal dysgenesis and with mental retardation [261].
patients with small duplications should be interpreted care- Keitges et al. reported dizygotic twins with the same pericen-
fully [183]. tric X inversion [inv(X)(p11;q22)] [261]. One twin was
Recurrent Xq duplications identified through chromo- phenotypically normal with normal intelligence and menses
some microarray have been reported for Xq25 and associ- and had random X inactivation. The other was mildly mentally
ated with hemihyperplasia and digital anomalies and also for retarded and had psychiatric problems, irregular menses,
Xq27 involving the SOX3 gene causing pituitary failure in minor anomalies, and selective inactivation of the inverted
males [247, 248]. Xq28 duplications involving MECP2 cause X. Proposed explanations for these findings include different
the Lubs X-linked mental retardation syndrome (MRXSL) in normal Xs, a nondetectable deletion or duplication in the
males [249, 250]. A different condition involving copy abnormal twin, or chance. This also raises the likelihood that
number gains at Xq28 has also been reported that involves the replication pattern of the inverted X is a better predictor
not MECP2 but FLNA and GDI1, with severity correlating of fertility than the breakpoints. Interestingly, females with
with number of additional copy numbers and causing nonrandom X inactivation are more likely to have normal fertility
syndromic intellectual disability [251]. Sanlaville et al. have than those with skewed inactivation of the inverted X [261].
reviewed distal Xq duplications [252]. Offspring of females with pericentric inversions are at risk
for inheriting a recombinant chromosome with associated
phenotypic abnormalities [260, 263–265].
Inversions of the X Chromosome Most males with inherited pericentric inversions of the X
have a normal phenotype and fertility [260, 265, 266].
Paracentric Inversions However, X-linked disorders have been found to segregate
Paracentric inversions of the X chromosome (Fig. 10.6) are with pericentric inversions of the X, presumably by disrup-
relatively rare. There has been a wide range of phenotypes tion or deletion of a gene by the inversion [267–270].
described. In general, when long-arm paracentric inversions Analysis of X-chromosome inactivation in women with
involve the critical region at Xq13-26, females have some apparently balanced pericentric inversions might determine
degree of ovarian dysfunction [253]. When the inversion is whether an imbalance is present at the molecular level.
outside the critical region, normal phenotype and fertility Random inactivation is usually associated with a balanced
have been reported, although there are exceptions to this inversion, whereas skewed inactivation is more likely associ-
[254, 255]. There has also been variability in mental function ated with an unbalanced inversion [266, 268]. Inactivation
in females, with some having mental retardation and others status of the mother might provide helpful information in
cases of prenatal detection of a male fetus with a maternally
inherited inversion [271]. Chromosome microarray is also
helpful (see Chap. 18).
Isodicentric X Chromosomes
Isodicentric Xp Chromosomes
Isodicentric X chromosomes are formed by the fusion of two
X chromosomes [272]. The phenotypic effects are variable
and dependent on whether the chromosomes are fused at long
or short arms, as well as on the location and extent of the dele-
tion. The isochromosomes that are composed of two copies of
Fig. 10.6 Distal paracentric inversion of Xq: inv(X)(q26q28) in a
woman with normal phenotype and fertility. Brackets indicate region the entire short arm [i(Xp)] are rare and have only been
involved in inversion reported in females; they are believed to be nonviable in male
194 C.M. Powell
conceptions [273]. These i(Xp) chromosomes are typically are thought to be derived from exchanges between sister
pseudodicentric with one active and one inactive centromere, chromatids. Analysis of breakpoints in cell lines with idic(X)
and the XIST locus is retained in at least one of the deleted (p11.2) has found that they lie within large inverted repeat
long arms [273, 274]. Most of these females have gonadal sequences associated with low copy repeats or highly repeti-
dysgenesis that could be explained by the mosaicism for a tive elements on Xp11.2, suggesting that the principal mech-
45,X cell line that is often present, loss of critical regions on anism in their formation involves nonallelic homologous
Xq, and/or meiotic pairing abnormalities. Emotional and recombination. The Xp11.2 region appears to be susceptible
behavioral problems and intellectual disability have been to rearrangements leading to isochromosome formation and
reported in these patients [273]. Stature ranges from tall to other rearrangements [280].
short. Tall stature could be related to the presence of three
copies of SHOX in those patients with long arms joined.
Structural Abnormalities of the Y Chromosome
Isodicentric Xq Chromosomes
Isodicentric X chromosomes consisting of two copies of the Structural abnormalities of the Y chromosome that lead to
long arm joined at the proximal short arms with two centrom- deletion of the proximal long arm might be associated with
eres are the most common structural abnormality of the X chro- azoospermia, infertility, and short stature. Marker chromo-
mosome seen in Turner syndrome. Although most look somes derived from Y chromosomes are important to detect
monocentric in a G-banded karyotype, they are actually dicen- due to the risk of gonadoblastoma in females with Turner
tric with one active centromere. The correct nomenclature (in syndrome. FISH probes and chromosome microarray have
the absence of direct evidence of a dicentric chromosome) is improved the ability to identify marker Y chromosomes.
46,X,i(X)(q10), with the understanding that in many instances
it would be more accurate to use 46,X,idic(X)(p11.2).
Females with isodicentric X chromosomes joined at their Translocations Involving the Y Chromosome
short arms exhibit short or normal stature, gonadal dysgenesis,
and, occasionally, Turner syndrome features [275]. Expla- The Y chromosome can be involved in translocations with
nations for the phenotype of short stature when the short any other chromosome (another Y, an X, or an autosome).
arms are joined include deletion of the distal short arm at the
region of SHOX and/or as a result of a 45,X cell line. There (X;Y) Translocations
are some males with Klinefelter syndrome who have one Although the short and long arms of the Y chromosome are
normal X and one isochromosome X [276] (see also the now divided into subbands, several references and the original
previous section on “47,XXY (Klinefelter Syndrome)”). cases cited in them designate “Yp11” or “Yq11.” In order to
Mechanisms to explain formation of terminal rearrange- avoid misrepresentation of the authors’ original work, this
ments between homologous chromosomes include the nomenclature will be retained here.
following: Hsu reviewed 51 reported cases of (X;Y) translocations,
1. Breakage and deletion of a single chromosome followed 47 with a derivative X and four with a derivative Y [281].
by rejoining of sister chromatids The (X;Y) translocations with a derivative X were divided
2. Breakage and deletion of two homologous chromosomes into seven types, with the most common types involving
at the same breakpoints followed by interchromosomal translocation of a portion of Yq11→Yqter onto Xp22.
reunion Patients with type 1, in which there is a normal Y chromo-
3. Terminal fusion without chromatin loss between sister some and a derivative X with a portion of Yq translocated
chromatids and homologous chromosomes [277] to Xp [46,Y,der(X)t(X;Y)(Xqter→Xp22::Yq11→Yqter],
The isodicentric X is almost always late replicating, suggest- were phenotypic males. For those with reported heights
ing nonrandom inactivation of the derivative X. The second (14 of 15 reported), all were short, presumably as a result of
centromere is usually nonfunctional, making it a pseudodi- nullisomy for SHOX on Xp22.3. Eleven cases with informa-
centric chromosome [278] (see also the subsections “Turner tion available on skin condition showed evidence of ich-
Syndrome,” “Isochromosome X” earlier). Studies have thyosis, presumably the result of nullisomy for the steroid
shown that those that are functionally dicentric tend to have sulfatase gene on Xp22. All 12 of the patients for whom
anaphase lag and, therefore, are more likely to be found in information was provided on intelligence were mentally
association with a 45,X cell line. Those that are functionally retarded. Minor facial anomalies, including flat nasal bridge
monocentric tend to segregate normally in mitosis [278, 279]. and hypertelorism, were also reported. Four patients had
The occurrence of a 45,X cell line also correlates with the short limbs compatible with the diagnosis of chondrodys-
distance between the centromeres, presumably due to less plasia punctata, presumed secondary to nullisomy for the
mitotic stability of the isodicentric chromosome [75]. Most X-linked chondrodysplasia punctata gene on Xp (ARSE or
CDPX). In two adult males, azoospermia and small testes (Xqter→Xp22::Yp11→Yqter). All three patients reported
were reported. The size of the Xp deletion varies, and pheno- were phenotypic males and had short stature and hypogonad-
types reflect which genes on Xp are missing. Short stature is ism or azoospermia [281].
a consistent finding; hypogonadism with infertility is Type 4 patients had a portion of Yq translocated to band
common. Patients can have short stature with or without p11 of the second X chromosome. Of one type 4 case
Leri-Weill dyschondrosteosis (as a result of SHOX deletion), reported, the patient was a phenotypic female, with short
chondrodysplasia punctata (ARSE deletion), mental retarda- stature, streak gonads, and secondary amenorrhea [281].
tion (presumed MRX locus deletion), ichthyosis (STS Types 5 and 6 patients had varying amounts of Yq mate-
deficiency), and hypogonadotropic hypogonadism in com- rial translocated to Xq22; of two patients described, both had
bination with anosmia (Kallmann syndrome) when the streak gonads [220].
deletion is large and encompasses all of the genes in this Type 7 has a dicentric chromosome: 46,X,dic(X;Y)
region [282]. (Xpter→Xq22::Yp11→Yqter), and the one case reported
With probes for the STS and Kallman syndrome regions was a phenotypic female with streak gonads, normal stature,
on Xp, it is now possible to use FISH (see Chap. 17) or and secondary amenorrhea [281].
chromosome microarray (see Chap. 18) to delineate the Four cases of (X;Y) translocations with a derivative Y
extent of deletions of Xp22. This will be important in were reported, which Hsu classified into four types. All
helping to predict phenotype, especially in prenatally diag- involved a portion of Xp22 (three cases) or Xq28 (one case)
nosed cases. translocated to Yq11, and all patients had normal stature,
Type 2 patients had a translocation of Yq11→Yqter hypogonadism with hypoplastic male external genitalia or
onto Xp22, with one normal X chromosome and a derivative ambiguous genitalia, mental retardation, and various dys-
X: 46,X,der(X)t(X;Y)(Xqter→Xp22::Yq11→Yqter) (see morphic features [281, 284].
Fig. 10.7). Most of these women were ascertained through One case has been reported of a 45,X male with an (X;Y)
sons with a type 1 translocation. All 25 reported cases were translocation, in which distal Yp was translocated to Xp:
phenotypic females, and 17 of 22 with height information 45,der(X)t(X;Y)(Xqter→Xp22.3::Yp11→Ypter). The patient
were short. Most had proven fertility or reportedly had had short stature, a short broad neck, broad chest, wide-
normal ovaries [ 281 ] . Most have normal intelligence, spaced nipples, short metacarpals and slight cubitus valgus,
but mild mental retardation has been reported [283]. normal male external genitalia but small testes, and normal
Type 3 patients had one normal X chromosome and intelligence [285].
a second sex chromosome that was dicentric, consisting It should be noted that the presence or absence of a 45,X
of major portions of both X and Y: 46,X,dic(X;Y) cell line in addition to one with an (X;Y) translocation can be
of significance concerning the development of external geni-
talia. When a 45,X cell line is present, there is an increased
likelihood of a female phenotype with features of Turner
syndrome [281].
(Xp;Yp) translocations involving the testis-determining
factor can be found in XX males or, rarely, XY females with
sex reversal (see Disorder of Sex Development with “Normal”
sex chromosomes later). These translocations are usually not
seen with cytogenetic analysis and require molecular probes
for diagnosis [216].
There has been a case described of translocation of Yp
sequences including the SRY gene onto the long arm of the X
in a patient who had both ovarian and ovotesticular gonadal
tissue [286].
The finding of different phenotypes of XX males and
patients with both ovarian and testicular tissue or ovotestes
who carry the same translocation has been explained by a
different pattern of inactivation of the Y-bearing X chromo-
somes [287]. Inactivation on the X chromosome spreading
Fig. 10.7 Derivative X chromosome consisting of a small terminal Xp into a translocated Yp fragment is the proposed mechanism
deletion and translocation of Yq:46,X,der(X)t(X;Y)(p22.3;q11.2) mat. for an intersex phenotype in some 46,XX (SRY+) individ-
This was seen in a 5-year-old girl with short stature who had inherited uals [288].
the chromosome from her mother, who also had short stature but was
otherwise normal. Brackets indicate regions on X and Y making up the Because most males with (X;Y) translocations will inherit
derivative X the translocation from their mothers, it is important to advise
196 C.M. Powell
women with such translocations of the risk for more severe In most cases, when the breakpoint in the Y chromosome
manifestations in their male offspring who inherits the deriv- is in Yq12, the heterochromatic region of the Y, there is
ative X. normal fertility. When the breakpoint involves the distal
Yq11.2 euchromatic region at the azoospermia factor locus,
there is usually infertility. Exceptions to this have been
Y;Autosome Translocations reported [290]. Studying meiotic configurations in a patient
with a Y;autosome translocation at Yq12 and infertility, the
Y;autosome translocations are estimated to occur with a authors found pairing abnormalities involving the compart-
frequency of 1 in 2,000 in the general population [289]. In a ment of the sex vesicle (the condensed sex chromosomes).
review of more than 130 cases of Y;autosome translocations, Possible causes of degeneration of spermatocytes after the
Hsu reported that the most common involved translocation pachytene stage and thus infertility in such patients include
of the fluorescent heterochromatic region of Yq to the short extensive asynapsis, spreading of X inactivation to the
arm of a “D group” (13–15) or “G group” (21 and 22) chro- autosomal segments partially included in the sex vesicle,
mosome [281]. Most of these are familial, and an otherwise autosomal genes involved in the different rearrangements,
normal 46,XX or 46,XY karyotype with this translocation is and the modifying factors of the genetic background [290]
associated with a normal phenotype. Chromosomes 15 and (see also Chap. 12).
22 are most commonly involved: t(Yq12;15p) and
t(Yq12;22p). When the translocation is familial, it is unlikely
to have any phenotypic effects, and fertility is not affected. Yp Deletions
When the diagnosis is made prenatally in a 46,XX,der(“D
group” chromosome) or der(“G group” chromosome) fetus Individuals with deletions of the short arm of the Y involving
and the translocation can only be found in male relatives, the band p11.3, the location of SRY, are usually phenotypic
possibility of the presence of Yp material in the derivative females. Most have streak gonads with Turner syndrome
chromosome cannot be ruled out [281]. There would be a features, especially lymphedema, but normal stature [281].
significant risk of masculinization or sex reversal in the These individuals are at risk for gonadoblastoma (see previous
female. Molecular studies using Yp probes are indicated in subsection). This is in contrast to females with 46,XY “pure”
such situations. gonadal dysgenesis who do not have features of Turner syn-
Translocations have been reported involving all auto- drome (see section “Disorders of Sex Development with
somes except 11 and 20. Twenty-nine of fifty cases that did “Normal” Sex Chromosomes”).
not involve a “D group” or “G group” chromosome involved Males with cryptic deletions of Yp involving the pseudoau-
a reciprocal translocation, of which 27 were associated with tosomal region (PAR1) usually have short stature with Madelung
a male phenotype and 2 with a female phenotype. Eighty deformity and other features of Leri-Weill dyschondrosteosis
percent of the adult males had azoospermia/oligospermia or secondary to haploinsufficiency of SHOX. These deletions are
infertility, although there was bias of ascertainment, making often associated with Yp translocations [291–293].
the true risk of infertility in males with a balanced Y;autosome
translocation unknown. Four of the patients were infants or
boys with mental retardation and/or multiple congenital Yq Deletions
abnormalities. The two patients with female phenotypes had
gonadal dysgenesis and streak gonads. A small Yp deletion Deletions involving the heterochromatic portion of Yq are
or 45,X mosaicism could not be ruled out in these patients. compatible with normal genital development and sexual
Hsu also reviewed 21 cases with unbalanced Y;autosome differentiation (see the section “Y-Chromosome Polymor-
translocations, of which 13 had a male phenotype [281]. Two phisms” later). Larger deletions involving the euchromatic
of five adult males had azoospermia or hypogonadism; the portion of Yq could cause azoospermia [294]. When detected
other three were phenotypically normal and fertile. Eight prenatally or in a young patient, the father should be tested
were infants or children with abnormalities secondary to to see whether the deleted Y is an inherited or a de novo
autosomal aneusomy. Six patients were phenotypic females, abnormality.
five with gonadal dysgenesis, and one with Turner syndrome Hsu reviewed 52 cases of Yq deletions. In all cases Yq12
features; three had developed gonadoblastoma. was deleted, but for most the cytogenetic findings did not pro-
Males with 45,X and Y;autosome translocations involv- vide specific information about the breakpoint on Yq11. Forty-
ing all of Yp or a portion of distal Yp might have azoospermia eight were phenotypic males, and most were infertile with
or infertility, although some have normal fertility [281]. The azoospermia or oligospermia [281]. Based on patients with Yq
presence of Yp in a Y;autosome translocation explains the deletions, the azoospermia factor (AZF) was identified (see
male sex determination. section “The Y Choromosome” earlier). Males with these
deletions could have short or normal stature. No patients had

gonadoblastoma. Of the three patients who were phenotypic
females, two had streak or dysgenetic gonads, and two had
normal stature. One patient had ambiguous external genitalia
with left testis and right streak gonad, normal stature, and
Turner syndrome features. Mosaicism for a 45,X cell line
could not be ruled out [281].
Interstitial microdeletions in the euchromatic portion of Fig. 10.8 (Left to right) Normal Y; isodicentric Y consisting of two
the Y chromosome occur in 10–15% of men with azoo- copies of the short arm, centromere, and proximal long arm (q11.2);
ring Y, and pericentric inversion of the Y: inv(Y)(p11q11). Dots indi-
spermia and severe oligozoospermia [295, 296]. AZFa in cate location of centromeres
proximal Yq (Yq11.21) contains two genes (USP9Y and
DDX3Y) whose absence or mutation causes spermatogenic
failure [297–299]. Complete absence of AZFa is associated The presence of a 45,X cell line in addition to any cell line
with complete absence of germ cells. AZFb (Yq11.23) con- with an isochromosome Y or isodicentric Y and the degree
tains seven Y genes (CDY2A, EIF1AY, PRY, RBMY1, of mosaicism in different tissues leads to variable phenotypic
KDM5D, TTTY5, and TTTY6). AZFb absence is associated manifestations, ranging from phenotypic male with azoo-
with a meiotic maturation arrest; that is, spermatogonia and spermia to ambiguous genitalia to phenotypic females with
spermatocytes are present in the patients’ testis tubules in typical or atypical Turner syndrome features [281]. Many
normal amounts, but postmeiotic germ cells are completely reported patients have short stature. Gonadoblastoma has
absent. AZFc (Yq11.23) contains seven genes (BPY2, CDY1, been reported in females with a dicentric Y cell line. Males
CSPG4LY, DAZ, GOLGA2LY, TTY3, and TTY4). AZFc dele- often have hypospadias. Azoospermia is common in pheno-
tions are the most common genetic abnormality in men with typic males with an isodicentric Y. Again, this has been
azoospermia or severe oligozoospermia. They are associated proposed to be due to loss of an AZF gene [281, 307, 308].
with variable testicular pathology and occasionally are inher- Mental retardation has been reported in a few patients, and
ited, although most are de novo in origin [300–302, 306]. schizophrenia in two patients, although there is a bias of
Polymerase chain reaction (PCR) techniques are needed to ascertainment in postnatally diagnosed cases and there are
identify various deleted regions (see also Chap. 12). very few reports of prenatally diagnosed cases [248, 308,
Short stature in males with Yq deletions might be the 309, 310, 314]. In a report of outcomes of nine cases ascer-
result of the loss of the proposed GCY (growth control tained prenatally with nonmosaic 46,X,idic(Yp) or with
gene[s] on the Y chromosome) locus near the pericentro- mosaicism for a 45,X cell line, one had ambiguous genitalia,
meric region of Yq [303, 304]. No gene has yet been identified two of the pregnancies were terminated but the fetuses had
in this region. Using FISH analysis, Kirsch et al. demon- normal male genitalia, and six had normal male genitalia at
strated 45,X cell lines in metaphase preparations from all birth and at follow-up examinations. The patient with ambig-
patients with terminal Yq deletions, suggesting that at least uous genitalia also had short stature, but in five cases carried
in some patients, short stature could be explained by mosa- to term and followed postnatally with available height infor-
icism for a 45,X cell line [304]. mation, stature was in the normal range [311]. Another study
reported that the likelihood of female anatomic development
increases proportionately to the intercentromeric distance in
Y Isochromosomes idic(Yp) chromosomes, presumably due to increased insta-
bility of the isodicentric chromosome with higher likelihood
In most cases of isochromosome for Yp or Yq, the abnor- of a 45,X cell line [305].
mal chromosome is dicentric and present in mosaic fash- Infertility in males with isochromosome Yp (despite
ion, usually with a 45,X cell line (see the section “Turner having two copies of SRY) might arise through lack of distal
Syndrome Variants” earlier). The dicentric Y is among the Yq genes that play critical roles in spermatogenesis, from
most commonly detected structural abnormalities of the Y meiotic pairing disruption as a result of duplication of the
chromosome (Fig. 10.8) [281]. Most dicentric Ys have the pseudoautosomal region of Yp and loss of the pseudoauto-
breakpoint in the long arm, with duplication of the proxi- somal region on Yq and/or from mitotic instability of the
mal long arm and entire short arm, while some have the isochromosome and resulting 45,X mosaicism [305].
break in the short arm with the proximal short arm and In a report of five phenotypic females with isodicentric Y
entire long arm duplicated. Most isodicentric Y chromo- and a 45,X cell line who had removal of their gonads, three
somes are thought to arise through homologous recombi- had microscopic gonadoblastoma, one had evidence of a
nation between opposing arms of palindromes on Y sister previous gonadoblastoma that according to the authors had
chromatids [305]. probably “burned out,” and one had a microdysgerminoma
198 C.M. Powell
(malignant tumor) [312]. Gonadal tumors have been reported one of whom had proven azoospermia. Other variable
in patients with isodicentric Y chromosomes with break- features described included small testes, small penis, hypos-
points in both the short and long arms. This could be padias, and short stature. One patient was a phenotypic
explained by the presence of multiple gonadoblastoma loci female with streak gonads and sexual infantilism. Of cases
(GBY) on both arms of the Y that may correspond to the with mosaicism, phenotype varied from normal male to
TSPY gene that has clusters in several locations on both arms ambiguous genitalia to normal female. Phenotypes were
[312]. This gene has recently been reported in gonadoblas- similar to the nonmosaic cases. Because of the different
toma tissue as well as in surrounding normal tissue in five of degrees of deletion of Yp and Yq in ring formation, pheno-
six gonadoblastoma specimens [313]. A cause-and-effect type-karyotype correlation is difficult [281].
relationship has not yet been proven. The type of Y isochro- In a series of five males with 45,X/46,X,r(Y) mosaicism
mosome does not predict the risk of development of gonado- and bilateral scrotal testes, the three adults had azoospermia,
blastoma in a female. and one of the three had short stature. All had normal puberty
A review of prenatally diagnosed cases of isodicentric and testosterone levels. Two prepubertal males had short stat-
chromosome Yp that compared the percentage of mosaicism ure, one of whom had testes removed due to concern for
in peripheral blood postnatally suggested that idic(Yp) may gonadoblastoma. Pathology exam of the gonadal tissue
be preferentially present in blood and that the proportion in showed calcinosis that was thought to be an early sign of
amniocytes is not a reliable indicator of sexual phenotype gonadoblastoma [317]. There has been a report of a male with
[314]. Fetal ultrasound is more important in predicting normal phenotype, oligozoospermia, and a 45,X/46,X,r(Y)
phenotypic sex [314]. karyotype who fathered a son with 46,XX/47,XX,r(Y). The
additional X was paternal in origin. Sperm analysis in the
Isochromosome Yp father showed a high percentage of XY aneuploid sperm
Without a demonstrable 45,X cell line, most cases with compared to controls [318]. There has been another report of
monocentric i(Yp) will have a male phenotype but have a man with ring Y having a son with ring Y through intracy-
infertility [281]. Cases with normal fertility typically have toplasmic sperm injection [319] (see also Chap. 11). Although
mosaicism for a 46,XY cell line. In one study, all seven cases men with a ring Y chromosome have a high incidence of
with an i(Yp) (one centromere) were phenotypic males. This infertility, those who father children appear to have a high risk
may be the result of greater mitotic stability with a single of passing the ring Y to their offspring and may have an
centromere [305]. increased risk of chromosome aneuploidy in their offspring.
Men with a 45,X/46,X,r(Y) mosaicism with scrotal testes
Isochromosome Yq have a relatively low risk of gonadoblastoma or other
Hsu reviewed seven reported cases with nonmosaic, mono- testicular tumors. Their testes are often functional in terms
centric isochromosome Yq. All were phenotypic females of testosterone production despite poor sperm production.
(expected due to the absence of SRY), with sexual infantilism Monitoring for tumors with physical examination, Doppler
and streak gonads. Approximately half had Turner syndrome ultrasound, and serum markers should be considered, as
features and short stature. The lack of Yp in a case with opposed to prophylactic gonadal removal.
monocentric i(Yq) without a demonstrable 45,X cell line
leads to a female phenotype with typical or atypical Turner
syndrome [281]. Y-Chromosome Polymorphisms
Heterochromatic Length
Ring Y Chromosome The Y chromosome varies in size in the normal male popula-
tion owing to variability in size of the heterochromatic
The brightly fluorescent heterochromatic region of Yq is portion of Yq (Yqh or Yq12). This is not associated with
usually deleted during formation of a ring Y [r(Y); see Chap. 3] phenotypic abnormalities or infertility (see the section “Yq
making Q-banding (see Chap. 4) an unreliable tool for Deletions” earlier).
identification (Fig. 10.8). The most accurate way to deter-
mine origin of a ring sex chromosome in a patient with a Satellited Y Chromosome
46,X,-X or Y,+r karyotype is with FISH, using probes for X The presence of satellites on the end of the long arm of
and Y (see Chap. 17), or with chromosome microarray (see the Y chromosome (Yqs) is considered to be a normal
Chap. 18). Because of the instability of ring chromosomes, variant not associated with phenotypic abnormalities.
multiple different cell lines might be seen [315, 316]. Transmission through several generations has been
In a review of 34 cases with r(Y), 25 had a 45,X cell line. reported. These chromosomes arise from translocations
Nine cases were nonmosaic: eight were phenotypic males, involving the short arm of an acrocentric autosome, most
commonly chromosome 15 [320, 321]. All have an active sexual development in which the sex chromosomes may
nucleolar organizer region. Loss of the pseudoautosomal appear structurally normal. Despite discovery of several sex-
region (PAR2) on distal Yq has been shown in several determining genes, the majority of patients with disorders of
cases of Yqs [321]. gonadal development, often referred to as intersex conditions,
At least two cases of satellited short arm of the Y chromo- still remain genetically unexplained. (For excellent reviews
some have been reported both in phenotypically normal of this complex process, see references [333] and [334]).
males, including one adult with normal fertility [322, 323]. Along with discoveries of genes involved in sexual differ-
In one case, the satellite Y was inherited from the father who entiation has come the realization that female development
had a satellited X chromosome, presumably from a meiotic is not merely the default state. Genes that are critical for
crossover event [323]. male sex determination include SOX9 (SRY-box 9) located
on chromosome 17 and thought to be the primary testis-in-
Inverted Y Chromosome ducing gene and SRY the sex-determining genetic factor in
Pericentric inversion of the Y chromosome—inv(Y) males on the Y chromosome. Disruption of the process that
(p11.2q11.2)—is estimated to occur as a normal variant in SRY initiates can lead to an XY female. Candidate genes for
0.6 in 1,000 males [324] (see Fig. 10.8). A very high fre- the sex-determining factor in females include RSPO1 (human
quency of 30.5% was found in the Gujarati Muslim Indian R-spondin-1 gene), NROB1 (nuclear receptor subfamily 0
population of South Africa [325]. In most cases, inverted Y which encodes DAX1), and WNT4 (wingless-type MMTV
chromosomes are inherited. These are not usually associ- integration site family, member 1). Additional as-yet-unknown
ated with any phenotypic or reproductive abnormalities, genes are also thought to be important in female sex differen-
although pericentric inversions of the Y have been reported tiation [335].
in males with infertility as a result of a small deletion in One in 20,000 newborns is 46,XX with sex reversal,
Yq11.1-2 or interruption of the DAZ gene area. Rivera et al. whereas 46,XY with sex reversal is more common, occur-
have characterized pericentric inversions of the Y chromo- ring in 1 in 3,000 newborns [336]. In approximately 75% of
some into two types: “true” inversions with a single active patients with sex reversal, the cause remains unknown [337].
centromere juxtaposed to Yqh and accounting for the major- For parents, genital ambiguity is one of the most stressful
ity and “false” inversions with a neocentromere at the most problems encountered at birth. Adding to this stress can be
proximal heterochromatin in addition to a classic centro- the well-meaning physician or nurse who makes premature
mere [325–329]. pronouncements about the infant’s sex [338]. Evaluation by
Knebel et al. demonstrated heterogeneity of the break- a team of specialists including pediatric endocrinologists,
points in nine cases of pericentric inversion Y using FISH geneticists, and urologists with appropriate endocrine, cyto-
probes. The inversion breakpoints in the short arm were all in genetic, pathology, and imaging studies is critical in differ-
a gene-poor region of X-transposed sequences proximal to entiating the various types of these conditions. It is best to
PAR1 and SRY. There were three different types of break- delay sex assignment until there is sufficient information
points in the long arm. One type was identified in both familial from these studies and after extensive discussions are held
cases and associated with infertility [330]. with the family. In the past, sex assignment in cases of genital
There have been rare reports of paracentric inversions of ambiguity was often based on what would give the most
the long arm of the Y [253, 331]. One of the cases had potential for sexual function and fertility. Early studies sug-
ambiguous genitalia with Müllerian structures present [331]. gested that sex of rearing different from genetic sex did not
The inverted Y was present in the father and grandfather who make a difference in terms of gender identification and
were normal fertile males. Abnormalities in SRY, ZFY, TSPY1, adjustment [339]. Many experts stressed the importance of
or NR0B1 were not found. The cause of the sexual ambiguity assigning gender as soon as possible in the newborn period.
could not be identified. A possible position effect related to Recently, however, there have been reports of major psycho-
the inversion or a coincidental cause was hypothesized. logical difficulties with gender identity for some adult
A case identified prenatally was inherited from a normal patients with disorders of genital ambiguity. The effect of
father, and the infant was reported as normal [332]. androgen exposure on the developing brain may be the
strongest predictor of sexual identity [340]. This has led
physicians to reexamine their treatment of such patients and
Disorders of Sex Development with “Normal” some experts to argue that the central nervous system dic-
Sex Chromosomes tates the sexual identity and that surgical procedures should
be postponed until the patient can participate in the deci-
Although visible structural abnormalities of the sex chromo- sion-making process [340]. These varying opinions empha-
somes are often associated with phenotypic abnormalities of size the need for more long-term studies [340, 341]. Recent
the internal or external genitalia, there are other disorders of reviews of patients with ambiguous genitalia have reported
200 C.M. Powell
that either male or female sex of rearing can lead to success- size, and there are interstitial fibrosis and hyperplastic Leydig
ful long-term outcome [342, 343]. cells and usually no spermatogonia [348]. There are also
Confusing and, to some, pejorative terminology such as 46,XX sex-reversed patients with both testicular and ovarian
“intersex,” “hermaphroditism,” and “pseudohermaphroditism” tissue in gonads, either separately or, more commonly, as an
has been replaced by the recommended overall term of ovotestis. They usually have ambiguous external and internal
“disorder of sex development” (DSD), defined as a con- genitalia depending on the amount of functional testicular
genital condition in which development of chromosomal, tissue present (see section “Ovotesticular Disorders of Sex
gonadal, or anatomical sex is atypical. These have been Development” later).
grouped into categories that include sex chromosome DSD, There are at least four different mechanisms to explain the
46,XY DSD, and 46,XX DSD [344]. The category known as male phenotype in XX DSD: (1) translocation of Y sequences,
sex chromosome disorder of sex development includes including the SRY gene, to an X chromosome or autosome;
47,XXY; 45,X; 45,X/46,XY (mixed gonadal dysgenesis); (2) gain of SOX9 through a duplication; (3) a mutation in an
and 46,XX/46,XY chimerism. This nomenclature recognizes as-yet-unknown X-linked or autosomal gene in the testis-
the importance of the karyotype in differentiating this often determining pathway; or (4) cryptic Y-chromosome mosa-
very complex and heterogeneous group of conditions and icism [349, 350]. The majority of patients (90%) fall into
should help in proper classification and diagnosis. 46,XX category 1, most often with Y sequences including SRY
DSD and 46,XY DSD will be summarized here [344–346]. translocated to the X chromosome. The pseudoautosomal
Accurate diagnosis of the underlying etiology in these regions of Xp and Yp pair during male meiosis, and there
conditions is critical for proper medical management and sometimes may be unequal interchange of material extend-
genetic counseling. Many have an increased risk of germ cell ing beyond the pseudoautosomal boundaries. This theory has
malignancy, other tumor types, and serious medical prob- been used to explain the origin of XX males with SRY and
lems [344]. The genetic etiology is extremely variable and other portions of Yp translocated to Xp [351]. Ten percent of
often difficult to determine. XX males have no detectable SRY or other Y sequences [350].
Most SRY positive XX males have normal male external
genitalia, while those lacking Y-derived sequences are more
46,XX Disorders of Sex Development likely to have ambiguous genitalia [352, 353]. However,
some 46,XX testicular DSD patients with SRY present may
Using the new classification system, this group consists of have ambiguous genitalia and evidence of ovotesticular
(1) disorders of ovarian development; (2) disorders of andro- DSD. This variability may be due to differential inactivation
gen excess, with the most common cause of 46,XX DSD of the X chromosome carrying SRY or the size of Yp present
being congenital adrenal hyperplasia; and (3) other disorders on Xp [354, 355, 359].
that include cloacal exstrophy, vaginal atresia, MURCS There have been familial cases of 46,XX testicular DSD,
(Müllerian, renal, cervicothoracic somite abnormalities), and suggesting autosomal recessive inheritance [360]. There
other rare syndromes. It is estimated that 1 in 20,000 new- have also been families reported with both XX testicular
borns has 46,XX sex reversal [336]. DSD and XX ovotesticular DSD, so that there may be a
common origin for both [357]. Others have also found
Disorders of Gonadal (Ovarian) Development evidence that full virilization requires the expression of a
This category includes gonadal dysgenesis in a 46,XX indi- second Y-linked gene, near SRY, which may be expressed
vidual, ovotesticular DSD (previously referred to as true outside the testis [361, 362].
hermaphroditism), and testicular DSD in a 46,XX individual
due to presence of SRY and duplication of SOX9 or RSP01. Ovotesticular Disorders of Sex Development
This term replaces that previously used of “true hermaphro-
46,XX Testicular Disorders of Sex Development ditism.” This is a rare condition where both testicular and
Previously termed XX male or XX sex reversal, this is a ovarian tissues are present either as separate structures or as
genetically heterogeneous group of conditions involving an ovotestis. Most patients have ambiguous external genitalia
individuals who have bilateral testes while lacking a Y chro- with a phallus of variable length and urogenital sinus and are
mosome. Most have normal male external genitalia, although reared as males. Secondary sex characteristics in each patient
10–15% have some degree of genital ambiguity, cryptorchidism, will be the result of the predominant steroid hormone pro-
and/or hypospadias and are more likely to be diagnosed in duced. Ovulation and pregnancy have been reported in a few
childhood [347]. Others present in adulthood with infertility cases [350]. A few patients who were chimeras with 46,XX
or gynecomastia. Most have small testes and some signs of and 46,XY cell lines arising from the fusion of two zygotes
androgen deficiency, similar to Klinefelter syndrome patients have been described, although not all 46,XX/46,XY individuals
[348]. The seminiferous tubules are decreased in number and have ovotesticular DSD [348, 351]. The SRY gene is present
in 33% of cases [352]. At least 50% of cases of ovotesticular Disorders of Gonadal (Testicular) Development
DSD are 46,XX with no Y DNA [348]. The cause of testi- This includes complete or partial gonadal dysgenesis associ-
cular tissue in these patients is unclear. Possible etiologies ated with mutations in sex-determining genes including SRY,
are gonadal mosaicism for a Y-bearing cell line and altera- WT1 on 11p, DMRT1 (doublesex- and MAB3-related tran-
tions in unknown X-linked or autosomal sex-determining scription factor 1) on 9p, and SF1 (splicing factor 1) on 11q
genes [350, 353]. in 46,XY individuals [334, 350]. Only 10–15% of cases of
Gonadal neoplasia and breast cancer have been reported complete gonadal dysgenesis are caused by mutations in
in these patients [73, 344, 351]. SRY. Some cases are due to loss of SRY [364]. Heterozygous
mutations of SF-1 (steroidogenic factor 1) appear to be a
Disorders of Androgen Excess relatively frequent cause of XY DSD without adrenal
This group of conditions includes those previously termed insufficiency [344]. There is also a dosage-sensitive region
female pseudohermaphroditism. Patients with disorders of on Xp that, when duplicated, leads to female external genita-
androgen excess with a 46,XX chromosome complement have lia in a 46,XY individual. DAX1 (dosage-sensitive sex
gonadal tissue of one sex but ambiguous external genitalia. reversal/adrenal hypoplasia congenita/critical region on the
This is most commonly the result of congenital adrenal hyper- X chromosome, gene 1) appears to be the gene responsible
plasia. It is critical to identify these patients early due to the risk for this [231, 365–367]. Mutations in this gene are associated
of hypovolemic shock in untreated 21-hydroxylase deficiency, with congenital adrenal hypoplasia and hypogonadotropic
the most common type of congenital adrenal hyperplasia that hypogonadism [367, 368]. Familial cases of disorders of
leads to salt wasting. This is an autosomal recessive condition, testicular development suggesting X-linked sex-limited,
and prenatal diagnosis and treatment are possible. autosomal recessive, or autosomal dominant inheritance
There are other causes of androgen excess such as have been reported [363]. Malformation syndromes such as
11b-hydroxylase (11beta-hydroxylase) deficiency and Smith-Lemli-Opitz and campomelic dysplasia also produce
3b-hydroxysteroid (3beta-hydroxysteroid) dehydrogenase female or ambiguous genitalia with a 46,XY karyotype.
deficiency. Exogenous hormones from the mother may also These are due to mutations or deletions in the autosomal
cause masculinization of genitalia in a 46,XX fetus [353]. genes DHCR7 (7-dehydrocholesterol reductase) and SOX9,
respectively. Other syndromes associated with gonadal dys-
genesis are alpha-thalassemia/mental retardation (ATRX)
46,XY Disorders of Sex Development and Denys-Drash syndrome, due to mutations in WT1
(Wilms’ tumor 1 gene) on 11p. Denys-Drash syndrome is a
This category includes (1) disorders of gonadal (testicular) condition with Wilms’ tumor, diffuse mesangial sclerosis of
development such as 46,XY complete gonadal dysgenesis or the kidneys leading to nephrotic syndrome, and gonadal
Swyer syndrome (previously known as XY sex reversal or dysgenesis with ambiguous genitalia or hypospadias (WT1)
XY females), partial gonadal dysgenesis (previously termed on 11p13 [354]. WT1 is also associated with WAGR syn-
male pseudohermaphroditism), and ovotesticular DSD (pre- drome (Wilms’ tumor, aniridia, genitourinary anomalies, and
viously termed true hermaphroditism); (2) disorders in andro- mental retardation) and Frasier syndrome (focal segmental
gen synthesis or action such as complete androgen insensitivity glomerular sclerosis, male-to-female sex reversal, and low
syndrome (the current term for what was previously known risk of Wilms’ tumor). Many other malformation syndromes
as “testicular feminization”), which is an X-linked condition associated with testicular DSD have been described including
due to mutations in the androgen receptor gene (AR), Smith- X-linked lissencephaly due to mutations in ARX [370–372].
Lemli-Opitz syndrome, an autosomal recessive condition due
to mutations in the gene DHCR7, and 5-alpha-reductase
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354. Eddy AA, Mauer M. Pseudohermaphroditism, glomerulopathy, 365. Zanaria E, Bardoni B, Dabovic B, Calvari V, Fraccaro M, Zuffardi
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355. Hughes IA. Disorders of sex development: a new definition and 366. McCabe E. Sex and the single DAX1: too little is bad, but can we
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The Cytogenetics of Infertility
11
with infertility are in those with primary amenorrhea.

Introduction Nonetheless, most women with infertility have experienced
normal menarche. In the evaluation of female infertility, if the
The World Health Organization has described infertility as a
serum estrogen level is low and the gonadotropins are high,
health problem of global concern. One in seven couples
ovarian failure is likely, and chromosome analysis is indi-
experiences infertility or subfertility [1]. Infertility is com-
cated. The gonadotropins include follicle-stimulating hormone
monly defined as absence of a pregnancy after a year of
(FSH) and luteinizing hormone (LH). They are secreted by
unprotected intercourse. For information about cytogenetic
the pituitary gland in response to gonadotropin-releasing
aspects of spontaneous abortions, please refer to Chap. 13.
hormone (GnRH), which in turn is secreted by the hypo-
Male factor and female factor infertility each accounts for
thalamus gland. If the estrogen level is low, and gonadotropins
about 40% of cases of infertility, and the remaining 20% is a
FSH and LH are also low, the likelihood is that hypothalamic
combination [2]. In this chapter, an overview of known causes
or hypopituitary issues are the cause. In this case, there are
of infertility will be presented so that the cytogenetic compo-
several gene abnormalities known to cause these problems,
nent’s relative contribution can be placed into context.
but the yield of cytogenetic investigation is very low.
Causes of Female Infertility Cytogenetic Findings in Female Infertility

Due to Ovarian Dysfunction
Female infertility falls roughly into four categories:
• Fallopian tube obstruction and/or adhesion
45,X and 45,X Mosaicism
• Anatomic abnormalities of the genital tract
As discussed in Chap. 10, one in about 2,500 baby girls is
• Endometriosis
born with 45,X or a mosaic variant thereof. Ninety percent of
• Ovulation disorders, which include hypothalamic, pitu-
women with 45,X or with 45,X mosaicism with 46,XY,
itary, and ovarian causes. Most cytogenetic abnormalities
46,XX, 47,XXX, or 46,X,i(Xq) cell lines present with pri-
fall into this category
mary amenorrhea and lack of pubertal development. Up to
In this chapter, only the latter two categories will be dis-
25% have some breast development [3]. Two to 3% of 45,X
cussed. An algorithm for the evaluation of delayed puberty/
women and 10–15% of women with mosaic 45,X experience
amenorrhea and secondary amenorrhea is shown in Fig. 11.1.
normal pubertal development and menarche but are highly
Primary amenorrhea is the condition of never having had
likely to undergo secondary amenorrhea [3, 4].
menses; secondary amenorrhea is described as discontinuation
Of those who undergo menarche, their only manifestation
of menses. The majority of cytogenetic abnormalities in women
of a sex chromosome abnormality may be short stature. Thus,
blood chromosome analysis is recommended when a woman
has short stature and reproductive failure.
L.M. Randolph, M.D., MA. (*) More than 100 pregnancies have been reported in women
Division of Medical Genetics, Keck School of Medicine,
with a 45,X cell line who did not use assisted reproductive
University of Southern California,
Los Angeles, CA, USA technologies (ART) such as in vitro fertilization (IVF).
Kaneko et al. reviewed the literature on 138 pregnancies in
Division of Medical Genetics, Childrens Hospital Los Angeles,
4650 Sunset Boulevard, MS 90, Los Angeles, CA, USA 62 women with a 45,X cell line, many of whom had a second
e-mail: [email protected] cell line [5]. Thirteen 45,X patients had 21 pregnancies, 22
214 L.M. Randolph
Fig. 11.1 Flow diagram for the workup of a woman with delayed follicle-stimulating hormone, LH luteinizing hormone, Asherman
puberty (Courtesy of Dr. Lawrence Layman.) PCOS polycystic ovary syndrome adhesions from prior uterine curettage
syndrome, CAIS complete androgen insensitivity syndrome, FSH
45,X/46,XX women had 50 pregnancies, 12 patients with mosaic 45,X. In this group, 12 nonmosaic 45,X women’s live
45,X/47,XXX had 20 pregnancies, 44 pregnancies were from born infants had no abnormalities.
14 patients with 45,X/46,XX/47,XXX, and one patient with Magee et al. described a patient with nonmosaic 45,X,
45,X/46,XX/47,XXX/48,XXXX had three pregnancies. based on several tissue studies, who appeared to have had
None was reported in women with a 46,XY cell line without seven pregnancies. Three of them were confirmed. These
IVF. three culminated in a missed abortion, a fetus with 45,X that
Among 13 women with 45,X without mosaicism, six of was terminated, and a healthy baby boy [6].
21 pregnancies ended in spontaneous abortions, and two of The approximately 30% incidence of fetal loss and other
21 ended in stillbirth, including one with hydrocephalus. abnormal outcomes among offspring of 45,X mosaic and
Another three culminated in a pregnancy and/or offspring nonmosaic women should be stressed when providing genetic
with a chromosome abnormality or significant birth defect counseling to these patients, and prenatal diagnosis by chori-
(one with Down syndrome, one with 45,X, one with a partial onic villus sampling or amniocentesis is indicated.
cleft of the soft palate).
Among the liveborn for 45,X and mosaic 45,X patients, Women with 45,X and 45,X Mosaicism
23/102 (23%) had chromosome abnormalities or birth and In Vitro Fertilization
defects. For 16 others in the mosaic group, no outcome infor- Pregnancy rates using ovum donors in centers specializing in
mation was available. Ten percent of liveborn of pregnant in vitro fertilization report pregnancy rates of 50–60%, with
women with a 45,X mosaic cell line had a female child with the endometrial response to estrogen treatment not
ovarian failure with reported karyotypes in their offspring significantly different from that of women with secondary
including 45,X (3%), 45,X/46,XX (4%), 45,X/46,XY (1%), ovarian failure [3]. Cardiovascular and kidney functions are
and 45,X/46,XX/47,XXX (2%). From these data, it appears to be assessed prior to instituting a pregnancy in these
that nonmosaic 45,X live-born offspring are less likely to patients, given the high baseline risks of heart and kidney
have abnormalities than live-born offspring of women with abnormalities in women with 45,X and mosaic 45,X.
11 The Cytogenetics of Infertility 215
Fig. 11.2 An idiogram of the X 22.3

chromosome with locations of
22.2
various deletions and the
corresponding clinical 22.1
characteristics (Reprinted with
21
permission from Simpson and
Rajkovic [9])
11.4
11.3
11.2
11.1
11
12 1° amenorrhea
13 (primary ovarian failure)
2° amenorrhea
(premature ovarian failure)
21
fertility or regular menses
22
23
24
25
26
27
28
Detection of Y Chromosome Sequences failure in about half of women, and the other half experience
in 45,X and Mosaic 45,X Patients menstrual irregularities. Fertility is rare even if menstruation
Among the hypotheses as to why all but one percent of 45,X occurs. If the deletion occurs more distally, such as at band
fetuses die in utero and why some women with apparent p21, patients usually have a milder phenotype with normal
nonmosaic 45,X have some fertility is that 45,X individuals menarche, even though secondary amenorrhea or infertility
may actually be cryptic mosaics for another cell line that sup- is common. Most women with Xp deletions are short, even if
ports survival. It is important to consider too that the detec- ovarian function is normal.
tion of mosaicism is limited by the numbers of tissues and Deletions of the long arm of the X chromosome generally
cells examined. Sometimes, mosaicism is inferred by cytoge- are associated with ovarian failure if they involve the so-called
netic findings in the offspring. In one such case, described by critical region—the region between Xq13 and Xq26. As with
Magee et al., a woman with 45,X had two pregnancies—one deletions of the short arm, more distal Xq deletions are asso-
ending in spontaneous abortion at 8 weeks of gestation and ciated with a milder phenotype. These women may have
the other resulting in a female with 46,X,del(X)(p21) [6]. menarche with or without ovarian failure. Women with dele-
In another case, a woman with apparent nonmosaic 45,X had tions in Xq13 have primary amenorrhea, no breast develop-
a baby girl with 46,X,der(X). Using fluorescence in situ ment, and ovarian failure with high levels of FSH and LH.
hybridization (FISH), one cell of 450 examined in maternal Davison et al. performed cytogenetic analyses on 79 women
lymphocytes showed a der(X). Kocova et al. note in their with primary or secondary amenorrhea, and two of the 79 had
paper describing Y chromosome sequences in Turner syn- an abnormal karyotype. One of them was a woman with
drome that when others evaluated both peripheral lympho- primary amenorrhea and a 46,XY karyotype. The other was a
cytes and fibroblasts, only about 21% of karyotypes of 87 woman with secondary amenorrhea and a deletion at Xq26.1.
live-born Turner syndrome patients were found to be 45,X. This woman had a family history of premature ovarian failure,
Kocova’s group evaluated 18 females with nonmosaic Turner and her mother, who had undergone premature ovarian failure
syndrome by performing chromosome analysis on blood and/ at 28 years, also had this deletion [8]. See Fig. 11.2, which
or skin fibroblasts. In six of these patients, presence of the shows locations of different deletions of the X chromosome
SRY (testis-determining factor, or gene) was detected [7]. and the associated phenotype. See also Chap. 10.
X Chromosome Deletions X Chromosome; Autosome Translocations

X chromosome deletions are usually sporadic, although In a balanced X-autosome translocation, the normal X is
familial cases have been reported. Deletions affecting the generally inactivated. If the abnormal X were inactivated,
short arm of the X chromosome at band p11 result in ovarian autosomal material would be inactivated along with it.
216 L.M. Randolph
Inactivation of autosome genes would probably be a tissue outside of the uterus in so-called chocolate cysts.
lethal event. These cysts occur in the ovary and elsewhere in the pelvis
In an unbalanced X-autosome translocation, the normal X and body. The name of the cysts is due to the appearance of
chromosome remains active, while the abnormal X is inacti- chocolate syrup within the cysts. Endometriosis causes pain-
vated in an attempt to compensate for the imbalance. ful menses as well as infertility and has been reported in
Translocations involving the X chromosome and an auto- sisters fairly often [10, 11].
some are rare, occurring in one in about 30,000 live births Endometriosis is characterized by monoclonal growth
[4]. This relates in part to the fact that all males and half of and can exhibit features of malignant behavior, including
females with this finding are infertile. For women, the local invasion and metastasis. Comparative genomic hybrid-
phenotypic effects depend on the breakpoint and the status ization (CGH), a molecular cytogenetic method that facili-
of inactivation of the X chromosomes. If the derivative X is tates screening of the entire genome for chromosome gains
active in all cells and the breakpoint does not interrupt a and/or losses (see Chap. 17), showed recurrent copy number
functional gene, about half have a normal phenotype and half losses on several chromosomes in 15 of 18 cases of endome-
have ovarian failure. In general, those with ovarian failure trial tissue. Losses of 1p and 22q were each detected in half
have breakpoints within the Xq13–26 region. of the cases. Chromosome 7p was lost in one-fifth of the
For women with an active derivative X, when the break- cases. These results were validated by selective dual-color
points interrupt important genes on either the X or the auto- FISH and were interpreted as indicating that genes localized
some, a single-gene disorder, such as Duchenne muscular to certain chromosome regions play a role in the develop-
dystrophy, may result. When the derivative X is active in ment and progression of endometriosis [12]. A subsequent
only a portion of cells, multiple anomalies and mental retar- case–control study showed that gene variants on chromo-
dation usually result. some 7 and chromosome 1 were found to be associated with
The breakpoints on the X chromosome vary widely in endometriosis [11].
X-autosome translocations. The most common autosomes
involved include chromosomes 15, 21, and 22. The pericen- Hypothalamic and Pituitary Causes of Female
tromeric regions of these chromosomes are predisposed to Infertility
pairing with the X chromosome. Several genes have been identified as the cause of infertility
For non-cytogenetic-inherited causes of ovarian dysfunc- involving malfunction of the hypothalamus or pituitary gland.
tion, please see Table 11.1. Because no cytogenetic testing is helpful in these cases, a
summary of gene-level conditions associated with hypotha-
Endometriosis lamic malfunction is provided in Table 11.2. Table 11.3
Endometriosis is a common disorder that accounts for indicates gene-level conditions associated with pituitary
infertility in 6–10% of women of reproductive age. It is malfunction, and Table 11.4 lists gene-level conditions asso-
characterized by the formation of collections of endometrial ciated with uterine development abnormalities.
Table 11.1 Gene mutations that affect ovarian function [4]

Gene Locus Phenotype Inheritance
FMR1 Xq27.3 Fragile X syndrome; ovarian failure X-linked dominant
SRY Yp11.3 Swyer syndrome; sexual infantilism, normal vagina and uterus, streak gonads with risk for Sporadic; Y-linked
gonadoblastoma +/− germ cell tumor
FSHR 2p21-p16 Primary amenorrhea, half with breast development; men have oligospermia Autosomal recessive
LHCGR 2p21 Anovulation in women; undermasculinization in men Autosomal recessive
CYP17A1 10q24.3 17-hydroxylase deficiency; delayed puberty in women, absent breast development, primary Autosomal recessive
amenorrhea, and elevated gonadotropins
CYP19A1 15q21.1 Aromatase deficiency; cannot convert androgens to estrogens; females with sexual ambiguity, Autosomal recessive
clitoromegaly; no breast development or menses
AIRE 21q22.3 Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED); Autosomal recessive
multisystem autoimmune disease; adrenal, ovarian, and testicular failure may occur
NR5A1 9q33 Steroidogenic factor 1; adrenal failure, sex reversal in men; presents as female phenotype; Autosomal recessive
rare
GALT 9p13 Galactose-1-phosplate uridytransferase; causes galactosemia; normal puberty; half with Autosomal recessive
primary and half with secondary amenorrhea; 67% of women have ovarian failure and
testicular function apparently not affected. Heterozygotes (carriers) not affected
Table 11.2 Gene mutations affecting hypothalamic functions in This chapter will provide an overview of causes but provide
females [4] detail only on cytogenetic and molecular cytogenetic causes.
Gene Locus Phenotype Inheritance
KAL Xp22.32 Kallmann syndrome; X-linked recessive
hypogonadotropic The SRY Gene and Genetic Sex
hypogonadism, anosmia;
affects only males
NR0B1 Xp21.3 Adrenal hypoplasia; X-linked recessive
The presence of the SRY (sex-determining region Y) gene on
congenital and hypogo- the short arm of the Y chromosome induces differentiation
nadotropic hypogonadism of precursor cells into Sertoli cells, which express anti-
LEP 7q31.3 Obesity, hypogonadotropic Autosomal recessive Müllerian hormone. Anti-Müllerian hormone, which is also
hypogonadism; delayed known as Müllerian-inhibiting substance, causes regression
puberty
of Müllerian structures—the fallopian tubes, uterus, and
LEPR 1p31 Obesity, hypogonadotropic Autosomal recessive
hypogonadism, and upper vagina—and the production of testosterone in the Leydig
elevated serum leptin cells. The Leydig cells are thought to differentiate because
of messages from the Sertoli cells. Testosterone leads to the
formation of internal male genitalia, such as epididymis, vas
deferens, seminal vesicles, and ejaculatory duct. The produc-
Table 11.3 Gene mutations affecting pituitary function in females [4]
tion of dihydrotestosterone results in the formation of the
Gene Locus Phenotype Inheritance penis, testes, prostate gland, and urethra. Secretion of insulin-
GNRHR 4q21.2 Hypothalamic Autosomal recessive like hormone 3 by the Leydig cells causes the descent of the
hypogonadism
testes [15].
HESX1 3p14.3 Septo-optic Autosomal recessive
dysplasia About 10% of infertile men have severe defects in sperm
LHB 19q13.3 Isolated luteinizing Autosomal recessive production, and it is in this group of men that many of the
hormone deficiency cytogenetic and genetic disorders are concentrated [16].
FSHB 11p13 Isolated follicle- Autosomal recessive Hackstein et al. note that in the fruit fly Drosophila, there is
stimulating hormone evidence that up to 1,500 genes contribute to male fertility
deficiency
[17]. Much more work remains to be done in humans, in
PROP1 5q35.3 Short stature, Autosomal recessive
hypothyroidism, and
whom several genes have been found to be involved in early
hypogonadotropic sexual development, but many remain to be discovered. In
hypogonadism this chapter, cytogenetic and molecular cytogenetic causes of
male infertility will be discussed. See Table 11.5 for infor-
mation on other genetic causes of male infertility.
Table 11.4 Gene mutations affecting uterine development [4] In general, men with infertility and a normal semen analysis
Gene Locus Phenotype Inheritance are less likely to have a cytogenetic or molecular cytogenetic
AR Xq12 Androgen insensitivity X-linked recessive basis for their infertility. However, men with normal sperma-
syndrome (male, 46,XY, tozoa concentrations but whose spermatozoa do not fertilize
phenotypic female)
also have an increased risk of a constitutional chromosome
HOXA13 7p15.2 Hand-foot-uterus Autosomal dominant
syndrome
abnormality. In a study of 400 men who were to undergo
intracytoplasmic sperm injection (ICSI), 6.1% of the azoo-
spermic men and 2.7% of the oligospermic men were found
to have constitutional chromosome abnormalities, and 7.4%
Causes of Male Infertility of the men with normospermic analysis also had constitu-
tional cytogenetic abnormalities [20].
A standardized approach to the evaluation of an infertile
man was published by the World Health Organization (WHO)
in 2004 [13]. This manual delineates several diagnostic Semen Analysis
categories for male infertility, including acquired and idio-
pathic causes. Among genetic causes are chromosome disor- Semen analysis is usually performed on a sample that has
ders, genetic disorders that affect reproduction only, and been ejaculated into a specimen cup. The volume and pH of
genetic disorders with other effects which also are associated the semen are measured, and the concentration, morphology,
with infertility. Although many advances have been made in and motility of the spermatozoa are analyzed under a
the field of male factor infertility, it is estimated that the microscope. Cellular debris is examined to determine
cause of about 30% of male infertility is still not known [14]. whether an infection is present, and fructose is measured as
218 L.M. Randolph
Table 11.5 Percentage of certain cytogenetic or microdeletion findings in various studies of men with infertility
% of men with
chromosome
Cytogenetic finding abnormality Sperm count Motility Morphology
Robertsonian translocations 6a; 0.7b; 1.6c £5.6 × 106/mLa; 0.8–12 × 106/mLb; Normal to very lowa; NSb; Poora; NSb; 11 of 15
1 of 15 with 0 and 14 of 15 with 13 of 15 with OA or OATc with OATc
<106 per mLc
Reciprocal translocation 0.97a; 0.3b; 0.94c 0a; 0–1 × 106/mLb; 4 of 9 with 0 5 of 9 with OA or OATc 2 of 9 with OATc
spermc
46,X,del(Y)(q11q22); 1.9a; 2.1b; 0.21c 0a; 0 in 7 of 17b; “oligo” in 10 of 2 of 2 with OATc 2 of 2 with OATc
del(Yq)b; 46,X,del(Y)(q12)c 17b; <106 per mLc
47,XXY 2.5b; 5.4c 0a; 0 in 49 of 52c; <106 per mL in 3 of 52 with oligo onlyc 3 of 52 with oligo onlyc
3 of 52c
47,XYY;46,XY/47,XXY; 0.8b; 1.0c 0 – –
47,XXY/48,XXXY;46,XX;
45,X/46,XY
46,XY,inv(1)—pericentric 0.97a; 0.1c 0a OAc –
46,XY,inv(9)(p23q33) 0.1b 6 × 106 per mLb
Other pericentric inversions: Together, 0.4c <106 per mL for all but inv(Y), inv(1) and inv(3), OAc inv(2) and inv(5), OATc
Two with inv(2)(p11q13), which had 0 sperm countc
one inv(3)(p13q25), one
inv(5)(p13q13), and one
46,X,inv(Y)(p11q11)
46,XY,inv(9)(p11q12) 2.7b 0 in 9 of 22b; “oligo” in 13 of 22b – –
Variants: Yqh+, 21 ps+, 7.2b 0 in 35 of 78b; “oligo” in 43 of 78b – –
22 ps+, 1qh+, 13 ps+,
14 ps+, 15 ps+, 16qh+
46,XY – 0a 0a –
OA oligoasthenospermia, OAT oligoasthenoteratospermia
a
Retrospective study over 5-year period of normally androgenized men with azoospermia, oligospermia, and normospermia with normal or
subnormal testicular volume [18]
b
Cytogenetic study of 820 men with 2-year infertility and who had azoospermia or oligospermia. The common inversion of chromosome 9 and
various chromosome variants were included in their study [19]
c
Bor et al [20]
an indicator of obstruction. Spermatozoa counts are designated of the azoospermic men (34.1%), and two of the oligosper-
as the number present per mL. A normal number as defined mic men (3.3%), had a constitutional chromosome abnor-
by WHO is 20 × 106 sperm per mL of semen [13]. However, mality. The most common abnormality was 47,XXY [22].
in a study of 430 couples in Denmark having unprotected In a review of the chromosomal contribution to male
sex, the probability of conception increased with increasing infertility, Van Assche et al. reported on the chromosome
spermatozoa concentration to 40 × 106 per mL. Above that constitution of about 8,000 infertile men and compared
level, there was no additional likelihood of pregnancy. The the findings to the chromosome constitution of a group of
authors suggested that the WHO guidelines should be used newborn children. In the infertile group, the incidence of sex
with caution, as some men above the normal range may be chromosome abnormalities was 27 times higher (3.8 vs.
subfertile [21]. 0.13%), and the incidence of autosome abnormalities was
five times higher (1.3 vs. 0.25%) [23]. When considering
Oligospermia, Non-obstructive Azoospermia, men with oligospermia only, pooled data show a frequency
and Teratozoospermia of chromosome abnormalities of 4.6%. For men with azoo-
Oligospermia, also called oligozoospermia, is defined as spermia, the pooled data show a frequency of 13.7% [23].
having a low spermatozoa count in an ejaculate. Azoospermia In a separate cytogenetic study of 1,007 infertile men,
is the absence of spermatozoa, and teratozoospermia indi- major chromosome abnormalities were seen in 62 (6.2%).
cates abnormally formed spermatozoa. The concentration, Of those, 38 (3.8%) had sex chromosome abnormalities, and
morphology, and motility of spermatozoa are important 24 (2.4%) had autosomal chromosome abnormalities. Of
factors in achieving conception. those with sex chromosome abnormalities, 28 were 47,XXY,
Gunduz et al. performed chromosome analysis on 41 men three were 47,XYY, and seven had a Y chromosome with
with azoospermia and 61 men with oligospermia. Fourteen a structural abnormality. Of the autosomal abnormalities,
10 were reciprocal translocations, eight had Robertsonian type are represented more frequently among infertile men
translocations, five had an inversion, and one had a ring chro- (0.26%) than in newborn males (0.07%). Their semen analyses
mosome. The likelihood of a chromosome abnormality was are usually normal, as previously noted, but in a minority of
higher in men with a sperm density of <5 × 106/mL, an cases, they have severe abnormalities of spermatozoa number,
FSH ³ 30.1 mLU/mL, an LH ³ 8.9 mLU/mL, and a testoster- motility, and/or morphology [26]. This was observed in a
one value £ 2.69 ng/mL [24]. study of 47,XYY males by Martin, in which no sperm was
In a review by Martin, the author noted that men with any disomic for sex chromosomes by chromosome analysis;
type of infertility, whether low count, abnormal motility, or 10,000 sperm studied by FISH showed an increase for XY
abnormal form, had an increased frequency of sperm chro- disomy to 0.6%. Martin noted other labs had reported
mosomal abnormalities, from 2 to 10 times higher than that increased frequencies of sperm aneuploidy for sex chromo-
of controls. She concluded that any abnormality of spermato- somes ranging from 0.3 to 15% [25].
genesis confers an increased risk of aneuploid sperm. The
highest risk was seen in macrocephalic, multinucleated, Autosomal Abnormalities
multiflagellate sperm, in which cases the risk of aneuploidy
and polyploidy is 50–100% [25]. The most common autosomal abnormalities seen in infertile
men are Robertsonian translocations (see Chap. 9). In the
aforementioned review by Van Assche, the incidence of
Sex Chromosome Abnormalities infertile men with this finding was 0.7% [23]. This was 8.5
times the incidence in the newborn survey used for com-
(See also Chap. 10.) parison. It appears that the increased frequency of the X-Y
Among men with infertility, the most frequent cytoge- bivalent and the trivalents formed by the chromosomes
netic findings are 47,XXY and 47,XXY/46,XY. Men with involved in Robertsonian translocations is correlated with
this chromosome constitution commonly have the clinical the extent of germ cell impairment [27] (see also Chaps. 2, 9,
features of Klinefelter syndrome, which include essentially and 10). Martin noted in seven men with Robertsonian trans-
normal appearance at birth but for a slightly small head; locations that the actual frequency of unbalanced sperm by
delayed puberty; higher incidence of gynecomastia than chromosome analysis is lower than theoretically expected,
other males have; and small, firm testes with hyalinization of with 3–27% of sperm being unbalanced because of the
seminiferous tubules. Intelligence is usually normal, with translocation. FISH studies showed 7–40% of Robertsonian
performance IQ normal and verbal IQ below normal on aver- translocation heterozygotes were unbalanced, with a mean of
age. Reading skills may be a problem [24]. These men have 15% [25]. When Huang et al. looked at characteristics of
hypergonadotropic hypogonadism and azoospermia or very embryo development in Robertsonian translocation carriers,
severe oligospermia. Although many of these men are diag- they noted that by day 3, whether or not the embryos have a
nosed as boys, others are not diagnosed until such time as chromosome abnormality could not be determined based
they are seeking the cause for their infertility. Given the inci- upon morphology, but by days 5–6, this was evident. On day
dence at newborn screening, it appears most males with 3 after fertilization, there were 161 high-grade embryos,
47,XXY or 47,XYY do not come to diagnosis. Studies of including 59 normal/balanced embryos and 102 abnormal
sperm chromosomes have shown that the frequency of aneu- ones, whereas by days 5–6, the blastocyst percentage in the
ploidy for sex chromosomes varies from 1.5 to 7% in sperm normal/balanced embryo group was significantly higher than
from 47,XXY mosaics and from 2 to 45% in the sperm of that in the abnormal embryo group (44 vs. 20%, p = 0.0000)
non-mosaic 47,XXY men [25]. The majority of the babies [28]. Huang also noted that several studies had shown a
born are chromosomally normal, but there is an increased strong prevalence of alternate segregation in the gametes of
risk for aneuploidy in the offspring of these men. Robertsonian carriers.
Men with 47,XYY or 47,XYY/46,XY karyotypes are The review by Van Assche, which pooled data from
usually fertile and typically have normal semen analyses [24]. several studies, also indicates that 0.5% of men with infer-
They are slightly taller than their chromosomally normal tility had reciprocal translocations, as compared to 0.1% in
brothers on average and on average have a normal IQ. About the newborn population. The association between reciprocal
half have learning disabilities requiring special education. translocations involving chromosomes 3–7, 9, 11, 13–15, 16, 17,
[24]. The incidence of men with 47,XYY is about the same and 19–22 and the impairment of sperm production has been
as that of 47,XXY in the general population; each is present documented in several studies [14]. Chromosomes from men
in about one in 1,000 newborns [24]. However, in infertility with reciprocal translocations involving these chromosomes
surveys—for example, the study by Gunduz et al., noted have been observed, at the pachytene stage of meiosis, to
previously—the finding of 47,XXY is about nine times as have a high frequency of centromeric contacts and chain
frequent as that of 47,XYY [22]. Men with a 47,XYY karyo- configurations between the translocation quadrivalent and
220 L.M. Randolph
the X-Y bivalent (see Chap. 10). These were not seen to any that were not present in the fertile fathers and brothers of the
significant degree in the chromosome preparations of the men. These were the first deletions found on the Y chromo-
men with reciprocal translocations involving other chromosome; some are detectable by conventional cytogenetic test-
somes. Martin confirmed this finding in her review, noting ing, whereas others are not (see Chap. 9). Thus, molecular or
that this significant number of quadrivalents was attached to molecular cytogenetic testing is required to detect these dele-
the sex body in the azoospermic carrier and not in the normo- tions. This deleted region was called AZF for azoospermia
zoospermic carrier. Her interpretation is that unpaired regions factor. The AZF region has 31 Y-specific genes, 14 of which
within the quadrivalent are likely to be detected by the are protein coding and the rest non-protein coding genes,
pachytene checkpoint, so asynapsed regions seek each other which have been subdivided as described later [33]. It is now
out and try to pair in order to escape the checkpoint and estimated that microdeletions of the Y chromosome are pres-
apoptosis of the cell. She also noted that sperm karyotyping ent in 8–15% of men with non-obstructive azoospermia or
studies of 37 reciprocal translocation heterozygotes have severe oligozoospermia—that is, men with a spermatozoa
shown that 19–77% of sperm are unbalanced. FISH analyses count of <5 × 106 per mL [34, 35].
in 99 reciprocal translocation heterozygotes have also The Y chromosome has been continually subdivided over
shown a large range in the frequency of unbalanced sperm, the years into more refined regions. These subdivisions have
from 37 to 91% [25]. been developed on the basis of nonoverlapping deletions in
patients with non-obstructive azoospermia or oligospermia.
Inversions The original AZF region now consists of AZFa, AZFb, and
It has been suggested that paracentric inversions are harm- AZFc [35]. These microdeletions are associated with various
less, but recombinant chromosomes have been observed in histopathologies and abnormal semen parameters, as shown
newborns, and the risk of viable recombinants has been esti- in Table 11.7. These are not strict categories, but some
mated at 3.8% [25]. Two men with paracentric inversions generalizations can be made.
were studied by sperm karyotyping. Neither showed recom- Many studies have been published about the incidence of
binant chromosomes in sperm; one case was studied by microdeletion of the Y chromosome in men with infertility.
FISH, and 1% of sperm were recombinant, with both dicen- The results have varied significantly, probably because of
tric and acentric chromosomes seen [29]. So the risk for selection criteria and because of differing numbers of
paracentric inversions appears to be low. sequence-tagged sites (STS), known stretches of DNA that
Sperm karyotyping has been performed in seven men can be amplified by polymerase chain reaction (PCR).
with pericentric inversions. Four had no recombinant chro- However, some findings appear consistent. These deletions
mosomes, and three had frequencies of imbalance from 11 to are found primarily in men with azoospermia or severe oli-
31%. FISH studies have been performed in 24 pericentric gospermia. The most common deletions are AZFc or AZFc
inversion carriers with the frequency of recombinant chro- plus AZFb, which together comprise more than three-fourths
mosomes varying from zero to 54%. The inversions that of deletions. AZFa deletions occur in fewer than 5% of men
produce recombinant chromosomes are large ones encom- with AZF deletions [15].
passing more than half of the length of the chromosome in These deletions are thought to arise de novo from fertile
most cases. An overall risk at prenatal diagnosis has been fathers with an intact Y chromosome; as such, they represent
estimated at 10–15%, but the risks are clearly dependent on one of the most frequent structural chromosome abnormal-
the individual inversion. ities, affecting one in about 5,000 males [36]. Previous
In Zuffardi and Tiepolo’s review of 7,277 men, the range reports have shown that boys born from oligospermic men
of autosomal abnormalities was 0.6–1.6%, with an average treated using ICSI have an increased risk of carrying a Y
of 1.1% [30]. Overall, the incidence of balanced translocations chromosome microdeletion [34]. This suggests that these
was 8.9 per thousand, which is six times greater than 1.4 per deletions can exist in a mosaic state in the testes of some
thousand newborns they used as a control population. For men. This was seen to a limited degree in a study by Le
Robertsonian translocations, the incidence in infertile men Bourhis et al. in a study of 181 infertile men with azoo-
was 10 times higher than in newborns—5.9 per thousand vs. spermia or severe oligospermia (sperm count <3 × 106 sper-
0.6 per thousand [30]. For a comparison of chromosome matozoa/mL) [36]. Of these, 18 had an abnormal karyotype,
abnormalities seen in studies of infertile men, see Table 11.6. and of the remaining 163, six (5.5%) were shown to have a
microdeletion of the Y chromosome. Two of the men, both
Microdeletions of the Y Chromosome with oligospermia, had germ cell mosaicism of 1.97 and
The fact that genes necessary for spermatozoa production are 4.13%, respectively, of spermatozoa with a deleted Y
on the long arm of the Y chromosome was first described in chromosome.
a study published in 1976 by Tiepolo and Zuffardi [32]. They A telling study was performed by Krausz et al., who studied
studied six azoospermic males and found deletions at Yq11 131 infertile males for the presence of a Y chromosome
11
The Cytogenetics of Infertility
Table 11.6 Constitutional cytogenetic studies of infertile men (includes men with azoospermia and oligospermia) [30, 31]
Other sex Y chrom
N 47,XXY (%) 47,XXY/46,XY(%) chrom abn(%) struct abn(%) 46,XX (%) Rob T (%) Rec T (%) Inv (%) + Mar (%) Other (%)
2,247 139a (6.2) – 6 (0.26) 15(0.67) 3(0.13) 25(1.1) 5(0.22) 1 (0.04) 2 (0.09) –
153 9 (5.8) 3 (2.0) 2 (1.3) 3 (2.0) 1(0.65) 2 (1.3) 2 (1.3) – – –
342 2 (0.58) 2 (0.58) 2 (0.58) – – 2(0.58) 1(0.29) 1 (0.29) – –
2,372 24 (1.0) – 7 (0.30) 2 (0.08) – 4(0.17) 10(0.42) – 4 (0.16) –
281 57(20.3) 1 (0.36) 6 (2.1) 1 (0.36) 2(0.71) 3 (1.1) 5 (1.8) – – –
57 9 (15.8) – 1 (1.8) 1 (1.8) – – 3 (5.2) 1 (1.8) 2 (3.5) –
1,000 21 (2.1) – ?b ?b – 2 (0.2) – – – –
2,542 147 (5.8) NA 7 (0.26) 18 (0.7) 3 (0.10) 26(1.0) 6 (0.24) 1 (0.04) 3c (0.12) 4d (1.5)
1,363 57 (4.2) NA 1 (0.07) 11 (0.81) 3 (0.22) 4c (0.29) 20e (1.5)
Total 10,357 465 (4.5) 6 (0.06) 31 (0.30) 40 (0.39) 10 (0.10) 75 (0.72) 35(0.34) 4 (0.04) 15(0.15) 24 (0.23)
Newborns 39/36,855 (0.11) – –f – – 51/59,514 (0.09) 55/59,514 (0.09) 7/59,514 (0.01) 13/59,514 (0.02) –
Abbreviations: N number of men studied in that series, Other sex chrom abn other sex chromosome abnormalities, Y chrom struc abn Y chromosome structural abnormalities, Rob T
Robertsonian translocation, Rec T reciprocal translocation, inv inversion, mar marker chromosome
NA not applicable; mosaics included in 47,XXY column in these surveys
a
Includes 47,XXY and 47,XXY/46,XY
b
Includes six cases described as XYY, deletions, and rings
c
Described as an “extra G”
d
del(14p); del(15)p; fragile site at 16q22; “ring E”
e
Includes several heteromorphisms and other variants not generally reported in other series
f
Frequency of XYY in newborn series was 33 in 36,855, or 0.09%
221
222 L.M. Randolph
Table 11.7 AZF gene regions and their usual phenotypes [35] DBY, and UTY) and DDX3Y, both thought responsible for
Gene region Phenotype the infertility phenotype observed. Men with deletions of the
AZFa Absence of germ cells, aka Sertoli cell-only syndrome AZFa region have lost the USP9Y gene, resulting in no germ
(SCOS) cells being present. These genes are expressed widely and
AZFb Maturation arrest at spermatocyte stage have homologs on the X chromosome [15].
AZFc Variable from SCOS to severe oligospermia In summary, the genes KDM5D in AZFb and DAZ and
CDY in AZFb/c are thought to represent key determinants for
spermatogenesis, in addition to DDX3Y in AZFa, although
microdeletion. Of this group, 46 were idiopathic and 85 were much is yet to be learned [33].
not. Nineteen percent of idiopathic males with normal 46,XY
chromosomes had microdeletions of the AZFa, b, or c region.
Of the group with known causes of infertility, 7% were found Intracytoplasmic Sperm Injection
to have microdeletions and a 46,XY chromosome comple-
ment, including deletions of the AZFb and c regions. They Intracytoplasmic sperm injection (ICSI) is the physical inser-
recommend that all males with reduced or absent sperm tion of a spermatozoan into the ovum in a laboratory setting.
counts seeking assisted reproductive technologies be Figure 11.3 is a photograph demonstrating ICSI. The sper-
screened for microdeletions of the Y chromosome [37]. matozoan may be extracted prior to the site of obstruction or
The presence of the deletions has been suggested to create from the testis, or it may be taken from several collections of
instability of the Y chromosome, as noted by Martin [25]. semen that have been ultracentrifuged to collect any sperma-
She described 12 mosaic 45,X/46,XY patients with Turner tozoa that might be present. Using micromanipulation tech-
syndrome traits or sexual ambiguity, and one-third had Y niques, an embryologist grasps the ovum with one instrument
chromosome microdeletions. In another study described in and the spermatozoan with another and injects the spermato-
the review, FISH studies were performed on 11 men with zoan into the cytoplasm of the ovum. This has been used for
AZFc deletions, and only 33% of Y-bearing sperm were several years for men with 47,XXY and for men with severe
found compared with 49% in controls. It was suggested that male factor infertility for other reasons.
men with a Y chromosome microdeletion might have an Although this technique has allowed people to have bio-
increased risk of 45,X and 47,XXY offspring as well as logical children who would not otherwise have been able to,
mosaic offspring because of loss of the Y chromosome. serious consideration of transmissible genetic conditions to
Several genes and gene families have been isolated from offspring must be given, and genetic counseling should be
the AZFb and AZFc regions [33, 38, 39]. One family is offered to couples prior to undergoing the procedure.
called RBMY, or RNA-binding motif. It consists of genes Some of the reasons for this have been discussed previ-
and pseudogenes that encode proteins involved in pre-mRNA ously. A man with a microdeletion of the Y chromosome
processing and transport [33, 40]. Unlike other such genes, would be expected to pass on the Y chromosome with the
however, it is expressed only in the testes. The functional deletion to his sons—in other words, to make his infertility
copies of RBMY are in the AZFb region [41]. A homolog for an inherited trait. One group studied the Y chromosome of
RBMY exists on the X chromosome [42, 43]. Delbridge 86 men who fathered 99 sons by ICSI to determine the
et al. suggested that RBMY and its homolog on the X chro- incidence of vertical transmission and de novo deletions in
mosome, RBMX, evolved from a gene on the mammalian the sons [45]. Two of the men, or 6.9%, were found to have
proto-X and proto-Y pair at least 130 million years ago an AZFd/c deletion, and identical deletions were seen in
before the divergence of eutherian and metatherian mam- their sons. No de novo deletions were detected in any of the
mals [42]. Other important genes are the KDM5D gene on remaining 97 sons of the other men, who did not have dele-
AZFb and in AZFb/c the CDY gene [33]. tions themselves.
Another family of genes is from the AZFc region. It is Levron et al. evaluated the risk for cytogenetic abnor-
called the DAZ (deleted in azoospermia)/SPGY (spermatogen- malities in offspring of men with nonmosaic 47,XXY or
esis gene on the Y) family. This group of genes, thought to Klinefelter syndrome [46]. They obtained testicular biopsies
have four copies on the Y) chromosome, also codes for RNA- from 20 patients and found testicular sperm in eight of them.
binding proteins. A homologous gene to DAZ called DAZL1, Four couples became pregnant following ICSI. Sperm chro-
for DAZ-like 1, is on chromosome 3 at band p24. This gene is mosomes were analyzed in five patients. Of 112 sperm, seven
expressed in ovarian cells as well as in the testes. Most of the (6.3%) had chromosome abnormalities, of which five were
genes on the Y chromosome are expressed more widely and sex chromosome abnormalities and two had monosomy 18.
have homologs on the X chromosome [33, 43, 44]. Six children were born, all with normal karyotypes. The
In the AZFa region, two main genes have been localized. authors discussed the probability of normal germ cell lines as
They are known as USP9Y (formerly known as DFFRY, the origin of sperm with normal chromosomes in these men.
Fig. 11.3 Intracytoplasmic

sperm injection (ICSI). In this
photograph, a technologist is
viewing an ovum and
spermatozoan under a
microscope and injecting the
spermatozoan into the cytoplasm
of the egg (Courtesy of
ViaGeneFertility.com.)
As for the studies that show a high risk of sex chromo- spermatozoa—showed an elevated Y to X ratio and elevated
some abnormalities in men with gonadal failure and low aneuploidy to diploidy rate. The authors concluded that
FSH undergoing ICSI, they postulate that their spermatozoa patients with the first three forms of teratozoospermia are
undergo meiotic errors as part of their underlying disorder good candidates for ICSI, and patients with macrocephalic
and therefore have a higher risk of having offspring with sex spermatozoa are not.
chromosome abnormalities. However, in his review of genetic risks of ICSI, Johnson
This suggestion was supported by the findings of Giltay cited a publication that suggested spermatozoa with amor-
et al., who examined semen specimens from seven severely phous, round, and elongated heads are associated with an
oligospermic ICSI candidates and compared results to three increased frequency (26%) of structural chromosome abnor-
normal specimens and to ten ICSI candidates with normal malities when compared with that of morphologically normal
constitutional karyotypes but with oligoasthenoteratozoo- spermatozoa [27].
spermia (OAT) [47]. Six of the ICSI candidates had a numer- Bonduelle et al. performed a study to determine whether
ical sex chromosome abnormality, including 45,X/46,XY prenatal cytogenetic abnormalities after ICSI could be related
mosaicism, 47,XXY/46,XY mosaicism, and 47,XXY. One to sperm parameters [49]. Of 1,586 fetuses, chorionic villus
man had an inversion of the Y chromosome. Chromosome sampling (CVS) was performed on 698, and amniocentesis
aneuploidy rates for chromosomes 18, X, and Y by FISH was performed on 888. Of these, 47 (3%) had abnormal
were high in the ICSI candidates with and without constitu- karyotypes; 25 of these (2%) were de novo. They found a
tional chromosome abnormalities, both for the sex chromo- 2.1% de novo prenatal chromosome abnormality rate for
somes and chromosome 18, compared to the normal controls. sperm concentrations, of <20 × 106 per mL and 0.24% abnor-
They conclude that males with sex chromosome abnormali- mality rate for sperm concentrations of 20 × 106 per mL or
ties have no higher risk of producing offspring with a sex greater. The likelihood of a chromosome abnormality was
chromosome abnormality by ICSI than do OAT males with associated with spermatozoa motility and concentrations, but
normal karyotypes. not morphology in this study. The de novo chromosome
Viville et al. examined the role of morphology of sperma- abnormality rate of 1.6% vs. 0.5% risk for women aged
tozoa and chromosome abnormalities of the spermatozoan 33.5 years (p < 0.007) was seen in ICSI offspring; most of the
[48]. They examined specimens from a patient with short- increase was in sex chromosome abnormalities, while some
ened flagella syndrome, a patient with globozoospermia, a were due to autosomal chromosome abnormalities.
patient with spermatozoa with irregular acrosomes, and a
patient with macrocephalic spermatozoa with associated
multiple flagella. From 1,656 to 5,000 spermatozoa were Effect of Chromosome Abnormalities
analyzed from patients and 5,064 to 7,423 spermatozoa from on Preimplantation Embryo Development
controls. They employed three-color FISH and found that
patients one through three had signals that compared with A study to evaluate the influence of aneuploidy upon preim-
normal controls. Patient 4—the one with macrocephalic plantation embryo development was conducted by Rubio
224 L.M. Randolph
et al. [50]. They evaluated 6,936 embryos from 1,245 women Fifteen of 578 newborns in this study had birth
undergoing PGD for various indications. Embryo biopsy was defects—nine major and six minor—and this frequency of
performed on day 3. Normal euploid embryos showed 2.6% compared to the frequency seen in the IVF frequency
significantly higher blastocyst rates of 68.2% compared to seen in that center. The major birth defects were Goldenhar
chromosomally abnormal (42.8%) and mosaic (53.7%) syndrome, ventricular septal defect (VSD), hypoplastic right
embryos (all p < 0.0001). Among autosomes, higher blasto- heart and pulmonary stenosis, pyloric stenosis, cleft palate,
cyst rates were seen in trisomies than monosomies, although aqueductal stenosis, spina bifida, and hydronephrosis [2]. All
statistically significant only in women over 36 years of age. of those were seen in multiple gestations except the VSD,
For sex chromosome aneuploidy, similar blastocyst rates which was present in a singleton. The minor birth defects
were seen between trisomies and monosomy X. were hypospadias, urethral obstruction, and double ureter
[4]. Hypospadias was seen in two singletons and a set of
twins, and the double ureter was present in a twin. The ure-
Chromosomal Mosaicism in Pregnancies thral obstruction was seen in a singleton. The conclusion of
from Couples with Infertility this study was that standard IVF and ICSI are similar in preg-
nancy evolution and in incidence of birth defects.
Huang et al. evaluated 5,337 consecutive chorionic villus More recently, Simpson et al. examined ICSI data from the
sampling specimens and found 1.29% were mosaic. Those United States in 1997 [54]. Of 6,077 ICSI cycles begun, there
from spontaneous pregnancies versus from infertility treat- were 17.5% pregnancy losses, and that same cohort showed a
ment were 1.22 vs. 1.32%, respectively. A subgroup of infer- malformation rate of 1.7% in the live-born babies. In their
tile couples comparing in vitro with in vivo fertilization paper, they note that the Swedish IVF Registry of 1,139 ICSI
showed rates of 1.84 and 0.41%, respectively, which is not babies listed a relative risk of 2.9 for hypospadias. Birth
statistically significant [51]. weight and prematurity rates in ICSI were found to be similar
to those of conventional IVF. In a cohort of 1987 pregnancies
in Brussels, de novo autosomal rearrangements of 0.36% and
Pregnancy Rates, Obstetric Outcomes, de novo sex chromosome abnormalities of 0.83% were seen,
Chromosome Abnormalities, and Birth both higher rates than are expected in the general population.
Defects After ICSI This raises the question of whether these abnormalities are
de novo in fact or would have been considered to be inherited
In 2006, 138,198 ART procedures were reported to the if more intensive genetic study of their parents had been car-
Centers for Disease Control, resulting in 41,343 deliveries of ried out before ICSI had been initiated.
liveborn and 54,656 babies. They accounted for 1% of live After Bonduelle et al. published a prospective follow-
births nationwide and 18% of multiple births. All but 12% of up study of 423 children born after ICSI in 1996, Kurinczuk
the centers providing ART provided outcomes data to the and Bower published a different interpretation of their data
CDC. The question of ICSI was evaluated separately. No [55, 56]. They applied the Western Australian classification
clear advantage was found of using ICSI when treating system of birth defects to their own population registry of
couples with no indication of male factor infertility. Cycles children and to the Belgian data reported by Bonduelle
using ICSI with no indication of male factor infertility were et al. They determined that the Belgian children were twice
less likely to fail before transfer but more likely to result in as likely as Western Australian babies to have a major birth
implantation failure, pregnancy loss, and a lower overall defect (7.4 vs. 2.3%, odds ratio 2.3) and almost 50% more
chance of live birth [52]. likely to have a minor birth defect (odds ratio 1.49). These
Palermo et al. performed a study in New York City of 751 reports highlight the importance of using a standard sys-
couples in whom 987 ICSI cycles were undertaken [53]. The tem of classification when reporting outcomes measures.
male partner was thought to be the cause of repeated in vitro As an example, the Belgian data considered coronal hypos-
fertilization (IVF) failures. The pregnancy rate was 44.3%, padias and renal duplication to be minor defects, while in
defined as the detection of a fetal heartbeat, with a delivery the Western Australian system these were considered to be
rate per ICSI cycle of 38.7%. In eight of the 11 spontaneous major defects. Results suggested an excess occurrence of
abortions for which cytogenetic information was available, cardiovascular, gastrointestinal, and genitourinary defects
an autosomal trisomy was found, and seven additional preg- generally. The authors report their findings with caution,
nancies were terminated because of a chromosome abnor- as the numbers are small.
mality after prenatal diagnosis. An equal number were The Centers for Disease Control reported that septal heart
delivered vaginally versus by Caesarean section; about half defects, cleft lip with and without cleft palate, esophageal
of those delivered by Caesarean section were multiple atresia, and imperforate anus were more common in babies
gestations. conceived by ART, with and without ICSI. The relative risks
were 2, 2.4, 4.5, and 3.7, respectively. The data were derived survey studies, whereas the only case–control study had
from mothers of about 13,500 babies born with birth defects shown the estimated prevalence of ART in BWS populations
and mothers of more than 5,000 babies without birth defects to be as high as 10.8% [61]. Their assessment based upon
born from October 1997 through December 2003 in 10 states. studies to date was that there is likely an association between
The findings applied to singletons only. About 1% of the BWS and ART. The association between AS and ART was
babies without birth defects were conceived through ART, described as more tenuous, and for Russell-Silver syndrome,
compared to 2.4% of those with birth defects [57]. it is inconclusive. For maternal hypomethylation, although
The contribution of male age to outcomes in ART was evidence is limited, there is no suggestion of an association
studied by Whitcomb et al., who reviewed 1,392 cycles from between ART and Russell-Silver syndrome. For retinoblas-
1,083 women and their male partners. They found no toma, only one study has shown a significant association,
significant association with the likelihood of live birth after although the sample sizes have been small.
adjustment for female recipient age [58]. Manipalviratn et al. agreed with the conclusions regarding
BWS, AS, and retinoblastoma and did not comment on the
other disorders [62]. Both expressed the need for prospec-
Imprinting, IVF, and ICSI tive, larger studies to address these questions, and neither
recommended testing of pregnancies for these disorders,
A review of the troublesome phenomenon of imprinting although counseling was recommended.
errors in the offspring conceived by in vitro fertilization, with This discussion makes it clear that more careful, prospec-
or without ICSI, was published by Gicquel et al. [59]. In this tive follow-up of children born after IVF and other assisted
report, they note that in their series of 149 patients of reproductive techniques must be carried out and that infor-
Beckwith-Wiedemann syndrome (BWS), six were born mation should be made readily available to people who are
following IVF, two of whom involved ICSI. All six showed contemplating such procedures. In the meantime, prospec-
demethylation of KvDMR1, a finding seen in 90 of the 149 tive parents should be told that the birth defect rate after ART
patients. Demethylation of KvDMR1 is an epigenetic, or may be twice that of the general population and that much
imprinting, abnormality. In the same review, they note other remains to be learned.
reports of BWS after IVF, and in all patients studied, the
same demethylation finding was present. They estimate an
odds ratio of 3.2 for the risk of BWS after IVF compared to And What About the Mitochondria
that of the general population. In addition, they comment on of the Spermatozoa?
three patients born with Angelman syndrome (AS) and
imprinting defects, which in Angelman syndrome is a rare Does the offspring inherit paternal mitochondria after ICSI?
finding. Menezo et al. studied some of the biochemical and After all, the entire spermatozoa is injected into the ovum.
metabolic pathways in oöcytes and embryos that might have Two groups have examined this question, and to a sensitivity
relevance to methylation and imprinting (see Chap. 20) level of 0.001 and 0.5%, no paternal mitochondrial DNA has
during IVF. They noted that ovarian stimulation leads to been detected in the offspring, placenta, or umbilical cords
elevated follicular homocysteine, which might affect methy- after ICSI [63, 64].
lation. This should be balanced by taking folic acid and other
B vitamins. In addition, they comment on a trend to culture
early human embryos in culture medium lacking essential References
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recommend the use of culture medium with essential amino
1990;54:978–83.
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In two reviews of imprinting disorders and ART, Owen 3. Saenger P. Turner’s syndrome. N Engl J Med. 1996;335:1749–54.
4. Layman LC. The genetic basis of female infertility. In: Rimoin DL,
et al. and Manipalviratn et al. summarize the literature, includ-
Connor JM, Pyeritz RE, Korf BR, editors. Principles and practice of
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Prenatal Cytogenetics
12
counseling of mothers with high risks of having children

Introduction and History with chromosome abnormalities or inborn errors of metab-
olism” [9].
Amniocentesis
Further refinement of the technique and timing of amnio-
centesis were demonstrated in a 1967 paper by Jacobson and
Amniocentesis, the transabdominal or transcervical puncture
Barter, and they proposed that the optimal timing of amnio-
of the uterus for the purpose of removing amniotic fluid, has
centesis is 16 weeks after performing the procedure from
been practiced since the 1930s [1]. It was used in the early
5 weeks to term in 85 women [10]. Of these 85, 57 were
1950s in the prenatal evaluation of Rh sensitization [2].
successfully cultured. In a thoughtful discussion after the
A key event that laid the foundation for prenatal cytoge-
paper, Edward C. Hughes noted that, “Speculation might go
netic analysis was the discovery of the ability to determine
so far as to suggest that, although chromosome constitution
gender on the basis of the incidence of the sex chromatin
cannot be changed, a specific DNA that would carry the
body observed in the nuclei of oral mucosa smears [3, 4]. In
coding information lacking in certain diseases might replace
1956, James described the use of amniotic fluid sediment to
the missing element,” and in the same discussion, S.R.M.
determine fetal sex by Papanicolaou and Giemsa stains, and
Reynolds pointed out that, “…in the future there will be even
Fuchs and Riis showed in amniotic fluid of term pregnancies
more refined methods of evaluating gene abnormalities in
that they could accurately determine the fetal sex in 20 of 21
which the karyotype appears normal.”
cases [5, 6]. It is of interest that they concluded,
In 1968, Nadler and Gerbie described the use of amnio-
Although transabdominal puncture of the uterus has been carried centesis for the detection of cytogenetic and biochemical
out often for therapeutic and experimental reasons without
abnormalities in 155 women at increased risk for these disor-
accidents, mere curiosity does not justify the procedure, and its
practical value is probably limited in the human. If the results ders. They reported a highly successful culture rate of 97%
are confirmed in animals, however, it might become of great and uniformity of timing of the procedure, from 13 to
significance in veterinary practice [6]. 18 weeks [11].
Other investigators confirmed the accurate determination By 1986, more than a quarter of a million amniocenteses
of fetal gender by similar procedures, staining amniotic fluid had been performed for cytogenetic analysis, and the number
obtained at term by various techniques [7, 8]. to date is undoubtedly in the millions [12]. Although other
In 1966, Steele and Breg demonstrated, in a study of means of prenatal cytogenetic testing have been developed,
amniotic fluid obtained from women because their fetuses amniocentesis is by far the most common technique per-
were at risk for erythroblastosis fetalis, that human amniotic formed today for prenatal genetic diagnosis. In the United
cells could be cultured and the chromosomes analyzed [9]. States in 2003, 66,901 procedures were reported from 31
They foresaw that this “…would allow more practical genetic states and the District of Columbia [13].
L.M. Randolph, M.D., M.A. (*) Chorionic Villus Sampling

Division of Medical Genetics, Keck School of Medicine,
University of Southern California,
Los Angeles, CA 90027, USA Although techniques for transcervical and transabdominal
placental biopsy, or late chorionic villus sampling (CVS),
Division of Medical Genetics, Childrens Hospital Los Angeles,
4650 Sunset Boulevard, MS 90, Los Angeles, CA 90027, USA were described in the 1950s and1960s for the diagnosis of
e-mail: [email protected] hydatidiform mole, the first paper describing a technique for
230 L.M. Randolph
fetal genetic diagnosis was published in 1968 [14–16]. Mohr flexible needle introduced through it could be operated under
developed techniques for sampling fetal cells no later than direct vision [25]. This required regional or general anes-
the third month of pregnancy by a transabdominal approach. thesia and an abdominal wall incision. Three of the women
Due primarily to the absence of real-time ultrasound, low had umbilical cord puncture, and the blood obtained was
culture success rate, and the risks of endoscopic approaches, shown to be of fetal origin. Hobbins and Mahoney performed
as described by Kullander and Sandahl in 1973 and by fetoscopy in 34 women scheduled for abortion [26]. Local
Hahnemann in 1974, the technique was not widely used in anesthesia was provided, and the cannula was smaller than
the United States [17, 18]. In Kullander and Sandahl’s expe- the endoscope used by Valenti. In eight of these, successful
rience, 19 of 39 specimens (48.7%) were successfully blood sampling of a placental vessel was attempted and
cultured, which they described as a “high percentage” [17]. achieved. However, in only one of these cases was the
In Hahnemann’s experience, there was a 38% success rate, composition of the blood 100% fetal. Placentocentesis was
with causes of failure being puncture or biopsy of the essentially replaced by cordocentesis thereafter.
amniotic membrane and bleeding [18]. The optimal time of Daffos et al. demonstrated in 50 women referred for
performing the procedure was the 10th week of gestation, abortion that by using local anesthetic, real-time ultrasound,
and although the procedure had a low success rate in terms and puncture of the umbilical vein, pure fetal blood was
of obtaining tissue, the culture success rate was 91%. All obtained in 46 cases [27]. Sixteen of the 50 women under-
but one of the pregnancies was terminated by previous inten- went abortion 2–10 days later, by which time none of these
tion, and in the one continuing pregnancy, the newborn fetuses had died. Twelve other women delivered healthy
was normal. babies, and 22 pregnancies were ongoing.
In China, transcervical CVS was widely used in the 1970s The technique was later applied, by the same group, in
as a method of fetal sex prediction and selection. A report of 606 samplings of 562 women with a variety of indications
the Chinese experience was published in 1975 [19]. The for prenatal diagnosis [28]. Complications were seen in 15%,
accuracy of their fetal sex prediction, based on X chromatin, including a 2% rate of fetal death or spontaneous abortion.
was 94%. Efforts to replicate this success were unsuccessful By obtaining larger volumes of fetal blood, these investiga-
for several years [20, 21]. tors were able to perform physiologic and hematologic
In their 1981 paper, Niazi and Loeffler reported an assays that helped provide the basis for normal values in fetal
improved technique using trypsin for culturing trophoblastic blood, and they showed that percutaneous umbilical cord
cells obtained by transcervical CVS, minimizing the risk of sampling (PUBS) deserved a place in the prenatal diagnostic-
maternal cell admixture in fetal cells [22]. testing world.
The first use of real-time ultrasound scanning in CVS was
reported in 1982 by Kazy et al [23]. Of their 165 patients,
139 had biopsies performed prior to induced abortion, and in The Incidence of Chromosome Abnormalities
26 patients, biopsy was performed for genetic reasons. Of
the eventual 13 continuing pregnancies, none was spontane- Combining surveys from 1969 to 1982 of 68,159 liveborn
ously aborted, and all 11 babies who had been born to date babies, one in 156 live births were found to have a major
were normal. Fetal sex prediction by X chromatin was accu- chromosome abnormality (see Table 12.1) [27]. The most
rate in all cases. This was the first study that brought CVS common is trisomy 21, or Down syndrome, with an inci-
out of the experimental category and into the world of a dence from these surveys of 1 in 833 live births. The next
promising prenatal diagnostic test. As of 1996, more than most common are sex chromosome aneuploidies, with one
200,000 CVS cases from more than 100 centers world- XYY or XXY per 1,000 male liveborns and one XXX per
wide had been entered into an informal World Health 1,000 female liveborns [29]. Because non-banded chromo-
Organization-sponsored CVS registry in Philadelphia, which some preparations were used in the early survey years
has not been continued [24]. (from 1969 to 1975), it was thought, when Giemsa banding
(G banding) was introduced, that the incidence of chromo-
some abnormalities would be found to be higher. However,
Percutaneous Umbilical Cord Sampling (PUBS) in a 1980 study by Buckton et al. of 3,993 newborns, no
significant difference in the frequency of rearrangements or
In the early 1970s, in an effort to develop a method for of other chromosome aneuploidies was found [30].
prenatal diagnosis of hemoglobinopathies, investigators It is clear that the incidence of most fetal chromosome
sought to establish safe techniques for fetal and/or placental abnormalities increases with maternal age. Data for women
blood sampling. In his preliminary report of 1973, Valenti ages 35–49 were compiled by Hook based on North American
demonstrated in 11 women scheduled for abortion that, in collaborative studies and the New York State registry [31].
the second trimester, a surgical “endoamnioscope” with a His analysis of the data indicated a 30% differential between
12 Prenatal Cytogenetics 231
Table 12.1 Chromosome abnormalities in surveys of 68,159 liveborn 47,XXY, 47,XYY, 45,X, and 45,X/46,XX, but not for
babies 47,XXX (see Table 12.3) [33]. Contrary to what was found
Type of abnormality Total abnormalities (%) in other studies, there was no significant maternal age
Sex chromosomes, males effect in the incidence of fetal death of chromosomally
47,XYY 45 (0.103) abnormally fetuses.
47,XXY 45 (0.103) The incidence of de novo balanced structural rearrange-
Other 32 (0.073) ments in 337,357 amniocenteses was reported by Warburton
Sex chromosomes, females [35]. Another survey of de novo balanced chromosome rear-
45,X 6 (0.024)
rangements in prenatal diagnosis was published by Giardino
47,XXX 27 (0.109)
et al. [36]. The results are shown in Tables 12.4 and 12.5.
Other 9 (0.036)
Autosomal trisomies
47,+21 82 (0.120)
47,+18 9 (0.013)
Spontaneous Abortions
47,+13 3 (0.004)
Other 2 (0.002) It is a well-established fact that the incidence of major chro-
Structural balanced arrangements mosome abnormalities is much higher in first-trimester spon-
Robertsonian translocation taneously aborted fetuses than later in pregnancy and at birth.
der(D;D)(q10;q10)a 48 (0.070) The incidences in various studies range from 20 to 60%, with
der(D;G)(q10;q10)b 14 (0.020) the average in pooled data of unselected spontaneous abor-
Reciprocal and insertional translocation 64 (0.093) tions being 41% (see Table 12.6) [29, 37]. A cautionary note
Inversionc 13 (0.019) in consideration of this high incidence range is that the tissue
Structural unbalanced arrangements cultured and analyzed may not represent the fetus. It has
Robertsonian 5 (0.007) been shown that 45,X cells and some lethal trisomies seen in
Reciprocal and insertional 9 (0.013) chorionic villus samples may not be seen in the fetus, so
Inversion 1 (0.001) this may lead to spurious elevation of estimates of chromo-
Deletion 5 (0.007)
some abnormalities in spontaneous abortion tissue [38].
Supernumerary 14 (0.020)
Notwithstanding this caveat, the following frequencies of
Other 9 (0.013)
chromosome abnormalities are reported in spontaneous
Total abnormalities 442 (0.648)
abortions: autosomal trisomies comprise the largest group of
Total babies surveyed
Males 43,612
52% of chromosome abnormalities, followed by 45,X at
Females 24,547 19%, triploidy at 16%, and tetraploidy at 6% [37].
The association between advanced maternal age and the
Data from reference [27]
a
der(D;D) refers to Robertsonian translocations involving chromosomes incidence of trisomies has been demonstrated in spontaneous
13, 14, and/or 15 abortions. Of interest is that in a study of 494 girls with
b
der(D;G) refers to Robertsonian translocations involving chromosomes Turner syndrome born in Sweden from a population of 1.6
13, 14, or 15 and 21 or 22
c million girls, among women older than 40 years, 3.2% gave
Excludes common pericentric inversion of chromosome 9
birth to an affected daughter, compared to 1.8% of the popu-
lation controls. This amounted to an odds ratio of 1.83 [39].
the rates observed at amniocentesis and those seen at birth, a In previous publications, 45,X appeared to be associated
figure that is still valid almost 20 years later. with younger maternal age, with, for example, about one-
In 1982, Schreinemachers et al. analyzed data on the third of 45,X spontaneous abortions coming from women
results of 19,675 prenatal cytogenetic diagnoses on women 20–24 years of age [40]. The distribution of trisomies is quite
aged 35 and older for whom there was no known cytogenetic different from that seen at birth or even at amniocentesis,
risk for a chromosome abnormality except parental age [32]. with 30% being trisomy 16, compared to almost negligible
The expected rates at amniocentesis of clinically significant rates of trisomy 16 at amniocentesis (see Table 12.7) [37].
cytogenetic abnormalities by maternal age were obtained This topic is also covered in detail in Chap. 13.
and compared with previously estimated rates by maternal
age in live births. A differential between amniocentesis and
live birth incidences was shown for trisomies 21, 18, and 13 Stillbirths and Neonatal Deaths
but not for 47,XXY and 47,XYY (see Table 12.2) [29–34].
In the following year, Hook confirmed and refined the Fetal loss from 28 weeks on in pregnancy is defined as still-
differences in the incidences for trisomies 21, 13, and 18 and birth, and neonatal death refers to death occurring within the
also found a difference between fetal and newborn rates of first 4 weeks after birth. Chromosome studies in such cases
232 L.M. Randolph
Table 12.2 Maternal age-specific rates (%) for chromosome abnormalities

From liveborn studiesa From amniocenteses From CVS
b
Maternal 47,+21 47,+21c All chromosome 47,+21b 47,+21c All chromosome 47,+21c All chromosome
age (years) abnormalitiesb abnormalitiesb abnormalitiesd
33 0.16 – 0.29 0.24 – 0.48 – –
34 0.20 – 0.36 0.30 – 0.66 – –
35 0.26 – 0.49 0.40 – 0.76 – 0.78
36 0.33 0.35 0.60 0.52 0.31 0.95 0.42 0.80
37 0.44 0.43 0.77 0.67 0.80 1.20 0.68 2.58
38 0.57 0.42 0.97 0.87 0.73 1.54 0.45 3.82
39 0.73 0.79 1.23 1.12 0.84 1.89 2.05 2.67
40 0.94 1.21 1.59 1.45 1.03 2.50 1.20 3.40
41 1.23 2.67 2.00 1.89 1.50 3.23 3.12 6.11
42 1.56 4.28 2.56 2.44 2.92 4.00 2.88 8.05
43 2.00 1.82 3.33 3.23 3.05 5.26 1.20 5.15
44 2.63 – 4.17 4.00 1.52 6.67 2.63 10.00
45 3.33 – 5.26 5.26 2.50 8.33 8.33 7.14
a
Estimated liveborn statistics [32]
b
Data compiled from 19,675 genetic amniocenteses [32]
c
Data compiled from 3,041 CVS; 7,504 amniocenteses; and 13,139 with no test [34]. These are observed prevalences
d
Data compiled by L. Hsu [29]
Table 12.3 Fetal deaths subsequent to amniocentesis have shown that the incidence of chromosome abnormality is
Fetal deaths about ten times that in the rest of the population. Combining
Abnormalities Number Proportion (%) 95% confidence three studies of stillbirths and neonatal deaths, of those in
interval (%) which chromosome analysis was performed, 52 of 823
47,+21 73 30.1 19.0–42.0 (6.3%) studied had a chromosome abnormality. Of these 823,
47,+18 25 68.0 46.5–85.1 59 macerated stillbirths were studied, of which seven (11.9%)
47,+13 7 42.9 9.9–81.6 had a chromosome abnormality. Of 215 nonmacerated still-
47,XXX 39 0.0 0.0–9.0 borns, nine (4.2%) were chromosomally abnormal, and of
47,XXY 37 8.1 0.8–11.0
549 neonatal deaths, 33 (6.0%) had a chromosome abnor-
47,XYY 33 3.0 0.08–15.8
mality [29]. Given the value it provides families in terms of
45,X 12 75.0 42.8–94.5
understanding more about their losses and in providing recur-
45,X/46,XX 19 10.5 1.3–33.1
rence risks, it is recommended that consideration of chromo-
Balanced translocations 71 2.8 0.3–9.8
and inversions some analysis be given in all such cases (see Table 12.8).
Markers, variants, 27 0.0 0.0–12.8
fragments
Data from reference [33] Prenatal Cytogenetic Diagnosis
Note: Proportion refers to the number of fetal losses compared to the
total number of fetuses diagnosed with the given abnormality Genetic Amniocentesis
With increased public awareness, number of practitioners,

Table 12.4 The incidence of de novo balanced structural rearrange-
ments in 337,357 genetic amniocenteses laboratory capacity, proportion of women older than 35 hav-
ing babies, and use of maternal serum screening, the utiliza-
De novo rearrangement Number of cases Percentage
tion rate of amniocentesis has grown. It was estimated that in
Reciprocal translocation 176 0.047
Robertsonian translocation 42 0.011
1974, 3,000 women underwent genetic amniocentesis, and the
Inversion 33 0.009 number now is in the millions. The increased utilization has
Supernumerary small marker 162 0.040 extended to women of lower socioeconomic status who previ-
chromosome ously did not have access to or finances for the procedure [41,
Satellited marker 77 0.020 42]. With improvements in laboratory procedures, including
Nonsatellited marker 85 0.023 sterile technique, plasticware, enriched cell culture media, and
Total 413 0.109 automated harvesting and imaging systems, the turnaround
Data from reference [35] time for reporting results of an amniocentesis has dropped
Table 12.5 The incidence of de novo balanced structural rearrangements in 269,371 prenatal diagnoses
Reciprocal Robertsonian Complex chromosome
Specimen translocation (%) translocation (%) Inversion (%) rearrangement (%) Total (%)
AF 160 (73) 38 (17) 15 (7) 7 (3) 220 (0.9)
CVS 15 (63) 7 (29) 2 (8) – 24 (0.8)
FBS 2 (100) – – – 2 (0.5)
Total 177 (72) 45 (18) 17 (7) 7 (3) 246 (0.9)
% Total prenatal diagnoses 0.7 0.2 0.1 0.03
Table 12.6 Frequencies of chromosome abnormalities in unselected spontaneous abortions

Different types of chromosome abnormalities (% of all chromosome abnormalities)
Number of Number of abortuses Autosomal 45,X Triploid Tetraploid Other Reference
abortuses (%) with chromosome trisomy
studied aberrations
8,841 3,613 (40.87%) 1,890 (52.29%) 689 (19.06%) 586 (16.21%) 119 (5.51%) 249 (6.89%) [29]a
3,300 1,312 (39.8%) 645 (49.2%) 201 (15.3%) 198 (15.1%) 78 (5.9%) 190 (14.5%) [37]
a
Data compiled from more than ten studies
dramatically, from several weeks in the 1970s and 1980s to provide patients, there has been only one large prospective
less than a week in some laboratories today. The cost of the controlled study performed regarding the risks of amniocen-
laboratory test has dropped as well due to increased efficiency tesis. In this paper, known as “the Danish study,” 4,606
and competition. Thus, prenatal diagnosis by amniocentesis women comprised the final study population [54]. Of these,
has become, and probably will remain, by far the most com- half were randomized to have amniocentesis, and the other
mon mode of prenatal diagnosis until such time as a reliable, half were randomly assigned to the control, non-amniocente-
cost-effective noninvasive procedure is developed. sis group. At the conclusion of the study, it was found that
The accuracy of amniocentesis for the detection of rec- the total rate of spontaneous abortion was 1.7% in the study
ognized chromosome abnormalities is greater than 99%. group and 0.7% in the control group (p < 0.05). When the
Diagnostic accuracy has been enhanced by the recent use women with a high maternal serum alpha-fetoprotein (AFP)
of fluorescence in situ hybridization (FISH) and were considered, it was found that they had a relative risk of
chromosome-specific probes. These are of particular value spontaneous abortion after amniocentesis of 8.3 compared to
in marker chromosome, translocation, and deletion cases, women with a normal maternal serum alpha-fetoprotein
when microscopic findings require further study for level. This equated to an overall relative risk of 2.3. Other
clarification [43–50] (see Chap. 17). factors found to increase the risk of spontaneous abortion
were transplacental passage of the needle (relative risk of 2.6)
Conventional Amniocentesis: and discolored amniotic fluid (relative risk of 9.9).
15–24 Weeks of Gestation Another study from Denmark in 2009 reported on results
Midtrimester, defined here as the 15th through the 24th week of a national registry-based cohort study, including all
of gestation, is by far the most common time period for per- singleton pregnant women having an amniocentesis or CVS
forming the amniocentesis procedure. Culture of amniotic between 1996 and 2006 [55]. The fetal loss rate was defined
fluid cells is optimal in this time period, both from the per- as that occurring before 24 weeks’ gestation. The miscar-
spective of rapidity of cell growth (and therefore sample riage rate after amniocentesis was 1.4% after amniocentesis
turnaround time) and because the culture failure rate is less and was not correlated with maternal age. The number of
than 0.5% in experienced laboratories [51, 52]. procedures performed at each center had a significant effect
The risks associated with midtrimester amniocentesis on the risk of fetal loss. In those performing fewer than 500
include leakage of fluid, cramping, bleeding, infection, and amniocenteses, the odds ratio for fetal loss was 2.2 (95% CI,
miscarriage. The risk of miscarriage following midtrimester 1.6–3.1) when compared to those performing more than
amniocentesis is related to practitioner experience, number 1,500 per year. There was no control group in this study.
of needle insertions, size of the needle, and other factors In reviews of procedure-related risks from many publica-
[53]. The appropriate risk figure to provide patients is still tions, in which five included a control group, the authors
debated. In spite of the millions of amniocentesis procedures concluded that the procedure-related miscarriage rate from
performed and the importance of an accurate risk figure to amniocentesis is 0.5–1.0% [56, 57].
234 L.M. Randolph
Table 12.7 Frequency of autosomal trisomy for each human

chromosome among aborted specimens
Trisomy chromosome Number of trisomies (%)
1 0
2 34 (5.2)
3 6 (0.93)
4 15 (2.3)
5 5 (0.78)
6 5 (0.78)
7 27 (4.2)
8 23 (3.6)
9 18 (2.8)
10 11 (1.7)
11 0
12 2 (0.31)
13 53 (8.2)
14 32 (5.0)
15 52 (8.1)
16 202 (31.3)
17 4 (0.62)
18 23 (3.6)
19 0
20 18 (2.8)
21 54 (8.4)
22 55 (8.5)
Total 645 (100)
Table 12.8 Frequencies of chromosome abnormalities in stillbirths and neonatal deaths: combined data from three studies
Macerated stillbirths Nonmacerated stillbirths Neonatal deaths Total
Number karyotyped Abnormal Number karyotyped Abnormal Number karyotyped Abnormal Number karyotyped Abnormal
59 7 (11.86%) 215 9 (4.18%) 549 33 (6.0%) 823 52 (6.31%)
Date from reference [29]
Odibo et al. reported on the fetal loss rate after second- a pregnancy loss rate after 8 weeks of 3.2% [59]. Other stud-
trimester amniocentesis in a single center in a retrospective ies have shown a maternal age factor in the loss rate [38].
cohort study comparing the fetal loss rate in women having The prevalence of trisomies is about 50% higher at 16 weeks
amniocentesis with those not having any procedure [58]. compared to term pregnancies (ibid.), so selection against
Of the 88% for whom complete outcome data were avail- chromosomally abnormal abortuses is still occurring at
able, fetal loss in the amniocentesis group was 0.4% com- 16 weeks. The incidence of spontaneous pregnancy loss after
pared with 0.26% in those without amniocentesis (relative 16 weeks is 1%.
risk of 1.6, 95% CI, 1.1–2.2). Fetal loss less than 24 weeks Some genetic counselors and amniocentesis practitioners
occurred in 0.97% of the amniocentesis group and 0.84% counsel patients regarding the risk of the amniocentesis rela-
of the no-procedure group, so the fetal loss rate less than tive to the risk of a fetal chromosome abnormality and in
24 weeks attributable to amniocentesis was 0.13% (95% effect use this as a decision point. In this way, a woman with
CI, 0.07–0.20%), or 1 in 769. The only subgroup with a a risk of fetal chromosome abnormality of 1 in 200 might be
significantly higher amniocentesis-attributable fetal loss inclined to decline amniocentesis if the risk of miscarriage as
rate was women with a normal serum screen (0.17%, a result of the procedure was quoted as 1 in 100 and the risks
p = 0.03). compared during the counseling session. A maternal age of
An important and often overlooked component of provid- 35 has traditionally been used as a cutoff for the definition of
ing risk assessments to patients is the underlying incidence advanced maternal age because the risk of a fetal chromo-
and timing of pregnancy losses. A prospective study of 220 some abnormality at this age is roughly equivalent to the
ultrasonographically normal pregnancies in women recruited originally reported risk of miscarriage as a result of the amnio-
prior to conception (in order to avoid bias of selection) found centesis. This is not sound reasoning because the burdens of
the risks are quite different—one burden being the potential is feasible but that other safety issues had not been adequately
lifetime task of caring for an individual with mental retarda- addressed, such as congenital orthopedic anomalies and neo-
tion and physical/health problems and the other being miscar- natal pulmonary compromise, which had been seen in some
riage of a potentially healthy fetus [60]. babies born after midtrimester amniocentesis [66].
Several other studies were published in the early 1990s
Early Amniocentesis [67–73]. In one paper, 505 amniocentesis procedures were
Interest in early amniocentesis (EA) rose in the 1980s, due in performed between 11 and 15 weeks’ gestation. In all but
large part to the continued desire to provide and receive pre- three pregnancies, follow-up information was available,
natal diagnosis at an earlier gestation without some of the including 16 fetal losses (3.1%)—ten in the 2 weeks after the
risks and limitations associated with chorionic villus sam- procedure and six within the 28th week of gestation. The
pling, which are outlined in the following paragraphs. An authors reported a significantly higher risk for fetal loss when
increase in sophistication in ultrasound technology has also the amniocentesis was performed at the 11th–12th week of
made earlier imaging of fetuses more feasible and has added gestation compared with the 13–15-week group. The fetal
to the confidence level of the physicians performing the pro- loss rate between the 12–13-week and the 14–15-week
cedure. Adding to this is the opportunity to measure amni- groups showed no statistically significant difference. They
otic fluid alpha-fetoprotein and acetylcholinesterase, which concluded that early amniocentesis is, “a valid alternative to
is not possible with CVS. One center reported a rise in EA traditional amniocentesis” [67].
procedures from 3.2% of their 495 procedures in early 1985 In their 1990 paper, Elejalde et al. performed a prospec-
to 6.5% of 980 procedures in late 1987 [61]. tive controlled study involving 615 amniocenteses performed
Early amniocentesis is usually described as one that between weeks 9 and 16 of gestation, and they reviewed pre-
occurs before 15 weeks’ gestation. It has been shown that the vious EA studies [68]. Their results showed that amniocente-
earlier a prenatal diagnosis procedure is performed, the sis after the 9th week of pregnancy does not appear to differ
higher the fetal loss rate is [62]. One should therefore further significantly in its complications and outcome from the
divide the periods at which amniocentesis is performed to results of the same procedure at 15–16 weeks or later. The
provide better comparative data for a variety of procedures issue of pseudomosaicism was also addressed and will be
since, “…true risks …appear to be a function of gestational covered more fully later in this chapter.
age and less related to the procedure performed” [62]. Penso et al. in 1990 performed amniocentesis in 407
Although the procedure by which EA is performed is sim- women between gestational ages of 11–14 weeks and com-
ilar to that of midtrimester amniocentesis, practitioners report pared the safety and accuracy with data obtained from col-
several challenges unique to EA. The placenta is more widely laborative studies of amniocentesis performed later in the
spread, the amniotic fluid volume is lower, and the amniotic second trimester [69]. Theirs was the first report to provide
membrane is not yet totally adherent to the uterine wall, lead- information regarding neonatal outcome associated with EA.
ing to the “tenting” reported by some physicians [63]. The spontaneous abortion rate within 4 weeks of the proce-
dure was 2.3%, and the fetal loss rate was 6.4%. Orthopedic
Background postural deformities, including club feet, scoliosis, and con-
In one study conducted from 1979 through 1986, 4,750 genital dislocation of the knees and hips, were seen in eight
amniocenteses were performed, 541 of which were performed newborns, three of whose mothers had post-amniocentesis
before the 15th week since the last menstrual period [64]. leakage of amniotic fluid. A total of ten women in the study
Outcome data were available for 298 women, of whom 108 (2.6%) had post-procedure fluid leakage. It appeared that the
were under 35 years of age. Fetal loss within 2 weeks of the orthopedic deformities might be related to a post-procedure
procedure was seen in five pregnancies, all in the 14th week, history of amniotic fluid loss. They concluded that the accu-
when 228 of the 308 women had the procedure. When all racy, risks, and complications were similar to those of tradi-
spontaneous fetal losses were accounted for, there were tional amniocentesis.
eleven spontaneous abortions (3.6%), two stillbirths (0.7%), In 1990, Hanson et al. reported their increased practitio-
and one neonatal death (0.3%), resulting in a total post-pro- ner experience and use of continuous ultrasonographic guid-
cedure loss rate of 14/298 (4.7%). No culture failures were ance in EA of gestations from 10 to 14 weeks [70]. The
seen. The needle gauge was 20, and no difference in outcome needle gauge was changed from the 20 gauge used in their
was seen in transplacental versus placental passage. 1987 study to 22, and the volume of fluid removed was gen-
In 1988, the combined experience of six groups, including erally less. Pregnancy outcome was reported for 523 patients,
the study previously mentioned, was reviewed [65]. The total of whom 12 (2.3%) had a post-procedural loss. This com-
loss rate in 1,240 pregnancies of known outcome ranged from pared favorably with their previously reported loss rate of
1 to 4.7%. Cell culture and amniotic fluid alpha-fetoprotein 4.7%. Of eight women with post-procedure amniotic fluid
measurements were satisfactory. The conclusion was that EA leakage, one had a baby at term with a dislocated knee.
236 L.M. Randolph
Another experienced fetal death 3 weeks after the amniocen- procedure but the risk may be minimally higher than that for
tesis, and the rest had normal term deliveries. standard amniocentesis.” There were no significant differ-
In a smaller series, 105 EA procedures were performed ences in congenital anomalies in the EA group (1.8%) versus
[71]. There were two pregnancy losses in the 64 patients for the traditional amniocentesis group (2.2%). Interestingly, in
whom outcome information was available at the time of pub- the EA group, 4 of the 12 abnormalities involved congenital
lication, and four congenital anomalies were seen in the 66 hip dislocation/subluxation or club feet, and three of the 30
delivered babies: one imperforate anus, one hemangioma of congenital anomalies seen in the traditional amniocentesis
the tongue, and two cases of positional talipes that required group were congenital hip dislocation or club feet. They con-
no treatment. These were apparently unrelated to amniotic cluded that EA is a, “relatively safe prenatal diagnostic test
fluid leakage. and an alternative to CVS and later amniocentesis.” See
Crandall et al. retrospectively studied 693 consecutive EA Table 12.9 for a comparison of fetal loss rates.
(prior to 15 weeks) cases, which had a spontaneous abortion In all these studies, the investigators concluded that, apart
rate (to 28 weeks’ gestation) of 1.5%, compared with a non- from a higher rate of pseudomosaicism seen in some EA
randomized, later control group of 1,386 women having tra- cases, the laboratory analysis of EA specimens compares
ditional amniocentesis, whose spontaneous abortion rate was favorably in validity and reliability compared to traditional
0.6%, a statistically significant difference [72]. In their amniocentesis specimens. This was confirmed in two labora-
review of background risk of pregnancy loss in the second tory studies of a combined 1,805 EA specimens of
trimester, they concluded that “at least some of the pregnancy 10–14 weeks’ gestation [73, 74]. The culture success rate
loss subsequent to early amniocentesis is independent of the was 99.8% for EA versus 100% for traditional amniocentesis
Table 12.9 Outcome in early (11–14 weeks) amniocentesis studies

# of patients Fetal loss rate Fetal loss rate (%)a,
with outcome within week(s) gestation at
data in EA 2 weeks of time of Total fetal
Group Study period group procedure (%) amniocentesis loss rate,% Needle gauge Comments
Hanson 1979–1986 298 1.7 5/80 (6.3), 4.7 20 Loss rate was 3.3% if
(1987) [64] 11–13 weeks; 5/228 patients with pre-amnio-
(2.2), 14 weeks centesis history of
bleeding were eliminated
Johnson and Review of 1,240 N/Ab N/Ab 1–4.7 22 in 5
Godmilow six studies, centers, 20 in
(1988) [65] including 1 center [64]
Hanson [64];
1979–1987
Stripparo 1987–1988 397 1.98c 9/208 (4.3), 3.9 22
(1990) [67] 11–13 weeks; 0/176
(0), 14 weeksd
Penso 1986–1989 389 0.8e 6/365 (1.6), 3.96 22 3 of 8 newborns with
(1990) [69] 11–13 weeks; 3/42 postural deformities born
(7.1), 14 weeks after post-amniocentesis
fluid leak
Hanson 1986–1987 517 0.8 6/272 (2.2), 2.5 20
(1990) [70] 11–13 weeks; 5/255
(1.96), 14 weeks
Crandall 1988–1993 681 0.9 13/681 (1.9), 1.9% for EA, 22, sometimes EA was compared to
(1994) [72] 11–14 weeks 0.97% for 25 conventional amnio;
13/1,342 (0.97), conventional spontaneous abortion
15–22 weeks amniocentesis rate was significantly
higher in EA group.
0.6% of EA group had
hip dislocation or
clubfeet compared to
.22% in conventional
amnio group
a
This figure includes spontaneous abortions, stillbirths, and neonatal deaths
b
NA not available
c
One hundred and eight 15-week-gestation amnios were included in this figure
d
Data based on status at 28 weeks’ gestation
e
Fetal loss within 4 weeks
in one study and 98.6% for EA versus 99.9% for traditional (TA CVS) from 1986 to 1988, and half of whom had EA
amniocentesis in the other study. The turnaround times for from 1987 to 1991 [79]. Of the EA specimens, all but 11
reporting results were 1–2 days longer in the EA group. In were obtained from weeks 12 to 14, and the rest were from
one study, the EA group showed a significant increase in the weeks 9 to 11. Of the continuing pregnancies, loss rates of
number of structural and numerical single-cell abnormalities 3.8 and 2.1% for EA and TA CVS, respectively, were seen.
and an increase in numerical multiple-cell abnormalities This was not statistically significant. The culture failure rate
compared to amniocenteses performed at 16–18 weeks. for both procedures was 0.8%. This study has limited appli-
These were dealt with by examining parallel cultures. cability inasmuch as the numbers were small and the patients
More recent studies are mixed in their conclusions. Diaz not randomized, and the time intervals were different.
Vega’s group performed 181 amniocenteses at 10–12 weeks’ Although all procedures were listed as initial cases, the rela-
gestation and reported a fetal loss rate within 2 weeks of the tive degree of prior individual practitioner experience in the
procedure of 0.5%, with a total fetal loss rate during preg- two procedures was not addressed.
nancy of 1.6% [75]. However, the culture success rate was In 1994, Nicolaides et al. reported on a prospective, par-
only 94.5% overall, with one culture failure out of three tially randomized study comparing EA and TA CVS in 1,870
10-week amniotic fluid specimens. women [80]. The spontaneous loss rate was significantly
Brumfield’s group performed a retrospective matched- higher after EA at 5.3% than with the CVS group (1.2%).
cohort study using a study group of 314 patients who had The rate of successful sampling was the same at 97.5%.
amniocentesis at 11–14 weeks versus a control group of 628 Culture failure occurred in 2.3% of the EA group, compared
women who had amniocentesis at 16–19 weeks [76]. With to 0.5% in the CVS group. Confined or true mosaicism was
the same practitioners, ultrasound equipment, and technique, seen in 1.2% of the CVS group, compared to 0.1% of the EA
they found a significant difference in the fetal loss rate within group. The authors concluded that although EA and CVS are
30 days of amniocentesis (2.2% vs. 0.2%) in the EA group equally likely to produce valid cytogenetic results, CVS
compared to the later-amniocentesis group. This was attrib- would probably become the “established technique” due to
uted at least in part to higher post-procedure amniotic fluid the 2–3% excess risk of fetal loss in the EA group.
leakage (2.9% vs. 0.2%) and vaginal bleeding (1.9% vs. In response to this study, Saura et al. stated that EA could
0.2%) rates. The culture success rates were not reported. be a “true alternative” to CVS after the 13th week, when the
Bravo et al. examined whether transplacental needle pas- disadvantages of culture failure and fetal losses decrease
sage is a factor in fetal loss after EA [77]. They reviewed 380 [81]. Bombard et al. reported one loss in 121 procedures
consecutive EA procedures performed for advanced mater- (0.83%) performed by one practitioner at 10–13 weeks using
nal age and found that transplacental needle passage had a 22-gauge needle [82]. They suggested that Nicolaides’
occurred in 147 cases (38.7%). Although the frequency of higher EA fetal loss rate could be related to the needle gauge
“bloody taps” was significantly increased in this group, there and the multiple practitioners in his study, compared to one
was no difference in fetal loss rates (3.4% in both groups, practitioner in Bombard’s center.
including stillbirths). Similar results were reported by Vandenbussche et al., who,
Wilson’s review states that there have been no studies that in a partially randomized study, reported eight fetal losses
have adequately addressed the critical question of the safety among 120 EA procedures, compared to none among the 64
of EA relative to traditional amniocentesis, pointing out that CVS patients with a follow-up of 6 or more weeks [83].
to date only two randomized trials had been performed, and Another response to these reports proposed the idea that
they differed in their methodologies and their conclusions the main drawback to the studies was the very small numbers
[78]. He also stated that procedures at less than 13 weeks’ of EA procedures performed and the evident greater practitio-
gestation should be considered experimental. Certainly, the ner experience with CVS than with EA. The authors reported
cumulative experience with 13–14-week EA procedures is a spontaneous abortion rate after EA of 1% up to week 24 on
much greater than that with under-13-week EA procedures. the basis of 1,800 pregnancies. The culture failure rate was
In addition, the two randomized EA studies he cited evalu- 0.3% for gestations ranging to 10 weeks 4 days [84].
ated 11–12-week gestations and thus are not comparable to An important consideration raised by some investigators
the 13–14-week-gestation studies. is that the banding quality of amniocentesis specimens of
any gestation is generally superior to that of CVS specimens,
Comparison of Early Amniocentesis which increases the informativeness of the cytogenetic anal-
with Chorionic Villus Sampling ysis [78, 84]. The fact that amniotic fluid AFP levels and
In order to compare first-trimester prenatal diagnostic modal- multiples of the median have been established in many labo-
ities, a number of investigators have published studies com- ratories down to 12 or 13 completed weeks of gestation adds
paring CVS with early amniocentesis. Shulman et al. reported another advantage to the diagnostic power of EA compared
on 500 women, half of whom had transabdominal CVS to CVS [85].
238 L.M. Randolph
A 14-center study of 3,775 women randomized to having Table 12.10 Volume of amniotic fluid (mL) calculated using all the
either CVS or EA was conducted to try to provide more values for a given week from published data
answers to the questions as to the safety and accuracy of EA Week n Mean SD Range
and transabdominal CVS at 11–14 weeks’ gestation [86]. 10 7 29.7 11.2 18–33
Both types of procedures were performed by the physicians 11 9 53.5 16.4 64–76
in each center. Early in the trial, reports of clubfoot at 12 13 58.0 23.4 35–86
11–12 weeks in EA patients caused procedures at these 13 13 71.4 21.3 38–98
weeks to be discontinued. 14 14 124.1 42.1 95–218
15 15 136.8 43.7 64–245
Criteria for inclusion included advanced maternal age,
16 16 191.2 59.7 27–285
serum marker screen positive, and prior trisomy [86]. The
17 20 252.6 98.5 140–573
primary outcome was deemed to be preterm delivery or preg-
18 4 289 150 70–410
nancy loss of a cytogenetically normal fetus at less than
19 14 324.5 65.2 241–470
28 weeks’ gestation. Secondary outcomes included total fetal 20 3 380 39 355–425
loss, including neonatal death; amniotic fluid loss; pregnancy
outcome; limb and other congenital defects; and cytogenetic
diagnostic success and accuracy. Multiple procedures were
required for EA at 11–12 weeks (2.4% vs. 1.2% for CVS). pooled, and the volume of amniotic fluid for weeks 10–20
Maternal cell contamination was seen in EA specimens at was calculated [68] (see Table 12.10). At gestations under
11–12 and 13 weeks’ gestation (0.6% in both cases vs. 0% in 15 weeks, many practitioners have adopted the practice of
CVS). Pseudomosaicism was seen in 1.2% of EA 11–12- removing 1 mL per week of gestation, and others have found
week specimens versus 0.6% of CVS specimens. CVS speci- excellent culture success rate and turnaround time with less
mens were harvested at 5.9–6.5 days across the sampling fluid removed. For example, one group withdrew 4–12 mL in
period, compared to 12.3–9.8 days for 11–12- or 14-week gestations of 9–14 weeks and obtained a 100% culture suc-
EA specimens, respectively. As for complications, the only cess rate in 222 specimens, while others withdrew 5–8 mL in
difference that reached significance at the p < 0.001 level was pregnancies of 10 weeks and 4 days to 13 weeks and 6 days
EA with a 9.6% amniotic fluid leakage rate. Gestational for an overall culture success rate of 99.7% [84, 87]. It has
hypertension/preeclampsia was seen in 5.4% of the CVS been observed that the total cell numbers rise exponentially
patients compared to 3.5% of the EA patients, for a p value of from 8 to 18 weeks’ gestation, but the number of viable cells
0.005. Of 1,914 CVS procedures, 34 had cytogenetic abnor- increases only slightly during that time [78]. This probably
malities, two were lost to follow-up, and 1,878 were cytoge- explains the comparable culture success rate of EA com-
netically normal. Thirty-nine, or 2.1%, were lost or delivered pared to midtrimester amniocentesis.
before 28 weeks. This compares to 1,861 EA procedures, of
which 38 had cytogenetic abnormalities, three were lost to
follow-up, and 1,820 were cytogenetically normal. Forty- Chorionic Villus Sampling
two, or 2.3%, were lost or delivered before 28 weeks. Clubfoot
was seen in 0.2% of CVS patients; in EA patients, it was seen Associated Risks, Limitations, Benefits,
in 1.2% of 11–12-week procedure offspring, 0.8% of 13-week Turnaround Time
offspring, and 0.2% of 14-week offspring for a relative risk of Risks associated with CVS have been extensively studied.
EA versus CVS of 4.1 (1.17–14.6). The authors concluded Perhaps the issue receiving the most attention in the past few
that, in general, CVS is the preferred prenatal diagnostic pro- years was raised by Boyd et al. involving one case and then
cedure between 12 and 14 weeks [86]. This conclusion was more extensively by Firth et al., who reported five babies
shared by Evans and Wapner and by Tabor and Alfirevic in with severe limb abnormalities out of 289 pregnancies in
their reviews of prenatal diagnostic procedures [56, 57]. which TA CVS had been performed at 56–66 days’ gestation
Tabor and Alfirevevic stated, “Amniocentesis should…not be [88, 89]. Four of these had oromandibular-limb hypogenesis
performed before 15 + 0 weeks’ gestation” due to increased syndrome. They hypothesized that CVS undertaken up to
fetal loss rates compared to conventional amniocentesis and 66 days’ gestation may be associated with an increase in the
the risk of talipes [57]. risk of oromandibular-limb hypogenesis syndrome and other
limb-reduction defects. This report generated many others,
Specimen Requirements with mixed conclusions.
The volume of amniotic fluid obtained for prenatal diagnosis A flurry of letters to the editor of Lancet in 1991 followed
varies with the stage of gestation, with 15–20 mL conven- Firth’s report. Reporting evidence to support the association
tionally removed by midtrimester amniocentesis practitio- between CVS and limb-reduction defects were Mastroiacovo
ners. In one report, data from several small studies was et al. and Hsieh et al. [90, 91]. Monni et al. suggested that the
incidence and severity of limb defects was related to the A five-center retrospective cohort study was performed
gauge of the needle since they used a 20-gauge needle while by the Gruppo Italiano Diagnosi Embrio-Fetali to examine
Firth used an 18-gauge needle [92]. In a series of 525 CVS this issue, with results published in 1993 [97]. Of 3,430 preg-
procedures done before 66 days’ gestation, no severe limb nancies in which CVS had been performed, outcome infor-
defects were seen, and only two mild defects were seen in mation was available for 2,759. Of these, three had transverse
2,227 procedures that were done later [92]. Mahoney then limb-reduction defects, two among 804 CVS procedures per-
reported on two multicenter studies that compared transcer- formed at 9 weeks and one among 1,204 CVS procedures
vical CVS with amniocentesis, and another comparing trans- performed at 10 weeks. There were no limb-reduction defects
abdominal CVS with transcervical CVS [93]. Of 9,588 noted in 2,192 amniocenteses with completed follow-up per-
pregnancies studied, 88% of the CVS procedures were performed during the same study period. The authors concluded
formed after 66 days’ gestation. Significant limb-reduction that performing CVS at less than 10 weeks’ gestation,
defects were present in seven babies. Two of these defects “should be discouraged until further evidence against this
were longitudinal, and five were transverse. Another baby association can be obtained,” while noting that their follow-
had minor reduction defects of the toes. They compared these up rate was only 80%.
abnormalities to those reported to the British Columbia reg- Hsieh et al. surveyed 165 obstetric units in Taiwan regard-
istry and found no significant increase in these birth defects. ing the incidence of limb defects with and without CVS [98].
The timing of the CVS procedures that resulted in babies Of these, 67 hospitals responded, representing 78,742 deliv-
with abnormalities ranged from 62 to 77 days’ gestation. eries. The incidence of limb defects was found to be 0.032%
Similar conclusions were reached in a study in which in the general population and 0.294% in the CVS population.
12,863 consecutive CVS procedures were performed [94]. The abnormalities seen in the CVS group included amelia,
Five limb-reduction defects were seen, which were found not transverse reductions, adactylia, and digit hypoplasia, much
to be significantly different from the incidence observed in like the abnormalities reported by Firth et al. [89]. The 25
the British Columbia registry of birth defects. Of the 12,863 limb abnormalities in the non-CVS group involved syndac-
procedures, 2,367 were done at 56–66 days, and one of the tyly or polydactyly. In addition, oromandibular-limb hypo-
limb defects was seen in this group. The authors observed no genesis was seen in four of 29 CVS cases with limb
gestational time-sensitive interaction related to CVS and abnormalities but in none of the non-CVS cases with limb
postulated that this was due to their larger experience base. abnormalities. The severity of the post-CVS limb abnormali-
In 1993, Jahoda et al. reported on 4,300 consecutive trans- ties appeared to correlate with timing of the procedure, and
abdominal and transcervical CVS cases for which newborn the authors recommended performing CVS only after 10 full
follow-up information was obtained [95]. Of the 3,973 gestational weeks to minimize the risks.
infants born in this group, three (0.075%) had a terminal In 1995, Olney et al. reported on a United States multi-
transverse limb defect. Two of these occurred in the transcer- state case-controlled study comprising the years 1988–1992
vical CVS group sampled before 11 weeks’ gestation (1,389 [99]. The case population was 131 babies with nonsyndro-
patients), and the other one was in the transabdominal CVS mic limb deficiency born to women 35 and older, and control
group, sampled after 11 weeks (2,584 patients). The authors subjects were 131 babies with other birth defects. These were
found the latter figure to be comparable to the prevalence drawn from a total of 421,489 births to women greater than
figure given in population studies. They concluded that post- 34 years of age. The odds ratio for all types of limb deficiency
ponement of CVS to the late first or early second trimester of after CVS was 1.7, and for transverse digital deficiency, an
pregnancy would contribute to the safety of the procedure. odds ratio of 6.4 after CVS was observed. They estimated
In the same year, a report of the National Institute of Child that the absolute risk for transverse digital deficiency in
Health and Human Development Workshop on Chorionic babies after CVS was one per 2,900 births (0.03%).
Villus Sampling and Limb and Other Defects was issued Froster and Jackson reported on outcome data in a World
[96]. The conclusions, based on a review of the literature, Health Organization (WHO) study on limb defects and CVS
were mixed; some concluded that exposure to CVS appeared in 1996 [100]. From 1992 to 1994, 77 babies or fetuses with
to cause limb defects, while others did not. All agreed that the limb defects from 138,996 pregnancies exposed to CVS were
frequency of oromandibular-limb hypogenesis appeared to reported to the WHO CVS registry. This group represented
be more common among CVS-exposed infants. This seemed the entire experience of 63 European and American centers
to correlate with CVS performed earlier than 7 weeks post- reporting to the registry. They found that the overall inci-
fertilization (9 weeks post last menstrual period). Whether or dence of limb defects in the CVS cohort did not differ from
not a distinctive type of limb defect was associated with CVS that in the general population, and they did not see a different
could not be determined, and it also was unclear whether the pattern of distribution of limb defects between the groups.
CVS-exposed infant had an increased frequency of other No correlation between limb-reduction defects and gesta-
malformations, including cavernous hemangiomas. tional age was identified. They indicated that other studies
240 L.M. Randolph
finding an association between limb defects and CVS are 35-and-older group, whether or not CVS had been per-
confusing because of different methodologies and interpreta- formed, and the under-35 group. The risk estimate for trans-
tions and that the numbers reported are too small to draw verse limb defects associated with CVS was 12.63 and did
firm conclusions. not change after stratification for maternal age or for gesta-
Larger numbers were collected by Kuliev et al., who sum- tional age.
marized the accumulated experience of 138,996 cases of After 1991, the utilization of CVS dropped significantly,
CVS from the same 63 centers that report cases to the World due in large part to the concern regarding limb deficiencies
Health Organization CVS registry [24]. They reported an [105, 106]. Although national utilization numbers are not
overall incidence of limb-reduction defects after CVS of available, a large national prenatal genetic counseling com-
5.2–5.7 per 10,000, compared with 4.8–5.97 per 10,000 in pany affiliated with a cytogenetics laboratory reported a
the general population. They also found no difference in the decrease of CVS of 3% per year from 36 to 14% from the
pattern distribution of limb defects after CVS and similarly years 2007 to 2001. In their patient population of 55,019
concluded that their data provide no evidence for any risk for women, 34% were offered CVS. The decline was statisti-
congenital malformation caused by CVS. cally significant. The acceptance rate increased again in 2008
to 24%, thought due to American College of Obstetrics and
Heterochromatin Decondensation Gynecology Practice Bulletin #77, 2007a, supporting first-
in Chorionic Villus Sampling trimester screening for fetal chromosome abnormalities, as
The spontaneous decondensation of the constitutive hetero- well as likely being due to such factors as increased access to
chromatic regions of chromosomes 1, 9, 16, and Y has been first-trimester screening [107].
observed in 46.6% of chorionic villus samples, per a study
by Perez et al. [101]. This type of decondensation is occa- Fetal Loss in CVS
sionally observed in amniotic fluid cells (9%) and has never In the first large controlled study of the safety of CVS,
been found in fetal lymphocytes. This decondensation can Rhoads et al. reported on seven centers’ experience with
lead to breaks, fragile sites, and loss of the chromosome, transcervical CVS in 2,235 women compared to that of 651
including, for example, the loss of 1q in culture. women who had amniocentesis at 16 weeks’ gestation [108].
They found an overall excess loss rate of 0.8% in the CVS
Maternal Age: A Confounder? group after statistical adjustments for gestational age and
Because CVS is usually performed on women 35 and older, maternal age. CVS procedures in which more than one
the issue of whether the limb deficiencies seen after CVS attempt was made were associated with a substantially higher
were related to maternal age was raised by Halliday et al. in loss rate, supporting the observation by Silver et al. and oth-
a study from Victoria, Australia [102]. A congenital malfor- ers that increased operator experience is a key factor in
mations registry maintained there was reviewed by a medical assessing the risks of CVS [109]. Silver’s group found that
geneticist, who classified all cases using the International the number of placental passes and increased sample weight/
Classification of Diseases, 9th revision [103]. All babies aspiration attempt ratio may be more sensitive indicators of
born with limb defects in 1990–1991 were identified, and the competence than the fetal loss rate.
number of those whose mothers had amniocentesis, CVS, or Results of a randomized international multicenter
no invasive study was known. Excluding babies with chro- comparison of transabdominal and transcervical CVS with
mosome abnormalities, recognized inherited syndromes, or second-trimester amniocentesis were reported in 1991 [110].
amniotic bands, the authors found a twofold relative risk of Outcome information was available for 1,609 singleton
having a baby with a limb deficiency of any type among pregnancies in the CVS group and 1,592 in the amniocen-
women at age 35 or older, compared to women under 35. tesis group. Thirty-one centers participated, and the numbers
They also discuss the difficulty in interpreting studies of limb of cases submitted ranged from 4 to 1,709. Significantly
defects and CVS, as others had, pointing out the importance fewer surviving newborns were seen in the CVS group than
of 100% follow-up, inclusion of all recognized cases of limb in the amniocentesis group (4.6% difference, p < 0.01). Most
deficiencies (induced abortions as well as all other births), of the difference was in the significantly greater number of
recognition of the heterogeneity of the condition, and the dif- spontaneous fetal deaths before 28 weeks: 86/1,528 in
ferent risk estimates at different gestational ages [100]. the successfully sampled CVS group and 25/1,467 of the
A subsequent study found no maternal age confounding successfully sampled amniocentesis group (rate difference
effect in interpretation of CVS/transverse limb deficiency of 2.9%, p < 0.02).
data [104]. The authors analyzed the maternal age-specific In a report from the Centers for Disease Control, an over-
rates of transverse limb deficiencies in the Italian Multicentric all risk of spontaneous abortion attributed to CVS is reported
Birth Registry and used a case-control model for maternal from a literature survey as 0.5–1.0%, compared to 0.25–0.50%
age. No difference in the relative risk was seen between the for amniocentesis procedures [111].
In the WHO study, registry participants reported a

spontaneous pregnancy loss rate after transabdominal or
transcervical CVS of 2.5–3.0%, with several large-volume
operators having loss figures of less than 2% [24]. This risk Placenta
was deemed comparable to that of amniocentesis. Tabor et al.
documented fetal loss rates in an 11-year national registry
study in Denmark of the outcomes of 31,355 CVS proce- Pubic
symphysis
dures at 24 weeks’ gestation in a 2009 report [55]. The over-
all fetal loss rate was 1.9% (95% CI, 1.7–2.0) and was not Bladder
correlated with maternal age. The number of procedures
done in each center had a significant effect on the loss rate; in
departments performing 1,500 or fewer during the 11 years,
the risk was 40% greater than in those performing more than
1,500 procedures per year. There was no control group.
In reviews of procedure-related risks from many publica-
tions, in which five included a control group, the authors
concluded that the procedure-related miscarriage rate from
CVS is 0.5–1.0% [56, 57].
Transabdominal Versus Transcervical CVS

Efficacy and risks associated with transcervical CVS (TC
CVS) and transabdominal CVS (TA CVS) have been studied
at several centers [110, 112–114] (see Fig. 12.1). The major-
ity of CVS had been performed transcervically until the late
1980s, when more centers began using TA CVS to avoid
cervical microorganisms and to reach placentas more easily.
In their pilot study in 1988, Smidt-Jensen and Hahnemann
reported on 100 TA CVS cases at 8–12 weeks’ gestation fol-
lowed to term, compared to 200 amniocentesis cases [114].
In all CVS cases, a sample was successfully obtained and
cultured, and the fetomaternal complication rates were found
not to be significantly different from those of previous TC
CVS reports.
Transabdominal CVS has been increasingly used in recent
years compared to TC CVS. Brambati et al. reported on
efficiency and risk factors in 2,411 patients; 1,501 of whom Fig. 12.1 Illustration of transcervical and transabdominal CVS.
had TC CVS and 910 of whom had TA CVS [112]. The two Upper: transcervical CVS. A flexible catheter is introduced into the
chorionic villi, or future placenta. Lower: transabdominal CVS. A spi-
approaches had comparable success rates and complication nal needle is inserted through the abdominal wall for sampling
rates, but TA CVS was considered easier to learn and less
likely to be contraindicated by clinical and anatomical condi-
tions. Subsequently, this group published results of a ran- one individual. Confined placental mosaicism (CPM) is
domized clinical trial of TA and TC CVS [113]. All CVS defined as a discrepancy between the chromosomal consti-
procedures were performed by the same practitioner, who tutions of placental and embryonic/fetal tissues. CPM
had prior similar experience in both techniques. The proce- results from viable mitotic mutations occurring in the pro-
dures were found to be equally effective, although TA CVS genitor cells of trophoblast or extraembryonic mesoderm
required significantly fewer insertions. The authors con- during early embryonic development. In 1983, Kalousek
cluded, “…transabdominal and transcervical CVS appear and Dill reported on numerical discrepancies between the
equally effective, and by and large the choice may be based karyotypes of fetal and placental cells, either full trisomies
on the operator’s preferences.” or mosaic aneuploidies, and similar reports followed [115,
116]. Based on six cases in which placental/CVS cells had
Confined Placental Mosaicism a different chromosome constitution from that of amniotic
Chromosomal mosaicism is characterized by the presence fluid cells, the authors concluded that the results of cytogenetic
of two or more karyotypically different cell lines within analysis from placental tissue may not be representative of
242 L.M. Randolph
the fetus. Their figures, though small, were similar to the 1,415 (1.5%) of the specimens. Table 12.11 shows the mosaic
2% incidence of this phenomenon as previously reported and nonmosaic chromosome findings seen. Updated CVS
[117]. Since then, others have found CPM to occur in 0.8– mosaicism reports are shown from other studies for specific
2% of viable pregnancies studied by CVS at 9–11 weeks’ chromosomes. Hahnemann and Vejerslev’s work on several
gestation and in 0.1% or less in amniocentesis specimens cell lineages indicated that mosaic or nonmosaic trisomies
[80, 118–125]. found in cytotrophoblasts, with a normal karyotype in the
The outcomes of pregnancies in which CPM is diagnosed villus mesenchyme, were not seen in fetal cells. However, if
vary from apparently normal outcomes to severe intrauter- such trisomies were seen on cultured preparations, a risk of
ine growth restriction (IUGR), although few follow-up fetal mosaic or nonmosaic trisomy existed. They recom-
reports are yet available in the literature. Kalousek et al. mended amniocentesis in all pregnancies involving mosaic
found six cases of IUGR among 17 gestations with CVS- autosomal trisomy in villus mesenchyme.
detected CPM, 5 in liveborns, and one associated with intra- A thoughtful study on this topic was published by Daniel
uterine death [126]. They noted that others had found a 22% et al., in which rare trisomies detected at the time of CVS
fetal loss rate among pregnancies with CPM. Wolstenhome and amniocentesis were analyzed [128]. The authors com-
et al. found 73 cases of CPM in 8,004 CVS specimens from ment on the likelihood of cryptic fetal mosaicism as the
women referred for advanced maternal age, previous child cause of abnormal phenotypic findings as opposed to CPM,
with aneuploidy, or family history thereof [125]. Comparison given the lack of phenotypic effect of maternal uniparental
at delivery with the control population did not show a disomy (UPD) for chromosomes 1, 7, 9, 10, 13, 21, and 22
marked increase in adverse pregnancy outcome. In 108 other or for paternal UPD for chromosomes 1, 5, 6, 7, 8, 13, 17,
cases referred for ultrasound detection of isolated IUGR, and 21. The effect of maternal and paternal UPD 20 is still
seven were shown to have CPM involving the following unclear, and for chromosomes 1 and 16, effects are less
chromosomes: 2 and 15 (1 case), 9 (1 case), 16 (3 cases), clear than previously believed. In the series, they reported
del(13) (1 case), and 22 (1 case). there was some evidence for cryptic fetal mosaicism and
Hahnemann and Vejerslev evaluated cytogenetic out- none for UPD. They describe other similar findings in the
comes of 92,246 successfully karyotyped CVS specimens literature and conclude that the finding of even very low-
from 79 laboratories from 1986 to 1994 [127]. CVS mosa- grade mosaicism in amniocytes should be regarded as
icism or nonmosaic fetoplacental discrepancy was found in significant.
Uniparental Disomy in Confined

Table 12.11 Distribution of specific single autosomal trisomies in Placental Mosaicism
each of the groups of mosaicism/discrepancy in chorionic villus tissue When a conceptus is trisomic, this aneuploidy is said to be
True fetal mosaicism
“corrected” if by chance there is early loss of one of the tri-
Trisomy CPM (# of cases)a (# of cases) somic chromosomes. Depending upon the parental origin of
2 11 the trisomy and of the chromosome that is lost, this can lead
3 10 to an apparently normal diploid cell line with uniparental
5 3 disomy (both chromosomes in a pair from one parent) for
7 32 that chromosome. Because most trisomies are maternally
8 11 1 derived, the disomy seen is often maternal, as was the case in
9 9 1 two previously reported cases of trisomy 15 mosaicism seen
10 6 at CVS in which the neonates subsequently manifested
11 1 Prader-Willi syndrome due to maternal disomy 15 [125].
12 2 1 The authors also note the reports of several cases of chromo-
13 15 2
some 16 CPM-associated IUGR in which maternal disomy
14 3
16 was seen in most of the cases. The presence of mosaic
15 11 1
trisomy 16 itself may be of most significance in such cases,
16 11
however. In this regard, Daniel et al. presented data question-
17 1
18 29 4
ing the clinical significance of UPD 16 and CPM versus that
20 12 1 of true cryptic fetal mosaic trisomy 16 [128].
21 22 9 The evaluation of parental disomy in all CPM cases
22 3 involving chromosome 15 should be offered, and this recom-
All 192 20 mendation has extended to other chromosomes as more
a
Includes all types of confined placental mosaicism, including direct information has become available.
only, long-term culture only, and both. Data from reference [127] For a thorough discussion of UPD, refer to Chap. 20.
Interphase FISH in Confined Placental Mosaicism versus long-term cultured tissues. Trisomy 2 is seen more in
Interphase fluorescence in situ hybridization (FISH, see cultured cells, and trisomy 3 is more often seen in direct prep-
Chap. 17) can be useful for the diagnosis of CPM, given that arations [124, 125]. False-positive trisomy 7 or 18 can occur
interphase FISH is rapid and has the great advantage of not with either technique. To add to the complexity, it should be
requiring growing, dividing cells to obtain results. Harrison kept in mind that true trisomy 2 and trisomy 7 mosaicisms
et al. examined the placentas of 12 pregnancies in which non- have been documented in liveborn children after having been
mosaic trisomy 18 had been diagnosed and found significant diagnosed prenatally by amniocentesis [132, 133].
levels of mosaicism, confined to the cytotrophoblast, in 7 of Maternal cell contamination (MCC) in CVS is generally due
the 12 [118]. Based on their observation that most of the mosaic to the lack of complete separation of chorionic villi from mater-
results were seen in stillborn or newborn trisomy 18 babies, nal decidua, and it is reported in an estimated 1.0–1.8% of cases
and on the fact that the great majority of trisomy 18 concep- [121, 123, 124]. The MCC reported in these studies is about half
tuses spontaneously abort, they suggested that a normal diploid of the figures above, reflecting the XX/XY admixtures, and is
trophoblast component in placental tissue may be necessary to doubled to account for the likely equal incidence of MCC in
facilitate the prolonged survival of trisomy 18 conceptuses. female fetuses. MCC occurs more often in cultured cells than in
Schuring-Blom et al. used FISH to document CPM in three direct preparations, thus underscoring the importance of using
pregnancies in which mosaic trisomy 8, mosaic trisomy 10, both methods in a full CVS cytogenetic analysis. In one report,
and nonmosaic monosomy X were observed following CVS the rate of MCC was significantly higher in specimens obtained
but which were found to be chromosomally normal at amnio- by the transcervical method (2.16%) than in samples obtained
centesis [129]. In all three cases, FISH showed the presence by the transabdominal method (0.79%) [121].
of the mosaic cell line confined to one part of the placenta. A note of caution is prudent here. Generally, when there
Henderson et al. performed a cytogenetic analysis using a is a discrepancy between the direct and the cultured prepara-
“mapping” technique of nine term placentas after CPM had tions, a subsequent amniocentesis is considered to provide
been diagnosed and found tissue-specific and site-specific the “true” result. However, a case of mosaic trisomy 8
patterns of mosaicism [130]. In addition to metaphase chro- reported by Klein et al. illustrates the fact that a true low-level
mosome analysis, they employed interphase FISH to examine tissue-specific mosaicism can exist [134]. In this case, the
several areas of the placentas. Noting that the outcomes of CVS showed a normal direct preparation and mosaic trisomy
pregnancies are highly variable after CPM is diagnosed, they 8 in culture. Subsequent amniocentesis showed normal chro-
proposed a wider study involving extensive analysis of term mosomes, but peripheral blood cultures of the newborn
placentas when this occurs in order to obtain more informa- showed trisomy 8 mosaicism. Therefore, when considering
tion regarding the outcome of such pregnancies. amniocentesis or PUBS as follow-up studies because of pos-
sible CPM observed in CVS, factors such as the specific
Direct and Cultured Preparations aneuploidy involved, the likelihood of detecting it using a
Direct CVS preparations involve the rapid metaphase analy- given sampling technique, and the risks of the additional
sis of villous cytotrophoblastic tissue. Cultured preparations invasive procedure need to be weighed.
involve the mesenchymal cells in the villi. Some laboratories
use only cultured cell preparations, and others utilize both Specimen Requirements
methods. Investigations into the outcomes of pregnancy after The minimum amount of chorionic villus material necessary
CVS support the use of both techniques to maximize the to obtain diagnostic results and the transport medium should
accuracy of the test [121, 123, 124]. These studies docu- be established in advance with the laboratory. In general, a
mented false-negative and false-positive results using direct minimum of 10 mg of tissue is needed to obtain both a direct
and cultured preparations, and the first two groups concluded and a cultured cell result, and 20 mg is ideal. If possible, the
that results from both direct and cultured techniques were specimen should be viewed through a dissecting microscope
necessary in a substantial number of cases to accurately pre- to ensure that villi are present. The specimen should be trans-
dict the fetal karyotype. In one study, long-term culture was ported at ambient temperature to the cytogenetics laboratory
advocated as having higher diagnostic accuracy, and the as soon as possible.
direct method was said to be a useful adjunct to the culture
method [121]. In a study by Los et al. of 1,829 consecutive
CVS procedures with direct and long-term cultures, one con- Percutaneous Umbilical Blood Sampling (PUBS)
clusion was that using both modalities decreased the neces-
sity for follow-up amniocentesis by 35% compared to that of Risks, Limitations, and Benefits
long-term culture alone [131]. In part at least, the finding that Percutaneous umbilical blood sampling (PUBS) is also
both techniques add to the diagnostic accuracy appears to be known as periumbilical blood sampling, fetal blood sam-
related to the nonrandom findings of some trisomies in direct pling, or cordocentesis. The largest series in the literature
244 L.M. Randolph
regarding risks of PUBS included outcomes of 1,260 diag- amniotic fluid and CVS cells. When pseudomosaicism or
nostic cordocenteses among three fetal diagnosis centers and mosaicism is seen in amniotic cell cultures, PUBS can pro-
25 practitioners[135]. A fixed needle guide was used in this vide valuable additional information regarding the likelihood
study, and prospective data was compared to the published of true mosaicism and thereby assist the couple in their deci-
experience of large centers that use a freehand technique, sion making [140–143].
where a 1–7% fetal loss rate has been reported. The proce- Although pseudomosaicism in amniotic fluid cell cultures
dure-related loss rate at a mean gestation of 29.1 +/−5 weeks is usually associated with normal chromosome analysis after
at the time of sampling was 0.9%, leading to the conclusion PUBS, the absence of trisomic cells in fetal blood does not
that technique is a variable in the loss rate for cordocentesis. guarantee that mosaicism has been definitely excluded [144].
PUBS experience at an earlier gestation was described by For example, fetal blood karyotyping is not useful for the
Orlandi et al. in 1990, who pointed out that, while cordocen- evaluation of mosaic or pseudomosaic trisomy 20. For fur-
tesis was a technique largely confined to the middle of the ther discussion of mosaicism, see “Special Issues” later, and
second trimester to term, in their experience it could be per- see also Chap. 8.
formed as early as the 12th week with acceptable results Because PUBS is associated with a significantly higher
[136]. They evaluated the outcomes of 500 procedures per- fetal loss rate than other prenatal diagnostic procedures, use
formed between 12 and 21 weeks for thalassemia study (386), of this technique should be recommended and provided with
chromosome analysis (97), fetomaternal alloimmunization great care and only in certain high-risk situations such as
(10), and infectious disease diagnosis (7). One practitioner those mentioned previously.
performed the procedures, and the volume of blood obtained
ranged from 0.2 to 2.0 mL, depending on the gestational age. Specimen Requirements
Of the 370 pregnancies not electively terminated and for Ideally, 1–2 mL of blood should be obtained and put into a
which outcome information was available, the fetal loss rate small sterile tube containing sodium heparin. Results can
was 5.2% for fetuses of 12–18 weeks’ gestation and 2.5% usually be obtained from 0.5 mL, and in some cases 0.2 mL,
between 19 and 21 weeks. Indicators of adverse outcome so even small amounts obtained should not be discarded.
included cord bleeding, fetal bradycardia, prolonged proce- A Kleihauer-Betke test may be useful in evaluating the
dure time, and anterior insertion of the placenta. Fetal brady- possibility of maternal cell admixture, particularly when a
cardia is a commonly reported complication after PUBS and 46,XX karyotype results.
is associated with a higher likelihood of fetal loss. In a review
of 1,400 pregnancy outcomes after PUBS, the overall inci-
dence of recognizable fetal bradycardia was estimated at 5% Indications for Prenatal Cytogenetic Diagnosis
[137]. It was significantly more likely to occur when the
umbilical artery was punctured. Boulot et al. performed 322 Advanced Maternal Age
PUBS and noted fetal bradycardia, usually transitory, in
7.52% of their cases [138]. Fetal bradycardia occurred in Advanced maternal age, generally defined in the United
2.5% of cases with normal outcome and in 12.5% of cases of States as 35 or older at delivery, is probably the most com-
fetal loss in one study, while in another, 11 of 12 fetal losses mon indication for prenatal cytogenetic diagnosis. For
were associated with prolonged fetal bradycardia [136, 137]. women in this age group, this indication alone provides the
The underlying fetal pathology is a significant factor in advantage of greater than 99% accuracy for detection of
fetal loss rate. Of these 12 losses, 10 were fetuses with a chromosome abnormalities. The chief disadvantage lies in
chromosome abnormality or severe fetal growth restriction. the fact that, overall, it results in the detection of only 20% of
In gestations from 17 to 38 weeks, Maxwell et al. compared chromosomally abnormal fetuses, given that 80% of chro-
the loss rates within 2 weeks of the procedure with the indi- mosomally abnormal babies are born to women under age
cations [139]. Of 94 patients having prenatal diagnosis with 35. Advanced maternal age is the most significant determi-
normal ultrasound findings, one pregnancy of the 76 that nant of the risk of a chromosome abnormality for all triso-
were not electively terminated was lost. Of the group with mies, structural rearrangements, marker chromosomes, and
structural fetal abnormalities, 5 in 76 were lost; and in the 47,XXY (Klinefelter syndrome, see Chap. 10). Maternal age
group of 35 with nonimmune hydrops, 9 were lost. It is is not a factor in 45,X (Turner syndrome), triploid (69 chro-
important to take this factor into account when counseling mosomes instead of 46), tetraploid (92 chromosomes instead
patients before the procedure. of 46), or 47,XYY karyotypes.
It has been said that no other fetal tissue “…can yield Very young women are also at increased risk of fetal
such a broad spectrum of diagnostic information (cytoge- chromosome abnormality. A 15-year-old has a 1 in 454 risk
netic, biochemical, hematological) as fetal blood” [136]. As of having a term infant with a chromosome abnormality,
a means of fetal karyotyping, it has the advantage of generat- compared to a 1 in 525 risk for a 20-year-old and a 1 in 475
ing results in 2–4 days, compared to 6–14 or more for risk for a 25-year-old woman [145] (see Fig. 12.2).
Fig. 12.2 Risk of chromosomally normal women to deliver chromosomally abnormal offspring [145]
Women 31 and Older with Twin Pregnancies Nuchal Thickening

Six causes have been proposed for nuchal thickening/folds:
A 31-year-old with a twin gestation of unknown zygosity has • Cardiac defects with heart failure related to abnormal
a risk comparable to that of a 35-year-old woman. This is ductus venosus flow velocity.
calculated as follows: given that two-thirds of such twins are • Abnormalities in the extracellular matrix of the nuchal
dizygotic, the risk that one or the other has a chromosome skin of fetuses, which may be the leading cause of this
abnormality is about 5/3 times that of a singleton pregnancy finding in fetuses with connective tissue disorders.
for that age. Thus, given that a 31-year-old woman’s risk is 1 • Abnormal lymphatic development and obstruction, which
in 384 at term for any chromosome abnormality, if she is car- appears to be the case in some fetuses with Turner
rying twins of unknown zygosity, the risk that one or the syndrome.
other has a chromosome abnormality is 5/3 × 1/384, or 1 in • Venous congestion in the head and neck due to constric-
231. This is between the risk of a 34-year-old (1 in 243) and tion of the fetal body in amnion rupture sequence or supe-
that of a 35-year-old. rior mediastinal compression or the narrow chest in some
The risk of a chromosome abnormality is not significantly skeletal dysplasias.
greater for monozygotic pregnancies compared to singletons. • Failure of lymphatic drainage due to impaired fetal move-
For pregnancies known to be dizygotic, the risk that one or ment in fetuses with neurologic disorders such as fetal
the other twin has a chromosome abnormality is about twice akinesia.
that of a singleton. See below for information on Down syn- • Congenital infection, acting through anemia or cardiac
drome risk calculations in twin pregnancies taking into dysfunction [146].
account the nuchal translucency in the co-twin. The fluid collects in the posterior neck fold, causing the
appearance of a nuchal membrane separation on ultrasound
examination (Fig. 12.3). With resolution of the fluid collec-
Abnormal Fetal Ultrasound Findings tion, a nuchal fold or thickening develops.
Nuchal membranes have been recorded as early as
Many fetal ultrasound findings are associated with an 9 weeks’ gestation. Measurement of the nuchal thickness,
increased risk for chromosome abnormalities. This list will with or without first-trimester serum screening, has become
continue to grow as the skill of practitioners and the resolu- the most sensitive first-trimester ultrasound finding used for
tion of ultrasound machines improve and also as the search Down syndrome detection [146–148]. Nicolaides, a pioneer
for indicators of increased risk other than advanced maternal of first-trimester nuchal thickness ultrasound scans, cites a
age continues. detection rate of 90% for chromosome abnormalities when
246 L.M. Randolph
Fig. 12.3 Ultrasound

image of increased nuchal
fold (NF) measuring
6.1 mm in a second-
trimester fetus (Courtesy
of Greggory DeVore, MD)
performed in conjunction with pregnancy-associated plasma There have been several first-trimester ultrasound studies
protein A (PAPP-A) and free ß-hCG at 11–14 weeks of preg- of nuchal thickening. Van Vugt et al. karyotyped 102 first-
nancy, with an invasive pregnancy testing rate of 5% [146]. trimester fetuses with a nuchal translucency of 3 mm or more
Nuchal folds and cystic hygromas have been known to be and found that 46% had an abnormal karyotype: 19 had tri-
associated with chromosome abnormalities since 1966, with somy 21; 9 had trisomy 18; 13 had 45,X; 1 had 47,XXX; and
an incidence of chromosome abnormalities ranging from 5 had other chromosome abnormalities [152]. Multiple logis-
22% to more than 70% in various series [149]. Based on 22 tic regression analysis was used to take into account data
other studies, plus their own data, Landwehr et al. found that modifiers such as gestational age and maternal age. The
32% of 1,649 karyotyped fetuses with nuchal folds or mem- authors examined the septated versus the nonseptated nuchal
branes and/or cystic hygromas had a chromosome abnormal- translucencies. Septa were seen in 45 (44%) of the fetuses, of
ity. These included 207 cases of trisomy 21; 108 cases of whom 36 (80%) had chromosome abnormalities. Of 57
trisomy 18; 30 cases of trisomy 13; 131 cases of 45,X; and fetuses with no septation, 11 (19%) had abnormal chromo-
48 other chromosome abnormalities. This study included somes. This compared to a 56% incidence of chromosome
first- and second-trimester ultrasound scans, which employ abnormalities in first-trimester fetuses with septation and
different criteria for nuchal thickness. 23% incidence of chromosome abnormalities in first-trimester
In a 12-center study designed to determine the sensitivity fetuses without septation in Landwehr’s study [149].
and specificity of second-trimester soft-tissue nuchal fold In 1,015 fetuses of 10–14 weeks’ gestation with nuchal
measurement for the detection of trisomy 21, 3,308 fetuses fold thicknesses of 3, 4, 5, and >5 mm, Pandya et al. found
of 14–24 weeks’ gestation were evaluated [150]. Using 6 mm incidences of trisomies 21, 18, and 13 to be approximately 3
as a cutoff, a nuchal skin fold was seen in 8.5% of chromo- times, 18 times, 28 times, and 36 times higher than the
somally normal fetuses and in 38% of those with trisomy 21. respective numbers expected on the basis of maternal age
A false-positive rate below 5% was obtained by 81% of the alone [153]. This corresponded to risks of one of these chro-
investigators. The authors concluded that this sign is useful mosome abnormalities to be 5, 24, 51, and about 60%,
in skilled hands in the second trimester, but it does not appear respectively.
suitable for population screening because of the high vari- Using a 4-mm cutoff in fetuses of 9–13 weeks, Comas
ability in the results among the investigators. et al. detected 57.1% of aneuploidies with a false-positive
A nuchal thickness cutoff of 4 mm was chosen by Nadel rate of 0.7% and a positive predictive value of 72.7% [154].
et al. in a study of 71 fetuses of 10–15 weeks’ gestation, of Szabó et al. evaluated 2,100 women under 35 years of age
which 63 were karyotyped [151]. Abnormal karyotypes were by ultrasound at 9–12 weeks’ gestation [155]. Women were
found in 31 of 37 hydropic fetuses and in 12 of 26 nonhy- offered CVS if the nuchal fold was 3 mm or greater. The
dropic fetuses. The nonhydropic fetuses also had no septations authors found an incidence of first-trimester nuchal fold to be
in the hygromas. Twenty-two of the fetuses with septated 1.28% in women under 35, with a corresponding percentage
hygromas had chromosome analysis, and 19 had abnormal of chromosome abnormalities being 0.43%. This indicated a
chromosomes. Of fetuses with hydrops and no septations, 11 1 in 3 risk for chromosome aneuploidy in this age group when
of the 14 had abnormal chromosomes. a thickened nuchal fold was seen.
with a nuchal translucency of ³95th percentile but less than

the 99th percentile. In fetuses with a nuchal translucency
measurement ³99th percentile or in which tricuspid regurgi-
tation and/or an abnormal ductus venosus flow pattern had
been found, an earlier fetal echo was recommended.
Sau et al. evaluated the significance of a positive second-
trimester serum screen in women who were screen negative
after a first-trimester nuchal translucency scan. Of 2,683
women screened, eight cases of trisomy 21 were detected, all
of which had a positive nuchal screen result. Serum screening
of 1,057 women who screened negative by nuchal translucency
showed 46 high-risk results, all of which proved to be false-
positive. The authors concluded that second-trimester bio-
chemistry screening following a negative nuchal translucency
screen did not increase the detection of trisomy 21 [159].
Cuckle and Maymon reported on a method whereby fetus-
specific Down syndrome risks in twins could be assessed
taking the other fetal nuchal fold into account [160]. This
was based upon the previous report by Wøjdemann et al. that
there is a correlation coefficient of 0.34 between the pairs of
Fig. 12.4 Fetal Echocardiogram: Normal 4 Chamber View of the NTs, expressed in log multiples of the median for crown-
Fetal Heart (obtained using an iE33 system and 5-MHz curvilinear rump length [161]. This was seen in both monochorionic and
probe; Philips Healthcare, Bothell, WA) in a breech fetus at 26 weeks dichorionic twins. Cuckle and Maymon found a correlation
gestational age. Ant, anterior chest wall; Ao, Aorta; Post, Posterior
Spine; RA Right Atrium; RV Right Ventricle; LA Left Atrium; LV Left coefficient in unaffected pregnancies of 0.45 (P < 0.0001)
Ventricle. Image courtesy of Jay Pruetz, M.D., Assistant Professor of and estimated to be 0.12 and 0.04 in discordant and concor-
Pediatrics, Division of Cardiology, Children’s Hospital Los Angeles, dant twins, respectively [160].
Keck School of Medicine, University of Southern California
Cystic Hygroma and Cytogenetic Evaluation
Given that nuchal thickening is clearly associated with of Cystic Hygroma Fluid
chromosome abnormalities, most commonly trisomy 21, and Women whose second- or third-trimester fetuses have large
that it is the most common abnormal ultrasound finding in cystic hygromas may not have an easily accessible fluid
the first trimester, ultrasound evaluation of nuchal thickness pocket in which to perform an amniocentesis. In such cases,
in the first trimester in combination with maternal serum paracentesis of the hygroma may yield a cytogenetic result,
markers has proven to be one of the most important early and at fetal demise or delivery, chorionic villus or placental
screening tools to evaluate an increased risk of aneuploidy cell cultures may prove beneficial in obtaining chromosomal
[156]. In a review of ultrasound diagnosis of fetal abnormali- diagnosis. The yield from amniocentesis is still the greatest,
ties in the first trimester, Dugoff cites the work of Hyett et al., so if it can be accomplished, this is still the procedure of
who reported on an association between increased nuchal choice for cytogenetic diagnosis in such cases [162].
translucency and heart abnormalities. In that study, the prev-
alence of major cardiac defects increased with nuchal thick- Heart Abnormalities
ness from 5.4 per 1,000 for translucency of 2.5–3.4 mm to Structural Heart Abnormalities
233 per 1,000 for translucency ³ 5.5 mm. The authors recom- Structural heart abnormalities are a well-established risk fac-
mended that when fetuses have a thickened nuchal fold and tor for chromosome abnormalities. Postnatal data indicate a
normal chromosomes, fetal echocardiography at frequency of chromosome abnormalities in infants with con-
18–22 weeks’ gestation is merited, besides close scrutiny of genital heart diseases to be 5–10%, and 2–8 per 1,000 live
cardiac anatomy in the first trimester [157] (Fig. 12.4). This births have a structural cardiac abnormality [163]. Prenatal
topic was reviewed by Clur et al. in 2009 [158]. They con- data indicate that up to 32–48% of fetuses with cardiac
cluded that an increased NT is associated with an increased abnormalities are chromosomally abnormal [163–165].
risk for congenital heart disease with no bias for one form or The difference between prenatal and postnatal data probably
another. The risk increases with increasing NT measurement. reflects the high incidence of in utero demise in fetuses with
In combination with tricuspid regurgitation and an abnormal chromosome abnormalities.
ductus venosus Doppler flow profile, however, it is a strong The most frequent prenatally and postnatally diagnosed
marker for congenital heart disease. The authors recom- heart abnormality is the ventricular septal defect, followed by
mended a fetal echo at 18–22 weeks’ gestation in fetuses tetralogy of Fallot (TOF), right or left hypoplastic heart, and
248 L.M. Randolph
Fig. 12.5 Fetal Echocardiogram: Abnormal 4 Chamber View of the is not formed. There was also mild mitral regurgitation on color
Fetal Heart (obtained using an iE33 system and 5-MHz curvilinear Doppler assessment (not shown). RA Right Atrium; RV Right Ventricle;
probe; Philips Healthcare, Bothell, WA) in a fetus with confirmed tri- LA Left Atrium; LV Left Ventricle. Image courtesy of Jay Pruetz,
somy 21 showing a complete atrioventricular (AV) canal defect with M.D., Assistant Professor of Pediatrics, Division of Cardiology,
large inlet ventricular septal defect and primum atrial septal defect (*). Children’s Hospital Los Angeles, Keck School of Medicine, University
Note the AV valves are located at same level and the crux of the heart of Southern California
transposition of the great arteries. Many investigators use the atrial isomerism with double outlet right ventricle. One of
four-chamber view (Fig. 12.4) to evaluate the fetal heart, with the fetuses had an absent kidney, and the others had isolated
an 80–92% sensitivity claimed by this method [166]. However, cardiac abnormalities [166].
the four-chamber view alone will not detect TOF or transposi- A population-based study of the 22q11.2 deletion was
tion of the great arteries, and only detects approximately 59% undertaken by a group from Atlanta, Georgia. They evalu-
of heart abnormalities. A complete atrioventricular canal ated data on babies born from 1994 to 1999 in the Atlanta
defect is seen in a fetus with trisomy 21 in Fig. 12.5. area and matched those records with the Metropolitan Atlanta
Extracardiac abnormalities are seen, depending on the Congenital Defects Program, a local heart center, and the
gestational ages at which the ultrasound evaluations are per- genetics division at Emory University in Atlanta. Among
formed and what is considered an abnormality, in 36% to 71% 255,849 births, 43 children were found to have 22q11.2 dele-
of fetuses with heart abnormalities [165–167]. The presence tions for an overall prevalence of 1 in 5,950 births [167].
of extracardiac abnormalities increases the risk of a chromo- Thirty-five of the children had heart abnormalities as shown
some abnormality from 32–48% to 50–71%. in Table 12.12. What the investigators found was that about
Conotruncal heart abnormalities are those related to faulty one of every two cases of interrupted aortic arch, one of every
conotruncal septation, or division, of the single primitive five cases of truncus arteriosus, and one of every eight cases
heart tube into two outflow tracts that in turn result from the of tetralogy of Fallot in the population were due to the dele-
fusion of two swellings that arise in the truncal region at tion. See Tables 12.12 and 12.13 for a listing of the data from
30 days’ gestation. With increasing awareness of the strong this study.
association between conotruncal heart abnormalities and
chromosome 22q11 deletions or microdeletions, it is now Intracardiac Echogenic Foci
recommended that FISH analysis of this region be performed Echogenic lesions within the fetal cardiac ventricles have
when a conotruncal heart abnormality is seen on fetal ultra- been recognized since 1987, when they were described in the
sound and fetal chromosomes are normal. In five patients left ventricles of 3.5% of fetuses examined by ultrasound
whose fetuses had fetal cardiac abnormalities and a prenatal [170]. The foci were attributed to thickening of the chordae
diagnosis of 22q11 deletion [del(22)(q11.2)], the heart tendinae. Others have reported a 20% incidence of left ven-
abnormalities included TOF with absent pulmonary valve, tricular echogenic foci and right ventricular foci in 1.7%
pulmonary atresia with VSD, truncus arteriosus, and left [171]. See Fig. 12.6.
Table 12.12 Cardiovascular abnormalities in children with 22q11.2 Table 12.13 Clinical findings amenable to ultrasound detection that
deletion in Atlanta study, 1994–1999 are consistent with 22q11.2 deletion
Total Percenta % of Finding Number Percent one in
Finding no. (%) totalb Any major diagnostic finding 43 100
Cardiac abnormalitiesc 35 100 81 Cardiovascular
Interrupted aortic arch type B 8 23 19 Heart and great arteries 35 81 1.2
Truncus arteriosus 4 11 9 Vascular (branch arteries 22 51 2.0
Tetralogy of Fallot and variants 15 43 35 and great veins)
Pulmonary atresia with VSD 6 17 14 Spina bifida 2 4.7 22
Tetralogy of Fallot, absent pulmonary 3 9 7 Brain stem anomaly 1 2.3 43
valve Communicating hydrocephalus 1 2.3 43
Tetralogy of Fallot, simple 6 17 14 Eventration of diaphragm 1 2.3 43
D-transposition of great arteries 1 3 2 Thoracic hemivertebrae 2 4.7 22
Valve pulmonic stenosis, apical VSDs, ASD 1 3 2 Rib abnormalities 1 2.3 43
Ventricular septal defect 7 20 16 Polydactyly of hands 1 2.3 43
Vascular abnormalities 22 63 51 Hydronephrosis 3 7.0 14
Right aortic arch 15 43 35 Renal atrophy 1 2.3 43
Mirror image of brachiocephalic vessels 5 14 12 Renal cyst 1 2.3 43
Vascular ring 2 6 5 Data from reference [167]
Aberrant origin subclavian artery 7 20 16
Left superior vena cava 4 11 9
VSD ventricular septal defect, ASD atrial septal defect
a
Percentage among children with 22q11.2 deletion and cardiovascular
findings (n = 35)
b
Percentage among all children with 22q11.2 deletion (n = 43)
c
One child had interrupted aortic arch and truncus arteriosus
Fig. 12.6 Fetal Echocardiogram: Normal 4 Chamber View of the Fetal the LV chamber and not within the myocardium itself. RA Right Atrium;
Heart (obtained using an iE33 system and 5-MHz curvilinear probe; RV Right Ventricle; LA Left Atrium; LV Left Ventricle. Image courtesy
Philips Healthcare, Bothell, WA) showing a single left ventricular echo- of Jay Pruetz, M.D., Assistant Professor of Pediatrics, Division of
genic focus or LVEF (arrow). Note the LVEF is brighter than the sur- Cardiology, Children’s Hospital Los Angeles, Keck School of Medicine,
rounding myocardium and is located in the mitral valve apparatus inside University of Southern California
250 L.M. Randolph
The association between left ventricular echogenic foci chamber view, and they were seen in all 89 with an apical
and chromosome abnormalities was noted in a study of 2,080 four-chamber view [175]. Wax et al., in a study of second-
fetuses at 18–20 weeks’ gestation; 33, or 1.6%, had an echo- trimester high-risk pregnancies, classified the foci by their
genic focus. Four of these had chromosome abnormalities echo amplitude and found that fetuses whose echogenic foci
(two trisomy 18, one 45,X, and one trisomy 13). All had images were lost at the same gain setting as that of the tho-
other abnormalities, including heart defects [170]. racic spine had a 40% risk of aneuploidy (two of five fetuses,
The natural history of echogenic intracardiac foci was p = 0.005) [176]. That some centers report an association
studied in a cohort of 1,139 patients [171]. Echogenic foci between the foci and an increased incidence of trisomy 21
were seen in 41 of 1,139 fetuses, or 3.6%. In 38, the foci were and other chromosome abnormalities, and others do not,
in the left ventricle; in two, they were in the right ventricle; may reflect differences in the populations studied—whether
and in one, they were in both. None of these fetuses had other small or large, whether high risk or not. Another difference
abnormalities. The echogenic foci were again seen in the 27 that has been documented is that an isolated echogenic
newborns having echocardiograms up to 3 months of age. intracardiac focus is more prevalent among Asian fetuses
The authors pointed out that the key clinical significance of compared to non-Asians, making it much less helpful in the
echogenic lesions is that they should be differentiated from risk assessment in this population [177].
intracardiac tumors and ventricular thrombi. In a review of second-trimester ultrasound evaluations
The outcomes of 25,725 ultrasound examinations were over the past 30 years, Benacerraf reviewed the literature
reported in a retrospective study from 12 to 24 weeks’ gesta- regarding EIF and concluded that the presence of an EIF
tion [172]. Echogenic intracardiac foci were seen in 44 cases alone in a patient previously found by screening to be at low
(0.17%). Of the 35 fetuses with left-sided isolated foci, all risk does not constitute an indication for prenatal diagnosis
had uneventful neonatal courses. In nine others, multiple foci [178]. She pointed out in her review that one-third of US
were seen, involving the right ventricle in five cases. Of practitioners in a survey indicated they do not tell their patients
these, two had uneventful courses, but the other seven had of the finding when it is the only ultrasound finding.
additional findings, including five with structural or func-
tional cardiac disease (including one with trisomy 13), one Nuchal Translucency and Echogenic Intracardiac Foci
with GM1 gangliosidosis, and one with echogenic bowel and To test the hypothesis that increased first-trimester nuchal
missed abortion. The paper includes a useful discussion of translucency is associated with isolated intracardiac foci in
the various possible causes of the echogenic foci, and the the second trimester, Prefumo et al. evaluated 7,686 normal
authors conclude by agreeing with the consensus that iso- singleton fetuses who had a nuchal translucency scan and
lated left ventricular echogenic foci are a benign finding, but either a subsequent normal follow-up scan at 18–23 weeks
other echogenic intracardiac findings may not be. (n = 7,447) or isolated intracardiac foci (n = 239) [179]. They
Two subsequent publications, in contrast, found a found that the prevalence of echogenic intracardiac foci in
significantly increased risk of trisomy 21 in fetuses with an fetuses with normal nuchal translucency was 2.9% versus
echogenic intracardiac focus. In a study by Bromley et al. of 8.1% in the fetuses with abnormal nuchal translucency. The
1,334 high-risk second-trimester patients, 66 (4.9%) had an adjusted odds ratio was 2.92. The authors concluded that an
echogenic intracardiac focus [173]. Four of twenty-two association exists between first-trimester nuchal translucency
(18%) trisomy 21 fetuses had an echogenic focus, compared and second-trimester echogenic intracardiac foci, so they
with 62 (4.7%) of 1,312 fetuses without trisomy 21. The should not be used independently in risk calculations.
presence of this finding increased the risk of trisomy 21 four-
fold. In two of the trisomy 21 fetuses, no other ultrasound Nasal Bone
abnormalities were seen. Hypoplasia or “absence” of the nasal bone is a more recently
In a retrospective blinded study of pregnancies at described ultrasound finding that appears to improve the
15–21 weeks’ gestation, Norton et al. found an echogenic detection of fetuses with trisomy 21 [182–186]. In 2001
focus of unspecified location in the heart in five of 21 (24%) Cicero et al. reported that in about 70% of fetuses with tri-
trisomy 21 fetuses compared to four of 75 (5%) controls, somy 21 from 11 to 14 weeks’ gestation, the nasal bone is
yielding an odds ratio for trisomy 21 of 5.5 (1.12 < OR < 28.4) not visible. In a follow-up study to determine whether fetal
when an echogenic focus is seen [174]. nuchal thickness and the level of maternal serum biochemi-
The variations in reported incidences of echogenic intrac- cal markers is independent of the presence or absence of the
ardiac foci (EIF) probably reflect the differences in definition nasal bone, Cicero’s group performed a retrospective case-
of echogenic foci and in ultrasound machines. Ranzini et al. control study of 100 trisomy 21 fetuses and 400 chromoso-
note that visualization depends on the orientation of the mally normal fetuses. The nasal bone was absent in 69 and
four-chamber view. In 89 fetuses with intracardiac echo- present in 31 of the trisomy 21 fetuses. There were no
genic foci, the foci were seen in only 29 with a lateral four- significant differences in any of the other study variables.
It was concluded that for a false-positive rate of 5 percent, in which different criteria exist for this measurement, the
screening with nuchal thickness, nasal bone, maternal free detection rate might be as high as 70%, but the false-positive
ß-hCG and PAPP-A would be associated with a detection rate rises to 5%. She cited a prevalence of 8.8% for small or
rate of 97%. For a false-positive rate of 0.5%, the detection absent nasal bone in the Afro-Caribbean population as
rate was 90.5% [187]. opposed to 0.5% of Caucasian fetuses, based upon Cicero’s
Another study by Cicero et al. attempted to answer the studies.
question concerning the association between “absence” of
the nasal bone at 11–14 weeks and chromosome abnormali- Renal Pyelectasis
ties [183]. In this study, 3,829 fetuses were studied. Maternal Renal pyelectasis is mild dilation of the renal pelvis. A pos-
ethnic origin was recorded. The fetal profile was success- sible link between fetal renal pyelectasis and trisomy 21 was
fully recorded in 98.9% of cases. In 3,358 of 3,788 cases, the described in 1990 [189]. This led to other studies with
fetal chromosomes were normal, and in 430, they were conflicting results. In 1996, Wickstrom et al. published a
abnormal. In the chromosomally normal group, the incidence prospective study of 7,481 patients referred for prenatal
of absent nasal bone was related primarily to the ethnic ori- ultrasound evaluation [190]. Of these, 121 (1.6%) had iso-
gin of the mother. It was absent in 2.8% of Caucasians, lated fetal pyelectasis (defined as ³4 mm before 33 weeks’
10.4% of Afro-Caribbeans, and 6.8% of Asians. gestation and ³7 mm at 33 weeks’ gestation). This compares
The nasal bone was absent in 66.9% of fetuses with trisomy with prevalences of 1.1–18% in other studies. Of the 121, 99
21. In trisomy 18 fetuses, it was absent in 57.1%, and with karyotypes were available. One of these was trisomy 21, and
trisomy 13, it was absent in 31.8%. In Turner syndrome and in the other was mosaic 47,XYY/46,XY. Based on maternal
other chromosome abnormalities, the rate was 8.3–8.8%. age and the baseline risk for trisomy 21 in the population, the
A study in Denmark showed the combination of nuchal authors calculated a relative risk of 3.9 for trisomy 21 when
translucency and visualization of the nasal bone between 11 isolated renal pyelectasis is seen and a 3.3-fold increase in
and 14 weeks to be as good a predictive marker as nuchal risk for all chromosomal abnormalities in the presence of
translucency and biochemical markers [186]. Zoppi et al. isolated fetal pyelectasis.
evaluated other fetal chromosome abnormalities with regard Corteville et al. studied 5,944 fetuses for the presence of
to nonvisible nasal bone and found the bone not to be visible pyelectasis, defined as an anteroposterior renal pelvic diam-
in four out of five trisomy 18 fetuses, in two out of three eter of 4 mm or greater before 33 weeks or 7 mm or greater
Turner syndrome fetuses, and in 0.2% of fetuses with normal after 33 weeks, the same definition as was used by Wickstrom
karyotypes [182]. et al. [190, 191]. Pyelectasis was seen in 4 of 23 (17.4%)
The literature to date suggests that when adequate visual- trisomy 21 fetuses and in 120 of 5,876 (2%) normal controls.
ization is possible, which occurs in 91.9–98.9% of series, This was statistically significant at p < 0.001. When fetuses
absent or hypoplastic nasal bone is seen in 60–80% of fetuses with other ultrasound abnormalities were excluded, the pre-
with trisomy 21. Bunduki et al. performed ultrasound exami- dictive value of pyelectasis fell from 1 in 90 to 1 in 340. They
nations on 1,923 consecutive singleton pregnancies at recommended that amniocentesis should be reserved for
16–24 weeks and noted that nasal bone length increased as a those cases presenting other risk factors such as advanced
function of gestational age, showing a linear relationship maternal age, abnormal maternal serum screening results, or
[187]. Screening for trisomy 21 using the 5th percentile as a other ultrasound abnormalities. They did not adjust the risk
cutoff value resulted in a sensitivity of 59.1% for a 5.1% for trisomy using maternal age.
screen-positive rate. The likelihood ratio was 11.6. In a literature review study, Vintzileos and Egan found
A review of “promises and pitfalls” of first-trimester sono- that isolated pyelectasis was not associated with an increased
graphic markers in the detection of fetal aneuploidy by Borrell risk for trisomy 21 unless other markers were present, such
in 2009 described the challenges inherent in imaging the fetal as those noted previously [191, 192]. In Benacerraf’s review,
nasal bone, including incorrect insonation angle and nonsag- she agreed, noting that the sensitivity for this marker is low,
ittal section [188]. He cited a detection rate of trisomy 21 with at 17–25%, with a false-positive rate of 2–3%, “making this
a 2.5% false-positive rate based upon Cicero’s studies. a minor marker, used almost exclusively in conjunction with
In Benacerraf’s review of current practice of second- others or in patients already at high risk” [178].
trimester sonographic markers for the detection of trisomy 21, Degani et al. evaluated the recurrence rate of fetal
she also notes the importance of proper insonation angle, pyelectasis in subsequent pregnancies [193]. They studied
as otherwise the nasal bone might appear short, thus increas- 420 women with two consecutive normal uncomplicated
ing the false-positive rate for this marker [178]. Using the pregnancies screened at 15–24 weeks by ultrasound.
criterion of absent nasal bone ossification, the detection Pyelectasis was defined as a fetal pelvis of 4 mm or more in its
rate in the second trimester is lower (30–40%), but the false- anteroposterior dimension. Of 64 women with fetuses with
positive rate is very low. If a hypoplastic nasal bone is used, pyelectasis, 43 (67%) had a recurrence in the next pregnancy.
252 L.M. Randolph
Compared with normal fetuses, those with pyelectasis had a ploid fetuses with CPC, trisomy 21, mosaic trisomy 9, trip-
relative risk of 6.1 to have a recurrence (95% confidence loidy, 47,XXY and 45,X/46,XX, trisomy 13, unbalanced
interval = 4.3–7.5, p < 0.001). This study has implications for (3;13) translocation, and cri du chat syndrome [del(5p)]
determining the clinical significance of pyelectasis. In this have also been seen in fetuses with CPC [196, 199, 202,
regard, Johnson et al. studied 56 pregnant women with fetal 204, 205, 208].
pyelectasis or cystic lesions identified from 7,500 ultrasound Shields et al. include mention of two issues in CPC,
examinations [194]. They found that none of 50 kidneys namely size and uni- versus bilaterality [199]. They con-
15 mm or smaller in anteroposterior diameter had obstruc- clude, based on a review of the literature, that neither size nor
tion, and 11 of 14 (79%) kidneys larger than 15 mm were laterality plays a part in the risk assessment. Size varies with
obstructed or showed vesicoureteral reflux on postnatal gestational age, and laterality can be difficult to determine
examination. Noting that other studies have found the need due to near-field artifact on ultrasound examination. These
for intervention in the child after a prenatal ultrasound finding conclusions were also reached by Meyer et al. in a retrospec-
of 10-mm dilation, they recommended complete radiological tive review of 119 pregnancies with CPC [209].
evaluation after birth for infants with pelvic diameters Demasio et al. performed a meta-analysis of eight pro-
exceeding 10 mm. For children with mild to moderate unilat- spective trials of 106,732 women under 35 years of age with
eral hydronephrosis, evaluation may be delayed for 1–2 weeks pregnancies affected by isolated choroid plexus cysts [210].
because oliguria in the first 2 days of life leads to an under- If serum screening was positive, the woman was excluded
estimation of the degree of hydronephrosis. from analysis, although those data were not available for all
in the study. A total of 1,235 fetuses had choroid plexus cysts
Choroid Plexus Cysts for an incidence of 1.2%. None had chromosome abnormali-
The existence of choroid plexus cysts (CPC) has become ties. The authors contend that amniocentesis is not warranted
recognized, along with several other fetal ultrasound in women with otherwise normal ultrasound examinations
findings, due to improvements in ultrasound imaging. CPC who are less than 35 years old or the equivalent by serum
were first described in 1984 [195]. The choroid plexuses are screening.
round or oval anechoid structures within the choroid plexus Another meta-analysis was performed by Yoder et al. to
of the lateral ventricle derived from neuroepithelial folds. assess the risk of trisomies 18 and 21 with isolated choroid
CPC are seen in 0.18–2.3% of pregnancies [196]. These plexus cysts [211]. Women of all ages were included in the
cysts usually disappear in the second trimester in normal 13 prospective studies, comprising 246,545 second-trimester
pregnancies but may also disappear in chromosomally scans. The likelihood ratio for trisomy 18 was 13.8 and for
abnormal pregnancies [197]. trisomy 21 was 1.87. The authors concluded that their data
The first association between CPC and fetal trisomy 18 support offering women amniocentesis to evaluate trisomy
was published in 1986 by Nicolaides et al. [198]. In the inter- 18 when maternal age is 36 or older or when the risk for tri-
vening years, many publications on the association between somy 18 detected by serum marker screening is greater than
CPC and chromosome abnormalities have come out. or equal to 1 in 3,000. In another study by Ghidini et al., a
Consensus has been reached as to the positive association likelihood ratio for trisomy 18 for isolated choroid plexus
between CPC and chromosome abnormalities. However, cysts in the second trimester was 7.09. They advocate multi-
investigators have differed in their conclusions as to whether plying the patient’s prior risk by this figure to decide on
an isolated CPC confers a risk of chromosome abnormality whether amniocentesis is indicated [212].
high enough to warrant amniocentesis or whether the risk is Benacerraf’s review of this topic concluded that the inci-
not high enough to routinely recommend amniocentesis dence of choroid plexus cysts in fetuses with and without
unless other risk factors are present [196, 199–206]. Gross trisomy 21 is the same [178].
et al. prospectively studied patients at their institution and On balance, counseling regarding isolated CPC clearly
reviewed literature to include a meta-analysis of other studies cannot be undertaken in a vacuum. A young woman with a
prospectively done with more than ten cases of CPC. From negative triple marker screen for trisomy 18 and no other
these data, they estimated the risk of trisomy 18 in fetuses ultrasound abnormalities is much less likely to be carrying a
with isolated CPC to be one in 374. From the incidence of fetus with trisomy 18 than is a 39-year-old woman with a
trisomy 18 and of isolated CPC, plus these data, they esti- triple marker screen result positive for trisomy 18 and no
mated the positive predictive value of CPC with trisomy 18 other fetal ultrasound abnormalities.
in the general prenatal population to be one in 390. [205] Even without other ultrasound abnormalities and with
Nyberg et al. reviewed 47 consecutive cases of trisomy normal chromosomes, CPC can be frightening to prospec-
18 and found that 12 of 47 fetuses (25%) had CPC, two of tive parents, who often are concerned about a “hole in my
whom had no other ultrasound abnormality [207]. Although baby’s head.” It is important to explain their significance in
trisomy 18 is the chromosome abnormality most often a balanced way, to indicate that in the majority of fetuses,
associated with CPC, seen in about three-fourths of aneu- they are an incidental finding and that they are likely to
disappear before birth. Results of a follow-up study (mean by several authors [219–222]. It is also associated with
35.5 ± 16.2 months) on 76 children who as fetuses were several adverse outcomes, including fetal chromosome
found to have CPC are also reassuring; no effect on develop- abnormalities, fetal cytomegalovirus infection, other infec-
ment was found as measured by the Denver II Developmental tions, cystic fibrosis (CF), intrauterine growth restriction,
Screening Test [213]. fetal demise, and intestinal obstruction possibly related to
CF [219, 220, 222–230]. The presence of coexisting ele-
Short Humerus or Femur vated maternal serum AFP increases the risk of adverse
Measurement of the long bones of the fetus does not require outcome, particularly fetal IUGR and demise [220, 230].
the same level of expertise as evaluating more subtle struc- See Table 12.14. The studies referenced previously describe
tural malformations. Thus, because shortness of the long the finding of HEB on second-trimester ultrasound exami-
bones is associated with an increased risk of chromosome nation. Third-trimester HEB associated with trisomy 21
abnormalities and because the length is relatively easy to has also been reported in a fetus in which the second-
measure, several investigators have focused on this finding trimester scan did not show HEB [231].
as a way of increasing or decreasing a woman’s a priori risk The incidence of HEB in second-trimester fetuses with
of having a fetus with a chromosome abnormality. trisomy 21 is 7% [232]. The relative risk of adverse outcome
Shortness of the humerus and the tibia might have greater in isolated HEB is 6.5 [230]. Benacerraf notes the sensitivity
sensitivity in detecting trisomy 21 than shortness of the femur of hyperechoic bowel in trisomy 21 as ranging from 3.3 to
and fibula, as was found in a prospective study of 515 patients 27%, likely due to the subjectivity in assessment of this
between 14 and 23 weeks’ gestation who were at increased marker, but as it has a very low false-positive rate of less than
risk for a chromosome abnormality because of age or triple 1%, it remains an important marker for the detection of tri-
marker screening results or both [214]. Tables of risk for trisomy 21 [178].
somy 21 for maternal age and maternal serum screening posi- Part of the reported variation in outcome of HEB is due to
tive status were developed that take into account all four long different degrees of brightness of the finding and also to
bones’ lengths being normal versus one, two, three, or four inter-machine and inter-observer variability (Fig. 12.7).
bone lengths being normal. Use of this approach led to the Grades of echogenicity, from 0 (isoechoic) to 3 (bone-like
conclusion that if all long-bone lengths are normal, amnio- density) have been used [222, 231], but even those compare
centesis may not be recommended to women under age 40. the finding to different fetal parts—liver versus iliac crest,
Others have not found femur length to be reliable in ultra- for example. The more hyperechoic, the higher the risks.
sound screening of trisomy 21, although humerus length does Another reason for variability in reported outcomes relates to
appear to be associated [215, 216]. The positive predictive the a priori risks. For example, Caucasian non-Hispanic
value for trisomy 21 in women with risks of 1 in 500 and 1 in patients have a much higher a priori risk of CF than indi-
1,000 was found to be 2.3 and 1.2%, respectively. viduals of other races.
A significant confounder, however, is that long-bone What causes the finding of HEB? One group commented
length varies with race, and this factor has not been taken on the decreased microvillar enzymes in amniotic fluid in
into account in most studies. In a fetal biometry study of pregnancies affected by trisomy 21, trisomy 18, and CF
Asians, the long-bone lengths were measured in more than [230]. It was thought that the low levels in CF may be due to
6,000 fetuses, and the conclusion was that the reference delayed passage of meconium, and in trisomy 18 and 21, the
charts derived should be used in all Asian fetuses [217]. In a delayed passage may be due to decreased bowel motility or
study of 110 Korean fetuses with trisomy 21 and 602 euploid abnormal meconium. Fetuses with intra-amniotic bleeding
controls, the femur length was found to be a poor predictor have a four- to sevenfold increase in HEB [222, 232]. These
of trisomy 21 in the second trimester, with a sensitivity of investigators hypothesized that swallowing of amniotic fluid
32.8% and false-positive rate of 5% [218]. Thus the use of containing heme pigments after intra-amniotic bleeding
fetal biometric measures should be cautiously interpreted seemed to be the cause of the echogenicity.
with racial factors in mind.
As Benacerraf has pointed out in her review of second- Other Ultrasound Markers of Aneuploidy
trimester ultrasound evaluation of trisomy 21, this marker A summary of several series of ultrasound studies indicat-
is best used in combination with other markers to predict ing risks of chromosome abnormalities in association with
risk [178]. specific ultrasound findings is shown in Table 12.15. Clearly,
some ultrasound markers in isolation indicate a significant
Hyperechoic Bowel risk of chromosome abnormality, and others may not achieve
Hyperechoic bowel (HEB), also known as echogenic bowel significance unless other ultrasound abnormalities or other
and hyperechogenic fetal bowel, is a qualitative ultrasound maternal risk factors are present. Less commonly used
finding of unclear significance. It has been described as a markers and abnormalities for trisomy 21 risk assessment
normal variant with an incidence of 0.2–0.56%, as reviewed include wide iliac angle, flat facies, sandal gap foot, short
254 L.M. Randolph
Table 12.14 Clinical outcome of second-trimester finding of isolated bright hyperechoic bowela
Scioscia [219]b Nyberg [220]c Bromley [222]b Slotnick [229]c Muller [224]b MacGregor [225]c
1. No. of cases withisolated 18 64 42 102 182 45
bright HEB
2. No. of cases with normal 13 (72) 41 (75) 26 (62) – 111 (67) 34 (76)
outcome (%)
3. No. of cases with chromosome 2 (11)d 7 (11)f 0 5 (4.9)g 8 (4.5) 0/16 (0)
abn. (%)
4. No. of cases with cystic fibrosis 0d/17 (0) NTc NTh 7/65 (11) 10/116i(8.6) 2/15 (13)
mutations (%)
5. No. of cases with infections (%) NTh 1 (1.6) – – 7/? (?) 2/45 (4)
6. No. of cases with IUGR (%) 1 (5.6) 6 (9.3) 8 (19) – 10/121 (8) NRj
7. No. of cases with nonelective 2 (11) 3 (4.7) 15 (36) – 24/104 (23) 3/45 (6.7)
demise (%)
a
This table excludes fetuses with ultrasound abnormalities other than isolated HEB
b
Retrospective study
c
Prospective study
d
Both trisomy 21
e
Seven CF mutations tested
f
Five trisomy 21, one 47,XXX, one trisomy 13
g
All trisomy 21
h
Not tested
i
One DF508 homozygote, 9 heterozygotes; 7 of the 9 were unaffected, and the other 2 had no follow-up information
One to eight mutations tested
j
Not reported
to increased detection of chromosome abnormalities while

not significantly increasing the use of amniocentesis since
some women 35 and older now have their a priori risks
altered downward and choose not to have amniocentesis as
a result. This was alluded to previously as well [106].
Several scoring indices have been developed to provide
individualized risk assessments [216, 233–239]. The fact
is that anyone with an ultrasound machine in the office
may do an ultrasound examination, and the range of exper-
tise and resolution vary significantly among practitioners
and machines. Optimally, each practitioner should develop
his or her own index based on the prospective evaluation
of a large series of patients. These indices will be much
more valid in that practice than those derived from the
literature.
Fig. 12.7 Ultrasound image of moderately hyperechoic bowel (indi-
cated by plus signs (+) and circle of dots) in a 17-week gestation fetus.
ST = stippling, referring to pattern of hyperechogenicity. (Courtesy of Limitations Imposed by Maternal Habitus
Greggory DeVore, MD.) It is universally recognized that women with high body
mass indices (BMI) present a challenge to visualization of
fetal findings. This was shown in the FASTER trial, in which
frontal lobe, clinodactyly/hypoplastic midphalanx of the a BMI of 30 or higher was the cutoff for this assessment
fifth digit, brachycephaly, small ears, and small cerebellar [240]. To quantify the degree of difficulty, Tsai et al. under-
diameter [178]. took a study to determine the degree of completeness of
In the past 15 years, medicine in the United States has ultrasound surveys for aneuploid markers in women
evolved from recommending amniocentesis to women 35 classified as normal, overweight, and obese [241]. Within
and older to refining risks based upon a variety of ultra- the obese group, women were further divided into three
sound and maternal serum screening markers. This has led classes based upon their BMI. The rates of completion of
12
Table 12.15 Ultrasound markers of fetal aneuploidy

Risk(s) of aneuploidy if other ultrasound
Finding Risk(s) of aneuploidy if isolated finding abnormalities are present Comment
Prenatal Cytogenetics
Structural heart defect 32–48% [163–165] 50–71% [163–165]

Echogenic intracardiac foci Not increased [170]; four- to fivefold 1.6-fold baseline risk [170] Isolated left ventricular foci appear more likely
baseline risk [173, 174] to be benign than multiple or right-sided foci [172]
Renal pyelectasis (³4 mm before Not increased [192]; 3.3-fold increase 1 in 90 [191] In Ref. [191], no adjustment was made for maternal age
33 weeks and ³7 mm at 33 weeks) over baseline [190]; 1 in 340 [191]
Choroid plexus cyst(s) Not increased [196, 204–206]; 1 in 374 82% [199]; 3.5% [200]; 5% [112]; 9.5% [201]; No adjustment for maternal age in Ref. 203]
for trisomy 18 [204]; 1–2% [199]; 0.6% one in three [106]
[200]; 2.4% [209]; 3.1% [201]; 4% [202];
1 in 82 [203] ; 1 in 150 [204]
Septated nuchal membrane, 56–60% [149] Not studied Ref. [149] is a retrospective database analysis. All
9–20 weeks; >3 mm, <15 weeks pregnancies included in study had isolated nuchal
and >5 mm, 15–20 weeks finding and karyotype
Simple nuchal membrane, 10–25% [149] Not studied
9–20 weeks; >3 mm, <15 weeks
and >5 mm, 15–20 weeks
Nuchal fold, >5 mm, 15–20 weeks 19–33% [149] NS*
Nuchal thickening, 10–15 weeks, 46% [151] 84% if hydrops and/or septations are Best outcome was in nonseptated, nonhydropic; worst
³4 mm present [151] was in septated/hydropic fetuses
Nuchal thickening, 9–15.5 weeks, 80% [152]
³3 mm
Septated 19% [152]; 27-fold risk for 34-year-old Other abnormalities only reported for
Simple women and ninefold risk for women 35 chromosomally normal fetuses
and older
Short humerus, femur Positive predictive value for trisomy 21 Increased to variable degrees If all long bones are of normal length and other
in women with risks of 1 in 500 and 1 ultrasound findings normal, some feel amniocentesis
in 1,000 = 2.3 and 1.2%, respectively, for is not indicated in women under 40. [214]
short humerus [214] Racial factors should be considered in any long-bone
For short femur, some studies found very measurement [217]
little increased risk [215, 216]
Absent nasal bone Absent or hypoplastic in 67–80% Appears to be independent finding from nuchal Also seen more frequently in other chromosome
trisomy 21 fetuses compared to 1–2% translucency, so can be used as independent abnormalities [183]
chromosomally normal fetuses [180–185] markers in multiple marker algorithm [185]
255
256 L.M. Randolph
evaluation among the groups varied from 64% for normal to substances, or markers, were combined now in what was
47% for class II, p < 0.001, and the screen-positive rates, commonly known as triple marker screening (TMS).
meaning more than one marker was found, differed Hundreds of thousands of women in the United States had
significantly as well. In this category 16% of normal women TMS in the second trimester of pregnancy, with a resultant
were positive compared to 10% of class III, p < 0.02. They increase in detection of trisomy 21 before age 35 and what
also noted an increased rate of return visits due to lack of appeared to be a decrease in the incidence of Down syn-
visualization of fetal structures. They recommended that drome births due to abortion of affected fetuses. The overall
ultrasound results in obese patients should be interpreted detection of trisomy 21 with TMS is about 65% with a
with caution and patients counseled accordingly. Aagaard- midtrimester risk cutoff of 1 in 190, with a much lower detec-
Tillery et al. evaluated a subset of participants in the tion in young women (about 44% in 18-year-olds) and a
FASTER trial in a separate study of the effect of BMI on much higher detection in older women (about 78% in
detection of fetal ultrasound abnormalities [242]. In their 36-year-olds) [247].
evaluation of 8,555 women, the detection rate for heart TMS detects 60% of trisomy 18 fetuses as well, when a
defects, echogenic foci, and choroid plexus cysts was midtrimester risk cutoff of 1 in 100 is used [248].
significantly decreased. In their discussion they point out Less recognized is the fact that TMS detects many chro-
that patients are often counseled that the risk of aneuploidy mosome abnormalities nonspecifically, for unknown rea-
is higher if two or more “soft signs” are present. If the likeli- sons. Thus for every trisomy 21 fetus found by TMS, a
hood of seeing such soft signs is decreased due to maternal fetus with a different chromosome abnormality is also
habitus, the rate of detection of aneuploidy among these detected [249]. This is important to keep in mind when
patients is also decreased. As with the study cited previ- counseling patients. Triple marker screening has been sup-
ously, they suggest this factor be taken into account in the planted by quadruple and integrated screening, with
counseling of patients. increased sensitivity and specificity of detection of trisomy
21 [250–255].
Positive Maternal Serum Marker Screen Quadruple Screening

Dimeric inhibin A, referred to as simply inhibin A or inhibin
High Maternal Serum Alpha-Fetoprotein in some studies, was added to the triple marker screen panel in
The association between an elevated level (2.0 or 2.5 mul- recent years and has been shown in several studies to increase
tiples of the median) of maternal serum alpha-fetoprotein the detection of trisomy 21 in the second trimester. In one
(AFP) and fetal neural tube defects has been known for study of 72 second-trimester fetuses with trisomy 21 and 7,063
many years. More recently, the presence of an unexplained unaffected fetuses, the detection of trisomy 21 at a risk cutoff
elevated level of maternal serum AFP has been found to be of 1 in 270 was 81.5% with a screen-positive rate of 6.9% and
associated with an increased risk for fetal chromosome a positive predictive value of 1 in 42 [250]. In other words, 1
abnormalities, with an incidence of 10.92 per 1,000 amnio- in 42 amniocentesis procedures yielded a result of trisomy 21.
centeses [243]. Of these, fetal sex chromosome abnormali- In a second, larger study of 23,704 women with unaffected preg-
ties were seen in 47%. Thus, although some practitioners nancies and 45 women with trisomy 21-affected pregnancies,
discourage patients from having an amniocentesis with an the sensitivity of the quadruple screen was 85.8%, with an
elevated AFP and a normal ultrasound study, the facts that initial screen-positive rate of 9.0%, corrected to 8.2% after
sex chromosome abnormalities other than 45,X and its gestational age error corrections [253]. The positive predictive
related karyotypes have no significant associated ultra- rate was 1 in 51. Women who were true-positives had very
sound abnormalities and that they are quite common (with high risks (median risk of 1 in 22) compared to risks in women
incidences of 47,XXX; 47,XXY; and 47,XYY each ³1 in with false-positive results (median risk of 1 in 111) [253].
1,000 liveborns) support consideration of amniocentesis in Hackshaw and Wald evaluated the increase in detection of
this group. trisomy 21 by performing the triple marker screen followed
by the quadruple screen in a series of patients. They found an
Low Maternal Serum AFP and Multiple increase in detection of 3–5% at a 5% screen-positive rate.
Marker Screening Their interpretation was that the “modest increase…is probably
The association between low maternal serum AFP and fetal not worthwhile in the light of the extra cost and delay” [252].
Down syndrome was established in 1984; and in 1987, the It should be pointed out, however, that similar arguments were
association between high maternal serum human chorionic made when unconjugated estriol was added to what at the time
gonadotropin (hCG) and low unconjugated estriol and fetal was a double screen. Based upon a study by Spencer et al. in
Down syndrome was established [244–246]. These three 45 cases of trisomy 18 and 493 control pregnancies at
10–14 weeks’ gestation, inhibin A was found not to add to the wise sequential screening, 95%; with serum integrated
detection of trisomy 18 over triple marker screening alone screening, 88%; and with fully integrated screening with
[253]. Nevertheless, second-trimester quadruple screening has first-trimester measurements performed at 11 weeks, 95%.
largely replaced triple marker screening. The authors concluded that first-trimester combined screen-
ing at 11 weeks’ gestation is better than second-trimester
First-Trimester Screening quadruple screening, but at 13 weeks has results similar to
In 1995 and 1996, first-trimester detection of trisomy 21 second-trimester quadruple screening. Both stepwise
using free ß-hCG and pregnancy-associated plasma protein sequential screening and fully integrated screening have
A (PAPP-A) was reported [254, 255]. Several more papers high rates of detection of trisomy 21, with low false-posi-
have been published since then that have shown first- tive rates.
trimester screening using those biochemical markers plus A study to determine patient choices and screening per-
maternal age alone or in combination with nuchal translu- formance when three trisomy 21 screening protocols were
cency measurements to be the most sensitive screening introduced was described by MacRae et al. [259]. In this
method for the detection of trisomy 21 and trisomy 18. study, called the SAFER study (second- and first-trimester
Chasen et al. studied a US population of 2,131 pregnancies estimation of risk), integrated screening protocols were cho-
in 2003 in New York [148]. By using nuchal translucency sen 4.6 times more often than four-marker screening (82%
measurement from 11 to 14 weeks’ gestation plus maternal vs. 18% uptake). Overall detection was higher and false-
age, the detection of trisomy 21 was 83.3% and the detec- positives lower. Overall, of 8,571 women screened and 23
tion of trisomy 18 was 90%. A large multicenter study cases ultimately diagnosed with trisomy 21, 21 were detected
called the BUN study—short for Biochemistry, Ultrasound, in the study (91%, 95% CI, 73–98%) at a 4.2% false-positive
Nuchal translucency was undertaken to screen pregnancies rate (95% CI, 33.3–5.1%).
between 74 and 97 days of gestation for trisomies 21 and 18
using maternal age, maternal levels of PAPP-A and free
ß-hCG, and fetal nuchal translucency measurements in Previous Pregnancy or Child with
8,514 patients with singleton pregnancies [257]. In this a Chromosome Abnormality
study, the detection rate for trisomy 21 was 85.2% with a
screen-positive rate of 9.4%. If the screen-positive rate was Having a previous pregnancy or child with certain chromo-
set at 5%, the detection rate of trisomy 21 was 78.7%. Of some abnormalities is associated with an increased risk of a
the trisomy 18 cases, screening identified 90.9% with a future fetal chromosome abnormality [259–262]. The risk is
screen-positive rate of 2%. For women 35 years or older, not only for a recurrence of the same trisomy, or homotri-
89.8% of fetuses with trisomy 21 were detected with a somy, but also for heterotrisomy [261]. The study that exam-
screen-positive rate of 15.2% and 100% of fetuses with tri- ined this question effectively showed that not only gonadal
somy 18 were detected [257]. mosaicism is responsible for trisomy recurrence, but some
women have a risk for nondisjunction higher than do others
Integrated and Combined Screening of the same age. The reasons for this are not known. Studies
The concept of integrated and combined screening exam- in this regard were summarized by Robinson et al. and
ines first- and second-trimester screening in combination included polymorphisms in genes involved in folic acid
or conjunction to improve the detection of aneuploidy metabolism and folic acid intake, mutations in MSH2, a mis-
while lowering the screen-positive rate. This can be done match repair gene, and environmental factors such as caf-
in varying ways, including using PAPP-A in the first tri- feine intake and cigarette smoking [262].
mester and the aforementioned second-trimester biochem- Chromosome abnormalities known to increase the
ical markers. This methodology was used in a 2003 study future risk of aneuploidy include all nonmosaic trisomies,
to detect 90% of trisomy 18 cases with a screen-positive structural rearrangements, and marker chromosomes. In
rate of 0.1% [258]. Warburton’s study of recurrence of homotrisomy and
A multicenter study called the First- and Second- heterotrisomy, there was no increased risk of X-chromosome
Trimester Evaluation of Risk (FASTER) published its aneuploidy [261]. Genetic counseling is suggested for
findings in 2005 regarding its comparison of first- and sec- couples who have had a pregnancy or child with any
ond-trimester screening modalities [240]. The rates of higher-risk karyotype, and ultrasound plus amniocentesis
detection of trisomy 21 in 38,167 patients, at a 5% false- or CVS are recommended for consideration in future preg-
positive rate, were as follows: with first-trimester combined nancies [259].
screening, 87, 85, and 82% for measurements performed at Also not known to be associated with an increased recur-
11, 12, and 13 weeks, respectively. With second-trimester rence risk are triploidy, tetraploidy, and 45,X. However, cou-
quadruple screening, rate of detection was 81%; with step- ples who have undergone the experience of having a
258 L.M. Randolph
pregnancy with one of these findings may wish to have significance. The others were two trisomy 18, two 69,XXX;
genetic counseling, ultrasound, and prenatal chromosome two 69,XXY; and one X-chromosome inversion. The control
analysis due to anxiety. population’s theoretical risk of chromosome abnormality
The recurrence risk in de novo structural chromosome was 0.3%. The combined findings were statistically
rearrangements is less than 0.5–2% and takes into account significant (p = 0.12). Other reports have noted mosaic tri-
recurrence by chance, gonadal mosaicism, and somatic- somy 14 and full trisomy 9 in association with nasal enceph-
gonadal mosaicism, but the numbers have not been derived alocele and spina bifida, respectively [268, 269]. Trisomy 13
by extensive study [259–262]. To evaluate this, Röthlisberger is also associated with increased risk for spina bifida [270].
and Kotzot performed a literature search and found 29 case NTDs have also been associated with 22q11.2 microdeletion
reports of recurrence of de novo structural chromosome rear- syndrome [271, 272]. Several other microdeletions and
rangements. Thirteen of them were due to a an i(21q) replac- microduplications detected by array comparative genomic
ing one normal chromosome 21, and in eight of them low-level hybridization (see Chap. 18) have also been reported in asso-
mosaicism in one of the parents was found. The authors ciation with various types of neural tube defects [273–276].
stated, therefore, that the recurrence risk should be reduced to
less than 1% for de novo i(21q) and to less than 0.3% for all Chromosome Rearrangement in
other de novo structural chromosome rearrangements. They Either Member of a Couple
recommended that prenatal diagnosis be performed only if Some balanced structural rearrangements (see Chap. 9) pre-
requested by parents after genetic counseling [263]. dispose a couple to an increased risk of fetal chromosome
Mosaicism presents complicated counseling issues. It is abnormality. The risk depends on the rearrangement and how
prudent to apprise the couple of this and offer them the it was ascertained.
opportunity for prenatal diagnosis since the risk of recur- For balanced reciprocal translocations, if the rearrange-
rence may be increased. Mosaicism is discussed in more ment was ascertained through multiple spontaneous abor-
detail later in this chapter. tions, the risk of having a child with abnormal chromosomes
is 1.4–4.8%, with the lower risk associated with a paternal
carrier. If it was ascertained by a previous child or stillborn
Other Indications for Prenatal with unbalanced chromosomes, the risk increases to 19.8–
Cytogenetic Diagnosis 22.2%. [277]
For balanced Robertsonian translocations, the risk of
Previous Pregnancy or Child with unbalanced chromosomes in the fetus is much less and
Open Neural Tube Defect appears to be negligible when chromosome 21 is involved
Rates of open neural tube defects (NTD) vary geographi- and if the translocation is paternal [277]. See Table 12.16.
cally. In California, NTDs occur in 1.05 per 1,000 Hispanic Most pericentric inversions (see Chap. 9), except the
women and 0.66 per 1,000 Asian women, with non-Hispanic population variant inv(9), are associated with an increased
Caucasians falling between [96]. The risk of recurrence of risk of unbalanced offspring due to deletions/duplications,
an isolated NTD is 3–5%. Folic acid supplementation of and individuals with such inversions should be offered
0.4 mg/day periconceptionally decreases the risk by 50–70%, amniocentesis. The risk of unbalanced offspring depends on
so the increased fortification of grains with 1.4 mg folate per the length of the inversion segments [278]. See Table 12.17.
pound of enriched cereal-grain products by the US Food and Whether this recommendation applies to individuals with
Drug Administration was announced in 1997 [264]. Having the common pericentric inv(2) is debatable. This inversion
a previous affected pregnancy or child merits offering is so common that some cytogenetics laboratories do not
genetic counseling, ultrasound, and amniocentesis. Such report it.
women are advised to take 4 mg folate periconceptionally. Paracentric inversions in a carrier parent may give rise to
All women of childbearing age, particularly those at acentric fragments or dicentric chromosomes, either of which
increased risk for NTDs, should receive information about would be expected to be lethal in utero. However, amniocen-
folate supplementation. tesis is generally to be recommended, given the possibility of
Chromosome abnormalities are associated with spina viability of a fetus with structurally unbalanced chromo-
bifida and encephalocele, but do not appear to any significant somes and the occasional difficulty in distinguishing between
degree to be associated with isolated anencephaly [265, 266]. a paracentric inversion and an insertion [279].
In Harmon’s study of 55,366 pregnancies in which isolated Because of the observation that marker chromosomes can
spina bifida was seen by ultrasound, outcome information interfere with meiosis, leading to aneuploidy, prenatal diag-
was available for 43 of 77 cases [267]. Of the 43, seven chro- nosis is also recommended to individuals with marker chro-
mosome abnormalities were detected. One was a balanced mosomes, even when these apparently confer no adverse
Robertsonian translocation and therefore of uncertain phenotypic effect.
Table 12.16 Prenatal results for Robertsonian translocations involving chromosomes 13, 14, or 15 and 21
Robertsonian
translocation type Maternal carrier Paternal carrier
Balanced Normal Unbalanced Total Balanced Normal Unbalanced Total Grand total
der(13;21)(q10;q10) 6 4 2 (16.7%) 12 5 4 0 9 21
der(14;21)(q10;q10) 36 25 10 (14.1%) 71 9 13 1 (4.3%) 23 94
der(15;21)(q10;q10) 5 4 0 9 4 2 0 6 15
Total 47 33 12 (13.0%) 92 18 19 1 (2.6%) 38 130
Table 12.17 Prenatal results for pericentric inversions (n = 173)

Method of ascertainment Maternal carrier Paternal carrier
Balanced Normal Unbalanced Total Balanced Normal Unbalanced Total Grand total
Through term unbalanced 6 1 1 (12.5%) 8 2 3 0 5 13
progeny
Through recurrent 10 4 0 14 4 2 0 6 20
miscarriages
Other 63 4 2 (2.9%) 69 68 3 0 71 140
Total 79 9 3 (3.3%) 91 74 8 0 82 173
Men with 47,XYY karyotypes usually have normal fertil- Advanced Paternal Age
ity and may be at increased risk for chromosomally unbal- A body of old literature in genetics suggests an increased
anced offspring. Some of the reported chromosome risk of fetal chromosome abnormality with advanced pater-
abnormalities occurring in pregnancies of 47,XYY males nal age, but the most carefully constructed analyses do not
include markers, trisomy 21, 47,XYY, and others [280]. support this association [283–287]. Advanced paternal age
is not definitively associated with fetal chromosome abnor-
Preimplantation Genetic Diagnosis malities. It is, however, associated with a linearly increased
Preimplantation genetic diagnosis, or PGD, involves the risk of some autosomal dominant new mutations in the off-
use of FISH or molecular testing on embryos for the pur- spring due to mutations in the form of single base-pair muta-
pose of implanting healthy ones into the uterus. Given the tions, particularly in the FGFR3 and RET genes [288]. Less
high risk of spontaneous abortion and of chromosomally common are mutations due to point mutations and base-pair
abnormal offspring for couples where one member carries deletions. Some autosomal dominant conditions show no
a balanced chromosome translocation, PGD increases the paternal age association, according to a policy statement on
likelihood of bearing an unaffected baby [281, 282]. Otani the subject issued by the American College of Medical
et al. studied couples with balanced translocations in whom Genetics [289]. In the statement, the authors point out the
no births had occurred after 117 pregnancies and an aver- four- to fivefold risk of some conditions in offspring of men
age of 3.5 spontaneous abortions had occurred. After PGD, in their 40s versus those of men in their 20s. The relative
18% of embryos were normal or balanced, and 5% of preg- increased risk for these defects is related to advanced age of
nancies were lost compared to 100% before PGC (p < 0.001) the father for autosomal dominant conditions and the mater-
[281]. Munné et al. evaluated 35 couples in whom one part- nal grandfather for X-linked conditions. Family histories
ner carried a chromosome translocation [282]. They noted will not provide clues, as these types of mutations are spo-
a statistically significant reduction in spontaneous abor- radic. Examples of autosomal dominant conditions associ-
tions from 95 to 13% after PGD. They also noted that the ated with advanced paternal age include achondroplasia,
chances of achieving pregnancy were correlated with 50% neurofibromatosis, Marfan syndrome, Treacher Collins syn-
or more of the embryos being chromosomally normal. drome, Waardenburg syndrome, thanatophoric dysplasia,
Robertsonian translocations caused fewer abnormal osteogenesis imperfecta, and Apert syndrome. Examples of
embryos than reciprocal translocations, resulting in higher X-linked conditions associated with increased maternal
rates of implantation [282]. grandfather’s age include fragile X syndrome (see Chap.
Also refer to Chaps. 11 and 18 for other discussions 19), hemophilia A (factor VIII deficiency), hemophilia B
regarding the use of PGD. (factor IX deficiency), Duchenne muscular dystrophy,
260 L.M. Randolph
incontinentia pigmenti, Hunter syndrome, Bruton agamma- In addition to the level of mosaicism, the chromosome
globulinemia, and retinitis pigmentosa [289]. Genetic coun- involved is an important consideration. True mosaicism has
seling is indicated so the expectant couple may understand been reported in liveborns with almost all trisomies [37].
the issues, and it is prudent to offer detailed fetal ultrasound However, true mosaicism for trisomies 8, 9, 21, 18, 13, 16,
examination in pregnancies involving men 40 years and X, and Y and for monosomies X and Y has potentially great
older [289]. The American College of Medical Genetics significance. For chromosomes 8 and 9, mosaicism is the
acknowledges the risk but says that ultrasound examination most common form in which trisomies occur in liveborns,
is usually of little benefit. perhaps because the full trisomy is not compatible with fetal
survival in the majority of cases [294, 295].
Three prenatal cases with mosaic trisomy 9 were reported
Special Issues by Takahashi et al., in which two showed severe IUGR and
had fetal demise in the third trimester and the third was born
True Mosaicism and Pseudomosaicism with diaphragmatic hernia and mild IUGR [296]. The authors
Mosaicism, or the presence of two or more cell lines origi- note that in nine previously reported continuing pregnancies
nating from a stem cell line, is one of the most complex and with trisomy 9 mosaicism, eight were live births and one
challenging issues in prenatal diagnosis. There are three lev- died in utero. Even one cell with trisomy 8 may be significant.
els of mosaicism in amniotic fluid and CVS culture: levels I, For trisomies of chromosomes 13, 18, 21, X, and Y and
II, and III. Level I is defined as a single-cell abnormality. monosomy X and Y, mosaicism has been fairly commonly
Level II is defined as a multiple-cell abnormality or (with an reported, and the clinical manifestations may vary from no
in situ culture method) a whole colony abnormality in one apparent abnormality, at least in the newborn period, to more
culture not seen in any other cell cultures. Level III mosa- characteristic features of the full trisomy. The degree of
icism is “true” mosaicism—the presence of a second cell line mosaicism is not related to the outcome [29]. See Table 12.18
in two or more independent cultures. The incidences of these for incidences of mosaicism for specific chromosomes.
in amniotic cell cultures range from 2.5 to 7.1% for level I, Schuring-Blom et al. evaluated first-trimester cytotro-
0.6–1.1% for level II, and 0.1–0.3% for level III mosaicism phoblast cell preparations—direct preparations—showing
[290–292]. full or mosaic trisomy 13 or 18, with the purpose of deter-
The origin of the mosaic cell line cannot be determined mining how often the result was a true-positive in the fetus
without molecular studies. In general, however, it appears or newborn [297]. Cultured mesenchymal tissue was avail-
that the majority of 45,X/45,XX cases occur after a normal able only for about half of the cases. Of the 51 cases, five
disomic fertilization, most mosaic trisomies are due to false-positives were seen in those with full trisomy 18 and
postzygotic loss of the trisomic chromosome, and, for tri- three with mosaic trisomy 18. One false-positive was seen
somy 8, most cases are due to somatic gain of the third chro- in full trisomy 13, and two false-positives were seen in
mosome 8 postzygotically [293]. mosaic trisomy 13. Their conclusions were that:
Table 12.18 Outcome of cases with rare autosomal trisomy mosaicism diagnosed in amniocytes
Type Abnormal outcomes/total no. of cases Abnormal phenotype (no. with IUGR)a Fetal demise or stillborn
46/47,+2 10/11 (90.9%) 7 (2) 3
46/47,+3 1/2 – 1 0
46/47,+4 1/2 – 1 0
46/47,+5 2/5 (40.0%) 2 (2) 0
46/47,+6 0/3 – 0 0
46/47,+7 1/8 (12.5%) 1 0
46/47,+8 1/14 (7.1%) 1 0
46/47,+9 14/25 (56.0%) 14 (2) 0
46/47,+11 0/2 – 0 0
46/47,+12 6/23 (26.1%) 4 2
46/47,+14 2/5 (40.0%) 2 0
46/47,+15 6/11 (54.5%) 6 (3) 0
46/47,+16 15/21 (71.4%) 15 (8) 0
46/47,+17 0/7 – 0 0
46/47,+19 0/1 – 0 0
46/47,+22 7/11 (63.6%) 6 (2) 1
a
IUGR intrauterine growth restriction
• Full trisomy 13 or 18 in a short-term culture preparation is a Outcomes of 144 cases of trisomy 20 mosaicism indicate
reliable result only in combination with abnormal ultrasound that 112 of 123 cases (91%) were associated with a normal
findings or trisomic cells in mesenchyme or amniotic fluid. phenotype; 18 of these were abortuses [29]. In most cases,
• Mosaic trisomy 13 or 18 in a short-term culture prepara- the cells with trisomy 20 are extraembryonic or largely
tion merits further prenatal testing by amniocentesis. confined to the placenta. Of the eleven abnormal outcomes,
In a multicenter study evaluating karyotype-phenotype three were in liveborns and eight in abortuses. Three abor-
correlations when mosaic trisomy 13, 18, 20, or 21 was seen tuses with urinary tract abnormalities and two with heart
at amniocentesis, Wallerstein et al. found an abnormal out- abnormalities represent the only consistent, serious abnor-
come in 40% of mosaic trisomy 13, 54% of mosaic trisomy malities associated with such mosaicism. Of 21 children fol-
18, 6.5% of mosaic trisomy 20, and 50% of mosaic trisomy lowed for one to two years, all were normal except for two
21 [298]. The risk of abnormal outcome in pregnancies with with borderline psychomotor delay. It was also apparent that
less than 50% trisomic cells and greater than 50% trisomic attempted cytogenetic confirmation of the finding should not
cells differed also, with better outcomes for lower levels of be limited to analysis of fetal blood, because trisomy 20 has
mosaicism, although the numbers were too small for statisti- not been observed in blood cells. Confirmation studies in
cal significance. Repeat amniocentesis was not useful in newborns should be done on placental tissues, skin, cord
predicting clinical outcome, although it might be useful blood, and urine sediment and in abortuses, on kidney, skin,
when there is insufficient number of cells or cultures to and placental tissues. Finally, true mosaic trisomy 20 may be
establish a diagnosis. The authors suggested PUBS as an associated with a mild phenotype. A case was reported in
adjunct study, as the risk for abnormal outcome increased which nonmosaic trisomy 20 was diagnosed by CVS, and the
with positive confirmation. One of five normal cases was term placental karyotype showed the same finding. Mosaic
confirmed versus five of eight abnormal cases. The authors trisomy 20 was seen in foreskin cultures and in a second skin
also recommended high-resolution ultrasound. culture, while lymphocyte culture chromosomes were 46,XY.
Mosaicism for trisomies 12 and 20 poses unique prob- Aside from diffuse hypopigmentary swirls along the lines of
lems. For both of these trisomies, mosaicism has been Blaschko on his extremities and trunk, he was considered
reported that appeared to have no discernible effect on the clinically normal at 8 years of age [300].
fetus or liveborn, and yet in other cases the mosaicism was Trisomy 16 mosaicism has attracted a great deal of inter-
associated with an abnormal outcome. A case report and sur- est in the past several years, inasmuch as it was previously
vey of a decade of literature showed a total of 13 reported thought that the finding of mosaic or nonmosaic trisomy 16
cases in which trisomy 12 mosaicism was observed in amni- was thought to result always in pregnancy loss; now it is
ocytes [299]. In nine cases, the pregnancy was terminated, known that this is not always the case. Of recognized con-
and in seven of the nine, no phenotypic abnormalities were ceptions that spontaneously abort in the first trimester, 6%
reported. One fetus was not described, and one had only two have trisomy 16 [37]. Most conceptuses abort between 8 and
lobes in each lung and appeared otherwise normal. In seven 15 weeks’ gestation, and the extra chromosome is usually of
cases, confirmatory cytogenetic studies on skin, blood, rib, maternal meiosis I origin. The mosaicism is thought to arise
placenta, kidney, liver, lung, and/or villi were performed, and from either failure of bivalent formation or the precocious
in the six cases in which fetal tissue was known to be cul- separation of bivalent homologs with or without crossing
tured, five showed confirmation of mosaic trisomy 12. over, during meiosis I. These unpaired univalents then enter
In five cases in which the pregnancy was continued after a second premature division, separating into constituent
diagnosis of trisomy 12 mosaicism in amniocytes, the diag- chromatids. During the second meiotic division, these chro-
nosis was confirmed in urinary cells or skin in two children. matids cannot take part in a normal anaphase and would
One of them had mild dysmorphic features with near-normal therefore be partitioned at random [301]. This would be mis-
development at 3 years, and the other was dysmorphic and interpreted as a maternal meiosis I error by DNA marker
died in the first weeks of life with cardiac abnormalities. In analysis. Virtually all mosaic trisomy 16 is thought to arise
the other three, the diagnosis was not confirmed in fetal skin from trisomic zygote rescue of error of maternal origin [302].
and/or blood; one had normal development at 5 months, and Nonmosaic trisomy 16 has not been observed in a liveborn
the other two died in the newborn period with heart, kidney, child, although it was documented in a third-trimester fetus
vertebral, tracheoesophageal, and other abnormalities. at 32 weeks’ gestation. That fetus was stillborn with a birth
It is interesting to note that the terminated fetuses were weight of 783 g, indicating severe IUGR, and the diagnosis
described as normal, and the liveborns were almost all abnor- was confirmed in skin chromosomes [303].
mal. This was not related to degree of mosaicism. It may be Mosaic trisomy 16 is commonly reported in CVS cultures
due to unrecognized abnormalities in second-trimester and has been reported to result in the birth of liveborn infants
fetuses, or there may have been a bias toward reporting live with maternal uniparental disomy or with normal biparental
births with congenital abnormalities. inheritance of the normal cell line [304, 305]. When CVS
262 L.M. Randolph
detection of mosaic trisomy 16 occurred, in one series of in affected fetuses and newborns—namely VSD, complex
continued pregnancies, 13 of 63 resulted in fetal death, with heart disease, hypospadias, imperforate anus, inguinal
three of those occurring after 37 weeks’ gestation. One baby hernia, clubfoot, and IUGR. The combination of an ele-
was stillborn. Preterm delivery occurred in 11 cases, often vated maternal serum hCG or AFP plus IUGR and one or
associated with fetal or maternal complications. Among the more of the structural abnormalities previously listed
50 liveborns, IUGR was seen in 27, or more than half. Birth merits the clinical suspicion of mosaic trisomy 16. [302,
defects or fetal abnormalities were seen in 13, or 18%, of 304–306, 309]
cases; multiple abnormalities were seen in six; and in seven,
the abnormalities were isolated to a single organ. Of the con- Other Mosaic Trisomies and Monosomies
tinuing pregnancies, only 17 of 60, or 28%, appeared to be Trisomy 22 mosaicism was reported in a collection of 11
full-term, normal pregnancy outcomes [305]. cases [29]. Of these, four continued and five terminated.
Finding mosaic trisomy 16 at amniocentesis appears to Four of eight reported cases showed a normal outcome, and
be associated with an elevated maternal serum AFP in the others, one fetal demise, one neonatal death with
(MSAFP, see above). Hsu et al. reported on a series of 11 IUGR, one liveborn with IUGR, and one abortus with mul-
cases diagnosed via amniocentesis ascertained after an tiple congenital abnormalities were seen. Another report of
elevated MSAFP [306]. In their series, 9 of the 11 pregnan- five cases was published by Leclerq et al. [310]. They note
cies affected with mosaic trisomy 16 were referred for this that 19 prenatal and 21 postnatal cases of mosaic trisomy 22
reason or because of elevated maternal serum human chori- have been reported. The phenotype of postnatal cases often
onic gonadotropin. includes growth restriction; dysmorphic features; mental
In another series of 29 amniocentesis-diagnosed cases of retardation; hemiatrophy; plus heart, eye, ear, and limb
trisomy 16 mosaicism not referred due to abnormal CVS defects. The five cases they added included four prenatal
results, the indication was elevated MSAFP in twelve; in cases diagnosed by amniocentesis and one diagnosed at
only three was the indication of abnormal ultrasound 4 years of age. One prenatal case showed normal results at
findings. Preterm delivery was seen in 12 of the 19 preg- 4 years of age. Two prenatal cases suffered in utero demise.
nancies, and IUGR was seen in 13 of the 19 continuing The others showed multiple congenital abnormalities, as did
pregnancies. Multiple abnormalities were seen in 18 of the the deceased fetuses. No predictive factors were helpful to
29 cases, or 62%, and isolated abnormalities were seen in determine the prognosis in such cases and those they
two other babies. Only four appeared to have a totally nor- reviewed, including confirmation of true fetal mosaicism. Of
mal outcome [305]. It is important to study skin fibroblasts, note is that the normal outcome reported was after a normal
as often the trisomic cell line does not appear in lympho- ultrasound reported during pregnancy. The others had had
cytes. Placental tissue should also undergo chromosome or IUGR and other findings.
FISH analysis [307]. In a study of chromosome mosaicism of chromosomes
Yong et al. evaluated 162 cases of prenatally diagnosed other than 13, 18, 20, and 21, one to 25 cases each of
mosaic trisomy 16 [308]. Among live births, 45% had at mosaic trisomies 2–9, 11, 12, 14, 15, 16, 17, 19, and 22
least one malformation, most commonly VSD, ASD, and were reported in one series [306]. The outcomes were
hypospadias. The level of trisomy on direct CVS, or cytotro- stratified by very high, high, moderately high, moderate, low,
phoblast, was associated with more severe IUGR and higher and undetermined. Most abnormalities were detectable
risk of malformation, while the level of trisomy on cultured by ultrasound. The authors also stressed the importance of
CVS, or chorionic villous stroma, was associated only with obtaining fibroblasts and placental tissues. See Table 12.19
more severe IUGR. The degree of trisomy in placental tis- for more information on these mosaic trisomies. Hsu
sues was independent of the degree of trisomy in amniotic reported on 13 cases of autosomal monosomy mosaicism
fluid and amniotic mesenchyme [308]. that had been prenatally diagnosed [29]. These included
A follow-up review of 17 patients diagnosed by amnio- five cases of monosomy 21, three of monosomy 22, two of
centesis and 19 by chorionic villus sampling with mosaic monosomy 17, and one case each of monosomies 9, 19,
trisomy 16 was published by Langlois et al. [309]. Of the and 20. Of seven cases with phenotypic information and
17 amniocentesis cases, 11 had congenital anomalies, and four cases with confirmatory cytogenetic studies, only one
almost all had growth delay. In 13 for which birth weight case with monosomy 22 was reported to have multiple
was available, it was low in 11, and catch-up growth occurred congenital abnormalities, including congenital heart dis-
in about 90%. Four of the 17 had global development delay. ease. Another case of monosomy 21 was confirmed but
Birth defects correlated with delay. Uniparental disomy did was reported to be phenotypically normal. If autosomal
not correlate with developmental delay [309]. mosaic monosomy is detected, particularly of chromo-
There is a phenotype associated with trisomy 16 mosa- somes 21 or 22, further workup, such as PUBS and/or
icism. Some abnormalities have occurred more than once ultrasound examination, is indicated.
Table 12.19 Rare trisomy mosaicism cases diagnosed in amniocytes specimens, since the number of nonviable cells in the fluid
involving autosomes other than chromosomes 13, 18, 20, and 21, along is very high, and they appear to slow the growth of the viable
with risk of abnormal outcome as determined by ultrasound and/or
physical examination at termination or birth cells. The usual counseling provided in such cases is that the
fetal outcome is not related to the lack of cell growth.
Chromosome Degree of risk for abnormal
number Number of cases outcome
However, there is a report describing 32 of 7,852 (0.4%)
2 11 Very high amniocentesis specimens that were classified as unexplained
3 2 Undetermined growth failures, and, in this group, ten women did not repeat
4 2 Undetermined the procedure while twenty-two did [311]. Of the ten who
5 5 High did not, a fetal bladder-outlet obstruction, two stillbirths, and
6 3 Undetermined one acardiac twin resulted. Of the twenty-two who repeated,
7 8 Moderate eighteen had normal fetal karyotypes, but four were aneu-
8 14 Moderate ploid. Of these, two had trisomy 21, one had trisomy 13, and
9 25 High one had Pallister-Killian syndrome, or tetrasomy 12p.
11 2 Undetermined
12 23 Moderately high
14 5 High Maternal Cell Contamination
15 11 High
16 21 Very high After cell culture and cytogenetic analysis of amniotic fluid
17 7 Low
specimens, maternal cell contamination (MCC) is rarely
19 1 Undetermined
found. Maternal cells were detected in 0.17% of 44,170 cul-
22 11 Very high
tured amniotic fluid samples in one study [312]. Since detec-
Very high = >60% risk; high = 40–59% risk; moderately high = 20–39%
tion of MCC would only be expected in male pregnancies (as
risk; moderate = up to 19% risk; low risk, no abnormalities seen; and
undetermined, no cases for evaluation. From reference [29] a mixture of XX and XY cells), the frequency of maternal
contamination was estimated at twice this rate, or 0.34%. If
in situ hybridization techniques are used on uncultured cells,
As Phillips et al. described, the common autosomal triso- thus identifying both maternal and fetal nuclei, the proportion
mies of 21, 18, and 13 make up a smaller number of cases of of MCC increases dramatically, being present at a level of
mosaicism detected on CVS but are confirmed in fetal tissue 20% in half of amniotic fluid specimens. This was found to be
in 19% of cases [307]. The uncommon autosomal trisomies strongly associated with the sampling technique in a survey
are more common but are less often confirmed in 3.2% of of 36 amniotic fluid specimens [313]. Maternal cell contami-
fetal tissues. They also confirmed that the type of chromo- nation of less than 20% was seen in 19 specimens in which
some abnormality is a predictive factor as to whether it will the placenta was posterior. In two other bloody specimens
be confirmed in fetal tissues. In 28 cases of mosaic polyploidy from pregnancies with posterior placentas, more than 20%
detected on CVS, fetal mosaicism was confirmed in one case, MCC was seen. In cases in which the placenta was anterior,
compared to marker chromosomes found on CVS, in which less than 20% MCC was seen in two cases and more than
mosaicism was confirmed in one-fourth of the fetuses [307]. 20% in 13 cases. It was thought that the maternal cells were
introduced into the amniotic fluid specimen as a result of
Mosaicism of an Autosomal Structural Abnormality placental bleeding during amniocentesis. The authors stated
In 78 reported cases of mosaicism for a balanced autosomal that molecular cytogenetic analysis, or FISH, should not be
structural abnormality, phenotypic information was available performed on uncultured amniotic fluid cells without prese-
in 16 cases, and all were associated with a normal phenotype lecting fetal cells. The preselection could consist of simulta-
[29]. However, for unbalanced autosomal structural abnor- neous analysis of the morphology of the nuclei and of the in
mality mosaicism, 25/52 (48%) were reported to be pheno- situ hybridization findings.
typically abnormal, and 28/48 (58.3%) were cytogenetically
confirmed. Such a finding thus merits consideration of PUBS
and ultrasound examination. Microarray Analysis
Microarrays are standard sets of short DNA sequences, or

Culture Failure targets, which are compared to an unknown DNA specimen
using computer software that reads and interprets the array
Rates of culture failure vary from lab to lab, and guidelines differences between the two. Arrays are made of short DNA
for acceptable rates exist (see Chap. 6). Cell culture failure sequences from 45 to 65 kilobases in length—in which case
is more likely to occur in advanced-gestation amniocentesis they are called oligonucleotide arrays, or oligoarrays—or
264 L.M. Randolph
shorter stretches of nucleotides of 1.8 million or more such Several commercial laboratories market oligoarrays designed
targets, called single nucleotide polymorphism (SNP) arrays. for prenatal use. An evaluation of 300 amniocentesis or CVS
These can be read and interpreted by allowing controlled specimens using bacterial artificial chromosome (BAC) or oli-
hybridization of the fragmented DNA specimen being stud- goarrays from women with advanced maternal age or abnor-
ied. The resulting report describes whether there is a deletion mal ultrasound findings showed 58 CNVs, or 19.3% [319]. Of
or duplication of any genetic material; its location; if any these, 40 of 58 (13.3%) were interpreted as likely benign, 15
disease-associated genes are involved; and, in the case of (5%) were clearly abnormal, and three (1%) were of uncertain
SNP arrays, whether loss of heterozygosity has occurred. clinical significance. For seven (2.3%), microarray contrib-
The advantage of arrays is clear—they have much greater uted important new information, and for two of these (1%),
resolution than conventional cytogenetic analysis, at 50 kb or the abnormality would not have been detected without
so, which is at least 100 times smaller than changes that can microarray analysis. In one case, after the microarray result
be seen by cytogenetic methods [314]. showed an abnormality, a targeted ultrasound showed an ana-
Although microarrays have been available for several years, tomic abnormality consistent with the genetic defect found by
their use in prenatal medicine has been more slowly adopted array; for this and other reasons, the authors stated that the use
due, in part, to concerns regarding the clinical significance of of prenatal array only for an indication of abnormal ultrasound
copy number variations in the human genome [315]. In 2009, might not be the optimal diagnostic strategy.
the American College of Obstetricians and Gynecologists Another group evaluated fetuses with abnormal ultra-
released a position paper indicating that the “…use of array sound findings and compared normal cytogenetic results
CGH technology in prenatal diagnosis is currently limited by with array results. They found clinically significant CNVs in
several factors, including the inability to detect balanced chro- one of 50 cases of those pregnancies [320]. In three other
mosomal rearrangements, the detection of copy number varia- cases, CNVs were seen that were inherited from a parent. As
tions of uncertain clinical significance, and significantly higher with the previous study, they note that many genetic syn-
costs than conventional karyotype analysis. Although array dromes present postnatally with growth disorders and facial
CGH has distinct advantages over classic cytogenetics in cer- abnormalities, and hypothesize that evaluating a subset of
tain applications, the technology is not currently a replacement fetuses with growth abnormalities and another anomaly
for classic cytogenetics in prenatal diagnosis” [316]. might yield more findings. They also raise an important
Friedman echoes concerns about adoption of array tech- question: if a parent has an identical CNV, does that mean it
nology, given the little that is known about the natural history is truly benign? Careful evaluation of the parent carrying the
and range of clinical variability associated with most patho- same CNV is vital in distinguishing true benign variants
genic submicroscopic copy number variants (CNVs). His from those that cause disease [320].
recommendation is that it be offered only if the pregnancy is Hillman et al. published a meta-analysis of 751 prenatal
at very high risk of having a pathogenic CNV or it is being array CGH cases, of which 409 had an ultrasound abnormal-
done as part of a clinical trial [314]. ity [321]. Array CGH detected an additional 2.9% of chro-
Increased use of prenatal microarray has nevertheless mosome abnormalities in 2.9% of cases, regardless of the
been increasingly occurring. A survey of genetic counselor indication, and when the indication was abnormal ultrasound,
current practice was reported by Smith et al. in 2009 [317]. the additional predicted causative chromosome abnormalities
In the survey responses, 84% indicated their employer had found on array CGH was 5.2%. Variants of unknown
no guidelines for the use of arrays in prenatal diagnosis, and significance were detected in 1.1% of specimens [321].
over 75% were not familiar with available literature. More An evaluation of low-level chromosome mosaicism by
than 50% were not familiar with ACMG guidelines. About array CGH was reported by two groups [322, 323]. In the
57% had offered array within the past 2 years, and pretest Ballif et al. study, mosaicism was detected in 14 cases of
counseling was performed by a genetic counselor in about 3,600 analyzed and was not detected by cytogenetic testing.
75% of cases. The type of array was approximately equal Because 20%, and possibly 10%, mosaicism may be detect-
among prenatal BAC, targeted oligoarray, and whole genome able by microarray, and the same degree of detection by
oligoarray. Of those who offered it, 77% only did so follow- cytogenetics would require analysis of 29 or 63 metaphases,
ing normal chromosome analysis, and all offered it for abnor- respectively, microarray might circumvent some of chal-
mal ultrasound findings. Fewer than 30% offered it for other lenges associated with detecting low-level mosaicism by
indications for prenatal diagnosis, and fewer than 1% offer it conventional cytogenetics. They also comment on the differ-
to everyone, regardless of indication [317]. ent percentages of mosaic cells found in stimulated cell
A comparison of the use of prenatal versus whole genome cultures compared to unstimulated blood smears, indicating
array was described in a study by Rickman et al. [318]. In their that the culturing process might introduce selection bias that
study, abnormalities were detected in 22 of 30 of specimens distorts the percentage of abnormal cells.
with genome-wide array and 29 of 30 with a prenatal array. Microarray technology is covered in detail in Chap. 18.
15. Alvarez H. Diagnosis of hydatidiform mole by transabdominal pla-

Conclusion cental biopsy. Am J Obstet Gynecol. 1966;95:538.
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17. Kullander S, Sandahl B. Fetal chromosome analysis after transcer-
The Holy Grail for prenatal diagnosis is the ability to detect vical placental biopsies during early pregnancy. Acta Obstet
fetal abnormalities through maternal blood testing. One of Gynecol Scand. 1973;52:355–9.
the limitations in the field has been isolating the small amount 18. Hahnemann N. Early prenatal diagnosis; a study of biopsy tech-
niques and cell culturing from extraembryonic membranes. Clin
of fetal DNA in maternal circulation. In a new model reported Genet. 1974;6:294–306.
by Chiu et al., 753 pregnant women at high risk for fetal 19. Department of Obstet and Gynecol. Fetal sex prediction by sex
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subjected to massively parallel genomic sequencing for J. 1975;1(2):117–26.
20. Horwell DH, Loeffler FE, Coleman DV. Assessment of a transcer-
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The Cytogenetics of Spontaneous
Abortion 13
Solveig M.V. Pflueger
may be uncomfortable with discussing the loss, if they are

Introduction even aware of it, and so may avoid the issue altogether. She
may have been told by her caregiver that such early losses
Pregnancy loss is quite common, with 15–20% of recognized are common and that there is no reason she cannot have a
pregnancies resulting in failure. The majority of these occur successful pregnancy, but this does not explain why the loss
early in gestation, though losses in the second and third tri- happened to her and usually does little to alleviate her sense
mester are not rare. Approximately 2–5% of women will of guilt and failure. These feelings of inadequacy are often
experience two or more losses. The majority of pregnancy amplified in the patient with recurrent losses [1–5]. Answering
failures are associated with cytogenetic abnormalities, with the patient’s questions, whether verbalized or not, will help
over 50% of early miscarriages and as many as 5% of still- bring about closure to the loss and may open dialog with the
births exhibiting abnormal karyotypes. patient and her partner about their specific concerns for the
Loss of a wanted pregnancy is always stressful for both future. This in turn may have significant impact on manage-
the patient and her partner. A number of questions and ment of future pregnancies. Thus, anyone caring for women
concerns may be raised regarding the loss, including: of childbearing potential should be familiar with the causes
What happened and why? How likely is it to happen and recurrence risks for pregnancy loss.
again? What can be done to improve the chances of a suc-
cessful future pregnancy? Is this even possible? Answering
such concerns is important in helping the patient through The Scope of the Problem
the grieving process and in facilitating resolution. The
answers that are provided may ultimately impact family When examining the chances for success of a given concep-
planning and management of any future pregnancies the tus, the results of human reproduction are quite poor.
couple may undertake. Approximately 78% of all conceptions fail to go to term [6].
Unfortunately, early pregnancy losses are often given less Combined data from three studies of women attempting
attention than they merit, both by medical care providers and pregnancy revealed a postimplantation loss rate of 42% in
by society. The patient who loses an older child or who expe- documented conceptions confirmed by positive human cho-
riences a stillbirth at term can expect an attempt at explana- rionic gonadotropin (hCG) levels [7–9]. A 4-year follow-up
tion for the loss from her health care provider. She will also of 3,084 pregnancies demonstrated a 23.7% loss rate follow-
be offered sympathy and support from family and friends. ing the first missed period [10]. The net overall fecundity for
Rituals associated with mourning and with disposition of the patients 20–30 years of age has been estimated at 21–28%, a
remains help bring closure. However, the patient who experi- level that is quite low compared with most other mammalian
ences an early loss often feels isolated and alone. Her friends species [11]. Leridon provides a useful summary table of
pregnancy survival from fertilization to term, with only 31
survivors among 100 ova exposed to fertilization
S.M.V. Pflueger, Ph.D., M.D. (*) (Table 13.1) [12]. Although most of the losses occur very
Department of Pathology, Baystate Medical Center,
early in gestation, losses continue to occur throughout the
Tufts University School of Medicine,
759 Chestnut Street, Springfield, MA 01199, USA second and third trimesters of pregnancy with a slight
e-mail: [email protected] increase in mortality at term.
276 S.M.V. Pflueger
Table 13.1 Intrauterine mortality per 100 ova exposed to fertilization Table 13.2 Chromosomal abnormalities and gestational age
[12]
Gestational age Number of Percent abnormal
Week after ovulation Embryonic demise Survivors (weeks) cases Abnormal cases (%)
– 16 (not fertilized) 100 2 23 18 78.0
0 15 84 3 374 258 69.0
1 27 69 4 203 125 61.6
2 5.0 42 5 139 85 62.2
6 2.9 37 6 302 211 69.9
10 1.7 34.1 7 56 27 48.2
14 0.5 32.4 Total weeks 2–7 1,097 724 66.0
18 0.3 31.9 8 36 8 22.2
22 0.1 31.6 9 42 6 14.3
26 0.1 31.5 10 14 7 50.0
30 0.1 31.4 11 8 1 12.5
34 0.1 31.3 12 8 3 37.5
38 0.2 31.32 Total weeks 8–12 108 25 23
Live births: 31 Total 1,205 749 62.2
Natural wastage: 69 Adapted from [13]
Relationship Between Cytogenetic age; 9.3% of cases were abnormal at 3–4 weeks, falling to
Abnormalities and Gestational Age 5.4% at 9–10 weeks.
The likelihood of detecting congenital anomalies in thera-
Multiple studies have suggested that approximately 50% of peutic terminations is variable and may be a reflection of the
early pregnancy losses are associated with cytogenetic abnor- thoroughness of the examination and the skill of the exam-
malities. Evaluation of 1,205 pregnancy losses of varying iner. However, identification of anomalous development may
gestational ages submitted to the author’s laboratory between have considerable impact upon future reproduction, and it is
1992 and 1996 revealed 539 (45%) cases with identified the opinion of the author that careful anatomic evaluation of
cytogenetic abnormalities (Yusuf and Naeem, 1997, personal aborted products of conception should be considered regard-
communication). The likelihood of a cytogenetic abnormal- less of whether the pregnancy is aborted spontaneously or
ity varies with the gestational age and morphology of the induced.
abortus. In evaluating products of conception, the develop- In the second trimester, ascending infection becomes
mental age at which growth arrest occurred is a more useful more frequent as a cause of spontaneous pregnancy loss.
parameter than gestational age at the time of miscarriage, Abnormal karyotypes become less prevalent as pregnancy
since products of conception are often retained in utero for progresses since many of the less viable abnormal gestations
several weeks following embryonic demise. have already undergone growth arrest and miscarriage.
Overall, the earlier the developmental age, the greater the Gaillard et al. studied 422 consecutive second trimester
likelihood of an abnormal karyotype in a spontaneous preg- losses [15]. Of these, 78.6% were recent demises without
nancy loss. Boué and colleagues found that approximately extensive maceration. Ascending infection could explain
two-thirds of losses under 8 weeks and nearly one-fourth of 85% of these. Structural anomalies were seen in 7.6% of
those between 8 and 12 weeks had abnormal karyotypes fetuses. Cytogenetic abnormalities were confirmed or sus-
(Table 13.2) [13]. pected in half of these. The majority of abnormal fetuses
It is also of interest to note that the earlier the pregnancy showed maceration consistent with long-standing intrauter-
undergoes growth arrest, the more likely it is for there to be ine fetal demise. This again confirms the observation that
anomalous development and that there will be an abnormal cytogenetic abnormalities are associated with early demise
karyotype (Table 13.3). but that there is also frequent retention of the products of
conception for some time prior to spontaneous abortion. The
macerated fetus is at significant risk for chromosomal abnor-
Gestational Age mality whereas the fresh fetal demise without gross congeni-
tal anomalies is more often due to other etiologies including
Examination of induced abortuses confirms the greater inci- but not limited to infection, endocrine disorders, abnormal
dence of karyotypic abnormalities earlier in pregnancy [14] uterine anatomy, and immunological factors.
(Table 13.4). A total of 1,197 pregnancies were examined. Although cytogenetic abnormalities are frequent in early
The rate of chromosomal abnormality varied with gestational pregnancy, they are much less common at term. Approximately
13 The Cytogenetics of Spontaneous Abortion 277
Table 13.3 Abnormal development and gestational age

4 weeks or less 5–8 weeks 9–12 weeks
Study Normal Abnormal Normal Abnormal Normal Abnormal
Milamo 0 48 21 40 71 10
Miller and 10 73 51 71 121 56
Poland
Total 10 (8%) 121 (92%) 72 (39%) 111 (61%) 192 (74%) 66 (26%)
Adapted from [13]
Table 13.4 Chromosomal abnormalities in induced abortuses [14] rearrangements [16]. The rate of unbalanced karyotypes
showing numerical or structural abnormality is much higher
Developmental age Number of cases Abnormal cases (percent)
in stillbirths, approximating 5–7% overall. Here, too, the risk
3–4 weeks 108 10 (9.3%)
5–6 weeks 570 37 (6.5%)
is greatest for macerated stillbirths, especially in the pres-
7–8 weeks 389 25 (6.4%) ence of congenital anomalies. Cytogenetic abnormalities and
9–10 weeks 130 7 (5.4%) associated congenital anomalies are also a significant factor
in neonatal deaths.
The likelihood of survival for a pregnancy with an
Table 13.5 Prenatal loss of chromosomally abnormal fetuses aberrant karyotype is a reflection of the particular cytoge-
Autosomal monosomy 100.0% netic abnormality and the extent of its deleterious effects
Tetraploid 100.0% on embryonic growth and development. Davison and Burn
Triploid 99.9% examined the likelihood of loss for various chromosomal
Monosomy X 99.8% abnormalities, confirming virtual 100% loss for autosomal
Autosomal trisomy 96.5% monosomies and tetraploids [17]. Autosomal trisomies
Mosaics 68.8% resulted in a 96.5% loss rate. Although greater than 99%
Structural rearrangements 53.4% of monosomy X pregnancies failed, only 11% of sex chro-
Sex chromosomal trisomy 11.0% mosome trisomies were lost spontaneously. Mosaic and
Adapted from [17] structurally rearranged karyotypes show intermediate loss
rates of 68.8 and 53.4%, respectively (Table 13.5).
Table 13.6 Percent of chromosomal anomalies among spontaneous Although there have been several reports of tetraploid
abortions and live births
conceptuses and near complete autosomal monosomies sur-
Spontaneous viving into the third trimester, these are exceptionally rare.
Anomaly abortions Live births
Summarizing data from several series, Kline and Stein
Autosomal trisomies
compared the frequency of chromosomal anomalies of spon-
13 1.10% 0.01%
16 5.58% 0.00%
taneous abortions and live births (Table 13.6) [18].
18 0.84% 0.02%
21 2.00% 0.11%
Other 11.81% 0.00% Summary
Total trisomies 21.33% 1.34%
Monosomy X 8.35% 0.01% These data indicate that the majority of chromosomally
Sex chromosome 0.33% 0.15% abnormal pregnancies fail, that the losses are selective rather
trisomies than random, and that the differing survival potential is
Triploids 5.79% 0.00% dependent upon the particular cytogenetic abnormality
Tetraploids 2.39% 0.00% involved.
Total abnormal 41.52% 0.60% Cytogenetic abnormalities are a significant factor in
Number karyotyped 3,353 31,521
human pregnancy wastage at all stages of gestation, as well
Adapted from [18] as into the neonatal period. However, the incidence of karyo-
typic abnormalities is greatest during early pregnancy, with the
majority of aberrant gestations resulting in early spontaneous
one in 200 live newborns exhibits readily identified aneu- loss. Very early pregnancy loss is most likely to be the result
ploid karyotypes, and one study estimates that with moderate of chromosomal abnormalities, especially when there is
levels of banding, 0.061% of infants will show unbalanced evidence of marked embryonic growth arrest at the time of
structural abnormalities and 0.522% will harbor balanced delivery. The clinical significance of the loss and the potential
278 S.M.V. Pflueger
impact on future reproductive risks for the couple is trisomic conceptuses due to formation of a gamete with
dependent upon the type of chromosomal error. fewer or greater than the usual number of chromosomes (see
Chaps. 2 and 8). With the exception of monosomy X, com-
plete, apparently non-mosaic monosomies are almost invari-
Types of Errors Leading to Chromosomally ably lethal early in gestation and are not usually identified in
Abnormal Conceptuses recognized pregnancies. Gene dosage effects or imprinting
failure may be factors contributing to the high embryonic
Although most chromosomal abnormalities are associated with lethality of the autosomal monosomies.
poor outcome early in pregnancy, the underlying mechanisms Trisomies, on the other hand, are relatively common and
leading to an aberrant karyotype and the risk for recurrence vary represent the most frequently encountered group of abnor-
considerably depending on the particular abnormal chromosomal malities leading to spontaneous pregnancy loss. Approximately
complement. Generally speaking, most karyotypic abnormalities 25% of karyotyped spontaneous abortions will be trisomic
fall into one of four classes: errors in meiosis (gametogenesis), [20–22]. All autosomal trisomies have been reported in mul-
errors in mitosis leading to mosaicism, errors in fertilization, and tiple studies with the unique exception of chromosome 1.
structural abnormalities and rearrangements. Trisomy 1 appears to be lethal prior to implantation and thus
A classic study of 1498 abortuses by Boué and colleagues would be unlikely to survive long enough to be seen in rou-
revealed 921 abnormal karyotypes (61.5%) [19, 20]. Among tine series of spontaneous abortions. The majority of trisomic
the chromosomally abnormal losses were 636 nondisjunc- conceptuses, even those with karyotypes that may be viable
tional events: 141 monosomies (15.3%), 479 trisomies in the neonate, result in miscarriage.
(52.0%), and 16 double trisomies (1.7%). There were 183
triploids (19.9%), 57 tetraploids (6.2%), and ten cases of Trisomies
mosaicism (1.1%). Structural abnormalities were identified The frequency of particular autosomal trisomies varies with
in 35 abortuses (3.8%). With improved cytogenetic and gestational age. At term, trisomy 21 (Down syndrome) is the
molecular methods being used today, the incidence of detect- most common and is seen in approximately 1 in 700 live
able abnormalities may have been even higher. However, the births. Trisomy 18 (Edwards syndrome) and trisomy 13
study clearly shows that cytogenetic abnormalities are pres- (Patau syndrome) are seen in approximately 1 in 6,000–8,000
ent in the majority of early spontaneous losses, and the data and 1 in 12,000 births, respectively. Trisomy 8 is much less
provide a useful breakdown of the types of abnormalities that frequent and most cases are mosaic. Although individual
are observed. Normal karyotypes were seen in 577 abortuses case reports indicate that other unusual autosomal trisomies
(38%), although there may have been a few undetected and rare autosomal monosomies do occasionally occur in the
underlying abnormalities such as subtle rearrangements, uni- neonate, these aneuploidies are typically seen only in a
parental disomy, or tissue-specific mosaicism that could have mosaic state and generally appear to be lethal when a normal
gone undetected in this sample. cell line is absent. See also Chap. 8.
Analysis of 1,205 products of conception of varying ges- The distribution of trisomies in spontaneous abortions is
tational ages received in the author’s laboratory between quite different from that seen at term. The most common tri-
1992 and 1996 revealed 539 (47.2%) abnormal karyotypes. somy observed in spontaneous abortuses is trisomy 16,
Of these, 50.6% were trisomies, 11.3% were monosomies, accounting for 31.0% of trisomic conceptuses and 7.27% of
4.2% were tetraploid, and 14.8% were triploid (Yusuf and all spontaneous abortions. This is followed by trisomy 22,
Naeem, 1997, personal communication). Although the total seen in 11.4% of trisomies and 2.26% of all spontaneous
percentage of abnormal karyotypes is lower in our series abortions. Trisomy 21 (Fig. 13.1) is third most frequent,
than that of Boué and colleagues, this can be explained by a accounting for 10.5% of trisomies and 2.11% of spontaneous
higher proportion of cases from later in gestation in our pop- abortions (Table 13.7) [17, 22].
ulation, giving a greater number of losses due to nonchromo- Double trisomies also occur and show a strong asso-
somal etiologies. The distribution of types of abnormalities ciation with advancing maternal age, even more so than
among the aberrant karyotypes is similar, however. the age effect seen with the viable trisomies such as trisomy
21 [13].
Identification of trisomic conceptuses is of clinical impor-
Errors in Meiosis tance because of the question of possible increased risk for
aneuploidy in subsequent pregnancies. The recurrence risk
During meiosis, the usual parental diploid chromosome for a couple with a previous trisomic infant is often cited as
complement of 46 is reduced to the haploid number of 23. approaching 1% [23, 24]. Verp and Simpson combined data
Nondisjunctional events during meiosis I or II of either from several smaller studies to suggest that the risk for an
oögenesis or spermatogenesis can result in monosomic or aneuploid liveborn following a trisomic abortus may also
Fig. 13.1 Trisomy 21 in a spontaneous abortion. Note the hydropic facial features and short hands with a single transverse palmar crease
appearance of the fetus, a frequent finding in trisomy 21. This may lead are typical of Down syndrome
to confusion with the Turner syndrome phenotype in some cases. The
Table 13.7 Distribution of individual trisomies among approximate 1% [25]. Connor and Ferguson-Smith offer an
trisomic spontaneous abortions [17, 22] empirical risk of 1.5% for a trisomy (not necessarily a viable
Chromosome trisomy) in any subsequent pregnancy following a trisomic
number % of trisomies % of abortuses abortus [26]. This raises the issue as to whether prenatal
1 Single case report 0 diagnosis should be offered to couples who have experienced
2 4.0 1.11 a previous trisomic abortus.
3 0.9 0.25 There may be increased susceptibility to trisomic concep-
4 2.4 0.64 tuses in some patients with a history of trisomic pregnancies.
5 0.3 0.04
The risk for these patients would be for nondisjunction in
6 0.9 0.14
general, not for a specific trisomy. The author’s laboratory
7 4.0 0.89
has seen several patients with three or more consecutive tri-
8 4.6 0.79
somies, each involving different chromosomes, suggesting a
9 2.3 0.72
10 2.0 0.36
population of couples who are at significant risk for recur-
11 0.3 0.04 rence. At this time, however, it is difficult to determine which
12 1.2 0.18 women with a trisomic abortus are more likely to experience
13 4.1 1.07 recurrent nondisjunctional events. Thus, couples may benefit
14 4.8 0.82 from genetic counseling following a trisomic or any other
15 7.4 1.68 chromosomally abnormal pregnancy (see Chap. 21).
16 31.0 7.27 The majority of autosomal trisomies are maternal in ori-
17 0.3 0.18 gin, with errors in meiosis I being more frequent than meio-
18 4.6 1.15 sis II, although there appears to be some variability depending
19 0.2 0.01 upon the chromosome involved. Of 436 informative cases
20 2.2 0.61 reviewed by Hassold and colleagues, 407 trisomies were
21 10.5 2.11 maternal in origin [27]. All cases of trisomy 16 and trisomy
22 11.4 2.26 22 were also maternal in origin, 19% of trisomies involving
280 S.M.V. Pflueger
chromosomes 2 through 12 were paternal in origin, and 27% Table 13.8 Effect of maternal age
of trisomies of chromosomes 13 through 15 were paternally Maternal age Number Percent Percent Percent non-
derived. Paternal nondisjunction was also associated with (years) karyotyped abnormal trisomic trisomic
sex chromosome aneuploidies, being responsible for 44% of 20 104 18.3 4.8 13.5
XXY and 6% of 47,XXX conceptions. 20–24 256 28.5 12.1 16.4
Examination of oöcytes reveals a significant percentage of 25–29 339 26.3 10.6 15.6
cytogenetic abnormalities. Kamiguchi and colleagues found 30–34 161 32.3 19.3 13.0
abnormal chromosomal complements in 24.3% of unfertil- 35–39 99 34.3 25.3 9.0
40+ 32 65.6 50.0 15.6
ized oöcytes [28]. Aneuploidy was most commonly observed,
followed by diploidy and structural abnormalities. A review Adapted from [35]
of 1,559 published cases revealed chromosomal abnormali-
ties in 24% of mature oöcyte karyotypes [29]. The majority effect on the smaller chromosomes, with mean maternal age
were aneuploid (22.8%); fewer had structural aberrations increasing with decreasing size of the trisomic chromosome
(1.2%). It is of interest to note that only one oöcyte with an [21]. Susceptibility to nondisjunction may not be the same
extra chromosome 16 was identified, although this is the for all chromosomes, and recurrence risks may be dependent
most common trisomy in spontaneous abortions. The differ- upon the particular chromosome involved in the trisomy, the
ence in distribution of trisomies suggests that postmeiotic parent contributing the extra chromosome, and the back-
viability may be as significant as meiotic error in determining ground risk associated with maternal age. Regardless of the
the incidence of particular trisomies in the human species. exact risk, many couples who have experienced a trisomic
Cytogenetic studies of human spermatocytes also reveal conceptus find the availability of prenatal diagnosis reassur-
abnormalities in paternal gametogenesis. The reported stud- ing in planning subsequent gestations.
ies have used several different methods for karyotype prepa-
ration. In 1987, Martin et al. reported that 3–4% of sperm Sex Chromosome Aneuploidy
exhibited aneuploidy due to nondisjunctional events, and Sex chromosome aneuploidies are among the most common
10% had structural abnormalities [30]. More recently, chromosomal abnormalities, both in spontaneous pregnancy
fluorescence in situ hybridization (FISH) techniques have loss and in liveborn infants. By far, the most frequent sex
been used, allowing for examination of far greater numbers of chromosome aneuploidy at conception is 45,X, accounting
sperm. FISH does have inherent limitations based on the par- for approximately 1–2% of clinically recognized pregnan-
ticular chromosome-specific probes utilized; only the specific cies. It is the single most frequent abnormal karyotype seen
aneuploidies being probed for will be detected. Using FISH in spontaneous abortions. The vast majority of monosomy X
techniques, Miharu and colleagues analyzed 450,580 sperm conceptuses terminate in miscarriage, less than 1% of
from 9 fertile and 12 infertile men [31]. Disomy for chromo- affected pregnancies surviving to term [17, 36]. The inci-
somes 1, 16, X, and Y ranged from 0.34–0.84% in infertile dence of Turner syndrome in surviving pregnancies is
subjects and 0.32–0.61% in fertile subjects. Guttenbach and approximately 1 in 1,000 female live births. No 45,Y karyo-
colleagues examined 16,127 sperm from eight healthy donors types have been reported. This is not an unexpected finding,
for disomy of chromosome 18 and found a range of 0.25– considering the important contributions of genes located on
0.5% [32]. Examination of 76,253 sperm from seven donors the X chromosome.
revealed a range of 0.31–0.34% of disomy for chromosomes The three sex chromosome trisomies, 47,XXX, 47,XXY,
3, 7, 10, 11, 17, and X [33]. Although FISH studies have and 47,XYY, are much less frequent than monosomy X in
inherent limitations, the data suggest that the rate of paternal spontaneous pregnancy loss but are similar to monosomy X
meiotic nondisjunction appears relatively constant for the in frequency at term, each affecting approximately 1 in 1,000
various chromosomes studied. infants of the appropriate sex. Affected infants with sex
Overall, maternal age is the best known predictor of risk chromosome trisomies are not usually markedly dysmorphic
for nondisjunctional events, in particular those resulting from and are often not identified unless cytogenetic studies are
errors in meiosis I. The association between maternal age performed for other reasons. These conditions are frequently
and risk for Down syndrome has long been established, and not recognized until later in life when behavioral changes or,
risk for trisomic abortuses also increases with advancing in the case of 47,XXY, infertility may cause the patient to
maternal age [18, 34, 35] (Table 13.8). present for evaluation. Some affected individuals may never
Not all chromosomal trisomies appear to have the same be identified. The mild phenotypic expression at birth
association with maternal age. Warburton et al. found that appears to reflect an absence of markedly deleterious effects
age-associated nondisjunction appeared to have a greater during embryogenesis. This would explain the relatively low
frequency of sex chromosome trisomies of 0.2% among or a Y. The non-mosaic 45,X conceptus appears unlikely to
spontaneous abortuses [22]. survive, whereas a mosaic gestation with a second sex chro-
Monosomy X gestations vary considerably in phenotype mosome, regardless of whether it is an X or a Y, has a better
and may exhibit marked dysmorphism. The majority undergo chance of undergoing orderly morphogenesis early in gesta-
early embryonic growth arrest and present as an empty ges- tion and of surviving to term [38, 39]. This second cell line
tational sac or as an umbilical cord ending with a small nod- may be absent from many tissues and is often difficult to
ule of necrotic embryonic tissue (Fig. 13.2). A lesser number detect with routine cytogenetic studies but can sometimes be
survive into the second trimester, at which time, the pheno- identified using multiple sampling sites or FISH techniques.
type is often that of a hydropic fetus with massive cystic Although extensive efforts at identification of a second cell
hygroma (Fig. 13.3). Renal and cardiac anomalies are fre- line may not be justified in routine evaluation of a mono-
quently seen as well. During the third trimester, the appear- somy X abortus, such techniques are often helpful in evalua-
ance may be similar to that seen in the second trimester, with tion of Turner syndrome patients with suspected low-level Y
cystic hygroma and dorsal edema over the hands and feet, the chromosome mosaicism. The presence of genes originating
classic Turner syndrome phenotype. There are also 45,X on the Y chromosome may place the patient at increased risk
infants who appear minimally affected and may not be rec- for gonadoblastoma.
ognized at birth, presenting later in childhood or adolescence Whereas the mean maternal age for most trisomic con-
with hypogonadism and short stature. ceptuses is increased over the normal population, this is not
Several explanations have been proposed for the wide the case with monosomy X. Rather, the mean maternal age
variability in phenotype. Although the majority of 45,X con- for monosomy X is the same or lower than expected for the
ceptuses surviving to term appear to have a maternally reproductive age population as a whole [13]. The evidence
derived X, parental origin of the monosomy does not appear that many cases of monosomy X are the result of post-zygotic
to affect phenotype or viability [37, 38]. Rather, survival of nondisjunction may possibly explain the difference in mater-
the early pregnancy may be dependent upon presence, in nal age between aneuploid pregnancies with monosomy X
some tissues, of a second sex chromosome, either another X and those with autosomal trisomies. Mitotic nondisjunction
Fig. 13.2 Gestational sac with very small embryo, consistent with an
underlying cytogenetic abnormality, often a nonviable trisomy or, as in Fig. 13.3 45,X spontaneous loss at mid-gestation. Note marked cystic
this case, monosomy X hygroma and generalized edema
282 S.M.V. Pflueger
during embryogenesis appears to be a different process, higher than estimates based on early series, since those cases
which may not exhibit the same maternal age effect; hence, were often examined using only a single tissue source, and
the maternal age for monosomy X would not be expected to some morphologically aberrant conceptuses classified as
be increased over the mean reproductive age of the popula- euploid may actually have been the result of undiagnosed
tion. Although patients who have experienced a pregnancy mosaicism.
with monosomy X often choose to have prenatal cytogenetic Recent molecular studies have shown that mosaic auto-
evaluation in subsequent gestations, the recurrence risk for somal trisomies can arise either from errors in meiosis, with
post-zygotic/mitotic nondisjunctional events has not been subsequent loss of one of the chromosomes leading to pro-
established. duction of a euploid cell line, or from the post-zygotic dupli-
cation of one of the chromosomes in an originally euploid
cell line. The likelihood of one or the other mechanism may
Errors in Mitosis vary depending on the particular chromosome involved.
Robinson and colleagues suggest that the mosaic trisomies
Malsegregation in the first mitotic division can give rise to involving chromosomes 13, 18, 21, and X most often result
tetraploidy. Tetraploid conceptions are usually lost relatively from somatic loss of a supernumerary chromosome that
early in gestation, although there are rare exceptions. arose from meiotic nondisjunction [44]. Mosaic trisomy 8,
Mitotic nondisjunction often results in mosaicism—the on the other hand, may be more likely to survive when the
presence of two or more cell lines with a different genetic aneuploid line is derived later, as a result of a post-zygotic
makeup. As has been suggested for Turner syndrome, mosaic error in mitosis in a conceptus that was originally chromoso-
aneuploidy may be better tolerated by the developing con- mally normal.
ceptus than complete aneuploidy, and there is evidence that Mosaicism in the placenta may be a significant determin-
survival of a trisomic fetus to term may be more likely if ing factor in survival of the trisomic conceptus. Those cases
there is a normal cell line present within the placenta. of trisomies 13 and 18 that survive to term appear to have a
The question of tissue-specific mosaicism has long been diploid cell line in the cytotrophoblasts, whereas those lost
an issue in prenatal diagnosis, especially with the advent of early in gestation are less likely to show a normal cell line
chorionic villus sampling (CVS; see Chap. 12). Early non- [45, 46]. Mosaicism does not appear to be necessary for sur-
disjunction can result in a generalized pattern of mosaicism, vival in trisomy 21, possibly due to a less deleterious effect
whereas divergence later in gestation can lead to mosaicism of this trisomy on placental function [45].
confined to either the fetus or the placenta. Mosaicism The presence of a euploid cell line in the fetus does not
confined to the amnion may present a dilemma in interpreta- necessarily imply a genetically normal fetus. If the mosa-
tion of amniotic fluid karyotype yet may not pose a problem icism is the result of “rescue” of a trisomic cell line, the pos-
for the fetus [40]. Within the placental chorionic villous tis- sibility of both remaining chromosomes of the pair originating
sue, there may be karyotypic differences between the direct from a single parent becomes a concern. This condition, uni-
preparation and long-term culture methods. This is a parental disomy, can often have severe consequences in the
reflection of the different origins of the trophoblast cells and affected fetus due to potential loss of heterozygosity with
the extraembryonic mesodermal cells. expression of recessive traits only carried by one parent or
Confined placental mosaicism is a potential concern even due to effects of inappropriate imprinting (see Chap. 20).
in the fetus with a normal karyotype. The presence of Thus, multiple sampling sites should be evaluated in cases
confined placental mosaicism has been associated with where a cytogenetic abnormality is strongly suspected, even
abnormal mid-trimester hCG levels, and with increased risk if a normal karyotype is identified on initial evaluation.
for adverse pregnancy outcome, including growth retarda- Molecular studies may be indicated to rule out uniparental
tion and fetal demise [41, 42]. Confined placental mosaicism disomy in ongoing pregnancies that have been identified as
may also be a factor leading to spontaneous abortion. A nor- mosaic. More study regarding the effects of uniparental dis-
mal fetal karyotype does not rule out a cytogenetic abnor- omy on embryogenesis is clearly needed.
mality in the placenta as a factor leading to pregnancy failure,
suggesting the need for karyotype analysis of both fetal and Chimerism
placental tissues in unexplained stillbirths [42]. Although the Another possible cause for the presence of more than one
incidence of mosaicism in CVS series is often cited in the cell line in a fetus is chimerism. The chimera of classical
1–2% range, Kalousek and colleagues detected confined pla- mythology was a creature with the head of a lion, the body
cental mosaicism in 11 of 54 spontaneous abortions studied of a goat, and the tail of a serpent. Although the mythical
and have suggested that the frequency may be especially chimera composed of several unrelated species is purely fan-
high in growth-disorganized embryos [43]. The cytogenetic ciful, individuals with cells derived from two separate fertil-
contribution to human pregnancy failure may thus be even ized eggs are known to exist in humans and other mammals.
Post-zygotic fusion of dizygotic twin zygotes results in a among the most frequent chromosomal aberrations in human
single chimeric individual. conception [53, 54].
Chimerism can explain the presence of two cell lines, in a Although the net result of either diandry or digyny is a
single individual, where neither can be derived from the other. pregnancy with 69 chromosomes, the phenotype of the pater-
This is the most likely mechanism underlying 46,XX/46,XY nal triploid is quite different from that of the maternal. On
hermaphroditism and may also explain a 45,X/69,XXY fetus microscopic section, paternal triploids will often show a
described by Betts and colleagues [47]. A number of diploid/ mixture of hydropic villi together with smaller, more normal
triploid mosaics have also been reported [48]. Some of these appearing villi, a phenotype sometimes referred to as a “par-
are probably chimeras, although another possible mechanism tial mole.” Most present as a “blighted ovum” with an empty
here is dispermy, in which a single maternal haploid pronu- gestational sac in the first trimester. Those that survive into
cleus is fertilized by a haploid sperm in the usual manner, the second trimester exhibit an abnormal fetal to placental
resulting in the diploid line. A second fertilization event then weight ratio with a very large placenta showing grossly
occurs in one of the daughter cells after the first cell division, hydropic villi (Fig. 13.4). Alpha-fetoprotein (AFP) and hCG
leading to the triploid cell line [49]. levels are characteristically elevated.
The maternal triploid fetus is growth retarded with a dis-
proportionately large cranium. The placenta is small and
Errors in Fertilization fibrotic in appearance, with none of the hydropic degenera-
tion seen in the paternal triploid (Fig. 13.5). In contrast with
Errors in fertilization can lead to pregnancies with an extra the paternal triploid, AFP and hCG levels are low.
complete set of chromosomes (triploidy; see Chap. 8) and The risk for triploid gestations appears to decrease with
also abnormal diploid pregnancies in which both sets of advancing maternal age. A decline in survival of aberrant
chromosomes come from one parent (hydatidiform or com- conceptuses, in older women, to the stage of recognized
plete molar pregnancies). Because paternal triploids may pregnancy is one possible explanation. Younger patients
exhibit changes in the villi that resemble hydatidiform moles, appear more likely to present with paternal triploids, whereas
these are sometimes referred to as partial moles. Both partial maternal triploids are more frequent in older patients.
and complete molar pregnancies have been instrumental in The complete mole is a pregnancy characterized by
advancing our understanding of imprinting (see Chap. 20) marked placental overgrowth with large, cystic-hydropic
and the role imprinting plays in fetal development and car- villi (Fig. 13.6). The fetus is absent and the villi do not exhibit
cinogenesis. Imprinting may have functions not only in gene fetal vascularization. The trophoblastic layers on the surface
expression early in embryogenesis but may also play a of the villi show varying degrees of proliferation. Patients
significant role in surveillance for chromosome loss later in usually exhibit markedly elevated hCG levels, although a
life and thus may help reduce the risk of cancer [50]. method-dependent artifact can result in falsely low levels
An extra haploid set of chromosomes from either the [55]. Despite the markedly abnormal phenotype, molar preg-
mother (digyny) or the father (diandry) can result in a trip- nancies usually exhibit a diploid karyotype of 46,XX in
loid conceptus. A 69,XYY karyotype is indicative of a pater- approximately 90% of cases and 46,XY in 6–10% of cases
nal origin for the extra chromosomal set, whereas a 69,XXX [56]. Both haploid sets of chromosomes are of paternal ori-
or 69,XXY karyotype could represent either digyny or dian- gin, however. Mechanisms are probably similar to paternal
dry. A variety of events can lead to the presence of an extra triploids but with fertilization of an “empty” egg. Duplication
set of chromosomes. of the chromosomes of a haploid sperm appears frequent and
The paternally derived triploid usually results either from may explain the preponderance of 46,XX karyotypes,
fertilization of a normal haploid egg by two separate sperm whereas fertilization by a diploid sperm could result in either
(dispermy) or from fertilization of the egg by a diploid sperm. a 46,XY or a 46,XX karyotype. The 46,YY karyotype
Fertilization by a haploid sperm with subsequent endoredu- appears to be nonviable [54].
plication of the paternal chromosomal complement is also a Hydatidiform moles pose a risk of undergoing malignant
possible mechanism. The latter process would result in isodi- transformation, becoming choriocarcinomas. Because of
somy for all paternal chromosomes, as would an error in this, the diagnosis is critical for patient management. The
meiosis II [51]. The maternally derived triploid, on the other triploid conceptus does not appear to have the same malig-
hand, most often originates from an error during maternal nant potential [57, 58]. The mechanism for malignant trans-
meiosis I or II, resulting in a diploid egg, although other formation in the complete mole appears to be relaxation of
mechanisms including fertilization of a primary oöcyte have imprinting with expression of genes that would normally be
also been postulated [52]. Together, triploidy accounts for repressed [59, 60]. Imprinting has also been suggested as an
1–3% of all recognized pregnancies and 15–20% of all explanation for the difference in phenotype between the
chromosomally abnormal miscarriages, placing the triploidy maternal and paternal triploids [61].
284 S.M.V. Pflueger
Fig. 13.4 Paternal triploid,

69,XXY karyotype. Patient
presented with markedly elevated
b hCG at 16 weeks. Note very
large placenta in relation to the
size of the fetus
crucial in order to prevent a malignancy in cases of complete

mole.
Both complete hydatidiform moles and most triploids
appear to represent random errors at the time of fertilization.
As such, a significant impact on the risk for other chromo-
somal abnormalities in subsequent pregnancies is not
expected. Berkowitz et al. studied 1,205 pregnancies follow-
ing a complete molar pregnancy and found no increase in
risk for stillbirth, prematurity, ectopic gestations, malforma-
tions, or spontaneous abortion [62]. However, there appears
to be a recurrence risk of about 1–2% for a future mole fol-
lowing a molar pregnancy [62–65]. Early sonographic sur-
veillance is suggested for future gestations to rule out
recurrent mole, and postnatal hCG determinations are rec-
ommended to rule out persistent trophoblastic disease [62].
Several pedigrees suggesting familial predisposition to molar
pregnancies have also been reported, although the significance
of family history on risk has not yet been established with
certainty [66–69].
Structural Rearrangements
Structural rearrangements are less common than the other

Fig. 13.5 Maternal triploid. Note very small placenta in relation to
types of chromosomal abnormalities in pregnancy losses.
fetal size and fetus with micrognathia, syndactyly, and disproportion- Approximately 1–2% of spontaneous abortions show struc-
ately large cranium in relation to body tural rearrangements. Jacobs summarized 5,726 karyotyped
spontaneous abortions, revealing 0.28% balanced and 1.54%
unbalanced rearrangements [70]. Balanced rearrangements
Although the experienced perinatal pathologist should include Robertsonian translocations, reciprocal transloca-
have little difficulty in recognizing the true hydatidiform mole tions, and inversions (see Chap. 9). A survey of the literature
based on the histologic appearance of the villi, cytogenetic by Dewald and Michels revealed translocations in 2.1% of
evaluation should be considered whenever there is a question couples with recurrent miscarriage [71]. Translocations were
of the diagnosis, since follow-up with serial hCG levels is found in 1.7% of male patients and 2.6% of female patients.
Fig. 13.6 Complete

hydatidiform mole with 46,XX
(paternal disomy) karyotype.
Note the hydropic villi with
absence of gestational
membranes and embryonic tissue
This compares with an incidence of 1 in 500 (0.2%) in new- Campana and colleagues note that the chromosomes and
borns [72]. The frequency of balanced rearrangements in breakpoints involved in structural rearrangements do not
spontaneous abortions is not markedly increased over that appear to be random [75]. Survival of pregnancies with
seen in live births. This is not unexpected, since balanced unbalanced chromosomal complements appears to be depen-
rearrangements are typically not associated with significant dent upon the particular chromosome and segment(s)
phenotypic alterations and are usually compatible with involved.
embryonic and fetal life. Structural rearrangements appear to occur with greater
The most frequent unbalanced rearrangements result from frequency in females than in males. Braekeleer and Dao
Robertsonian translocations (see Chaps. 3 and 9). These may found translocations or inversions in 2.6% of females with a
occur de novo or be familial in origin. The incidence of history of reproductive failure compared with 1.4% in males,
unbalanced Robertsonian translocations is much higher in and Gadow and colleagues found that 3.5% of women and
spontaneous abortuses than live births, reflecting the uterine 1.7% of men with recurrent loss had balanced translocations
mortality of trisomic conceptuses. [76, 77]. Both reports suggest increased risk for sterility in
Other unbalanced structural rearrangements seen in spon- male carriers as a possible explanation. Chromosomal rear-
taneous abortions include abnormal chromosomes with extra rangement appears to be associated with increased risk for
or missing material, ring chromosomes, and small supernu- infertility as well as for pregnancy loss.
merary chromosomes. De novo rearrangements are more fre- The risk for poor pregnancy outcome when one member
quently paternal in origin [73]. Analysis of human sperm of a couple carries a structural rearrangement varies consid-
revealed considerable variability among donors (0–17.8%) erably depending upon the particular type of rearrangement
with a median of 9.3% abnormal sperm, consisting primarily and the chromosome(s) involved. Counseling must be indi-
of breaks, fragments, and small deletions. Increased suscep- vidualized for each family with attention given to potential
tibility of sperm to chromosomal damage could explain the viability of any unbalanced meiotic products.
paternal origin of the majority of rearrangements. The risk figures that are used in counseling are often based
Although many structural rearrangements arise de novo, on pooled data from translocations involving various chro-
the majority appear to be familial. Numerous studies of mosomes and breakpoints. Generally, it has been suggested
patients experiencing recurrent pregnancy loss have shown that a male carrier is at lower risk for abnormal offspring
that these individuals are at increased risk of carrying a bal- than a female carrier. However, such generalizations may not
anced chromosomal rearrangement. Cytogenetic analysis to be applicable in all cases and more specific risks figures
rule out structural rearrangements and genetic counseling are based on the particular chromosomes involved may be
indicated for couples who have experienced two or more beneficial in evaluating reproductive options for a family in
losses. Since most balanced rearrangement carriers can which a balanced translocation has been identified [78].
produce both balanced and unbalanced gametes, a combina- The cause of reproductive failure in patients with bal-
tion of normal and abnormal conceptions is frequently seen anced translocations is most likely the production of unbal-
in such couples, and rearrangements may be more likely in anced gametes as a result of abnormal segregation during
those who have experienced both miscarriages and live births meiosis. Inversions can also lead to unbalanced gametes
than in those with only miscarriages [74, 77]. through crossover events involving the inverted segment.
286 S.M.V. Pflueger
A discussion of the implications of specific rearrangements the frequency of their contribution to poor pregnancy
with regard to abnormal segregation products can be found outcome.
in Chap. 9; see also [24].
Specimens for Cytogenetic Studies

Chromosomally Normal Pregnancy Loss
Although cytogenetic studies may be very helpful in manag-
Identification of the cytogenetically normal spontaneous ing patients with recurrent pregnancy loss, fetal karyotypes
abortion may be more important clinically than identification are infrequently performed. Cowchock and colleagues
of the aberrant gestation. The risk of repeat miscarriage is reported a success rate of 84% in a series of 100 samples,
higher when the prior loss is chromosomally normal [79]. showing that chromosome analysis is indeed feasible in most
Boué and colleagues found a risk of repeat loss of 23% after specimens [79]. Chorionic villi are often the tissue of choice,
a chromosomally normal miscarriage compared with 16.5% as skin biopsies from deceased fetal tissue can be associated
following a chromosomally abnormal loss [13]. Morton and with a higher failure rate. As previously mentioned, with
colleagues found that in women under 30, the risk for miscar- spontaneous pregnancy loss, it is frequently the case that the
riage was 22.7% following a chromosomally normal loss, tissue is retained in utero for several days or even weeks fol-
15.4% following a trisomy, and 17% following other chromo- lowing embryonic or fetal demise. Because of this, fetal tis-
some abnormalities. In women over 30, these risks were 25.1, sue is often autolyzed and is unlikely to respond to standard
24.7, and 20.3%, respectively [80]. Cytogenetic study of culture methods, although chondrocytes appear to survive
repeated spontaneous abortions suggests that those patients longer than skin and other soft tissues following fetal demise
who experience a chromosomally normal pregnancy loss are and may offer a greater chance of success [93]. Placental tis-
more likely to show normal karyotypes in subsequent losses sue, on the other hand, often remains viable for a much lon-
[81, 82]. ger period of time, since necessary substrates for survival are
Women with recurrent pregnancy losses and normal fetal provided by contact with the maternal blood supply. Ideally,
karyotypes may be more likely to have underlying uterine both fetal and placental sources should be utilized. The
abnormalities or endocrine dysfunction (see Chap. 11). advantage of the fetal tissue is that there is little risk for
Menstrual irregularities and elevated luteinizing hormone maternal cell contamination. If the fetus appears macerated,
levels are more common in women with normal fetal karyo- however, a high success rate is not to be expected. Placental
types than in women with abnormal fetal karyotypes [83]. tissue usually grows well but adds the risk of maternal cell
Immunological disorders have also been linked with contamination. This risk is reduced if the technologist pro-
recurrent normal pregnancy loss [84]. Systemic lupus ery- cessing the sample is experienced in the identification of
thematosus is perhaps the best known, but other autoim- membrane and chorionic villi.
mune conditions have also been implicated [85]. Since Direct preparations using the in situ method of tissue
patients with antiphospholipid antibodies and pregnancy culture work well with cells derived from spontaneously
failure frequently respond to treatment with prednisone and aborted tissues and have the advantage of rapid results
low-dose aspirin or heparin, it is important to recognize with a high success rate and minimal risk for maternal cell
autoimmune disease as a frequent cause of recurrent chro- contamination [94, 95]. However, if maternal cells are
mosomally normal pregnancy losses [86–88]. Alloimmune present in the original sample, trypsinization of slow-growing
disorders are less well understood but also appear to play a cultures to increase cell yield appears to increase the risk
role in recurrent pregnancy failure. Several therapies includ- for maternal cell overgrowth. Careful tissue selection and
ing immunization with paternal white cells and administra- washing to decrease the number of maternal cells may be
tion of intravenous immunoglobulin have been suggested helpful in decreasing the likelihood of maternal cell contami-
[89, 90]. nation [96].
Mutations that are lethal in the embryo are known from Fluorescence in situ hybridization (FISH, see Chap. 17)
animal models and may also be a factor in recurrent euploid using either tissue sections or disaggregated cells may be
abortion in man [91]. Mutations in genes responsible for used in cases in which the tissue was accidentally fixed in
early organization of the embryo can have devastating effects formalin prior to receipt in the cytogenetics laboratory, since
on embryogenesis, with resultant pregnancy failure. Parental it does not require dividing cells [97]. It must be remem-
sharing of HLA antigens may also increase risk for sponta- bered, however, that this method will detect only those chro-
neous abortion, although the mechanism is not yet clearly mosome abnormalities for which specific probes are
understood [92]. More study of such genes and their effects available. FISH can be useful in diagnosing suspected aneu-
on embryonic development is needed in order to determine ploidies, similar to its use in prenatal screening of uncultured
amniocytes, but the resulting information is limited to those
specific chromosomal regions for which probes are applied. Because of this, peripheral blood cytogenetic studies
Chromosomal rearrangements not involving numeric changes should still be considered for any couple experiencing recur-
are not generally amenable to this type of FISH analysis in rent pregnancy loss. In examining parental chromosomes,
interphase cells. structural rearrangements including translocations and inver-
Flow cytometry can also provide useful information in sions are the obvious focus. Such rearrangements may have
cases that are not amenable to cell culture, as it allows significant impact on the couple’s risk for miscarriage or
quantification of DNA [98]. This may be especially useful infants with anomalies.
for products of conception with hydropic changes seen with Cytogenetic abnormalities that are less clear in terms of
a histological examination, as it can differentiate between their implications for future reproduction may also be seen.
complete hydatidiform moles (paternal diploids) and partial It is not uncommon to find mosaic aneuploidy in couples
moles (usually triploid), an important distinction with regard with recurrent pregnancy loss. Low-level sex chromosome
to patient management because of the risk for persistent tro- aneuploidy is sometimes seen in lymphocytes but is not usu-
phoblastic disease with complete moles. DNA image cytom- ally found in cultured fibroblasts. The risk appears to increase
etry has also been shown to be useful in the diagnosis of with age but does not appear to be correlated with reproduc-
molar pregnancies [99]. tive history [105]. Discussion with a cytogeneticist can be
Newer methodology that has been proven useful for diag- invaluable in interpreting whether unexpected findings are of
nosis of unbalanced karyotypes in cases for which dividing potential clinical significance or artifact unrelated to the
cells are not available uses microarray-based technology, reproductive history.
either comparative genomic hybridization (CGH) or single
nucleotide polymorphism (SNP) analysis [100–102]. These
methods are dependent upon DNA extraction but do not Evaluation of Pregnancy Losses
require viable or intact cells and thus may be used for
formalin-fixed frozen or paraffin-embedded tissues as well Although a complete evaluation of a pregnancy loss requires
as fresh samples. Although both methods are useful for extensive specialized testing, including cytogenetic studies,
detection of aneuploidy, they do differ and may not always such tests are costly and labor intensive. With increasing
provide the same results. See also Chap. 18. emphasis on delivery of cost-effective health care, cytoge-
In CGH, test specimen and reference DNA samples are netic studies simply cannot be justified for every unsuccess-
hybridized together using different fluorochromes. The ful pregnancy. However, a careful examination by a
intensities between the test and reference samples are com- pathologist can often go a long way toward answering the
pared, enabling identification of gains or losses of individ- patient’s questions about the loss, and a more thorough eval-
ual chromosomes or chromosomal regions. This technique uation by a pathologist with training and interest in develop-
can detect unbalanced karyotypes such as trisomies, the mental anatomy can often provide considerable information
largest group of chromosomally abnormal pregnancies. without significant increase in cost.
However, CGH is not useful for the detection of balanced Such an examination can establish how far the pregnancy
structural rearrangements or polyploidy. Thus, a triploid or proceeded prior to developmental arrest and whether the
tetraploid conceptus would not be recognized by CGH pregnancy appears to have been developing normally. The
unless the sex chromosomes differed because the ratio of developmental age is especially helpful since the earlier the
intensity between the sample being tested and the control growth arrest, the more likely it is that the conceptus will
would be constant across the genome. However, additional exhibit an abnormal karyotype.
studies such as flow cytometry could be used if abnormal Any embryo or fetus should be examined closely for evi-
ploidy were of concern clinically [103]. SNP array may be dence of congenital anomalies. Embryos with malformations
an alternative to consider. and growth-retarded embryos are more likely to exhibit
Unlike CGH, a SNP array compares the patient sample abnormal karyotypes. Some isolated anomalies, such as cleft
with a control database rather than a single control sample. palate or neural tube defects, may be associated with
Like CGH, gains and losses are readily detected using SNP significant recurrence risks yet may have normal karyotypes.
arrays. However, SNP technology offers the advantage of Specific anomalies may also be indicative of an underlying
being able to detect loss of heterozygosity that may reflect syndromal process, with or without an abnormal karyotype.
uniparental disomy and will also permit the detection of trip- Single gene defects with significant recurrence risk can
loid gestations, conditions that may be missed using CGH sometimes be identified from a careful fetal examination.
methods [104]. However, as with CGH, a balanced translo- Evidence of infectious processes or teratogen exposure may
cation or inversion would not be recognized using this also be present, with their own implications for future preg-
technique. nancy management. Anatomic evaluation can therefore play
a useful role when traditional cytogenetic studies either are
288 S.M.V. Pflueger
Table 13.9 Phenotype of abortus and incidence of abnormal karyo- morphology when there is question of a skeletal dysplasia or
types [35] other anomalies. These can be reviewed later by a specialist
Appearance of abortus % Chromosomally abnormal in fetal dysmorphology if there is any question of anomalous
Incomplete specimen, no embryo 47.3 development. Additional special studies for congenital infec-
Incomplete embryo/fetus 40.0 tion, hematologic disorders, or metabolic disease may also
Intact empty sac 64.3 be indicated in some cases. Overall, a cause of death can be
Severely disorganized embryo 68.6 assigned in approximately 80% of cases [107, 109].
Normal embryo 54.1 A wide range of problems can result in decreased fertility
Embryo with focal abnormalities 55.0
or pregnancy failure, and the work-up for an infertile couple
Normal fetus 3.3
can be extensive and costly [110]. Identifying those couples
Fetus with malformations 18.2
whose losses are explained as being due to karyotypic abnor-
malities may be a cost-effective alternative. Cowchock and
not indicated, as in a first loss with no other risk factors, or colleagues argue that if cytogenetic studies cost $500, with
are not possible, for example, a formalin-fixed or otherwise an 84% chance of culture success and a 40% chance of
nonviable specimen. detecting a chromosomal abnormality that would explain the
Although most chromosomal abnormalities are not asso- loss, one of every three women with multiple miscarriages
ciated with distinct phenotypes, especially in very early would be spared further costly and invasive evaluations for
losses, there does appear to be some correlation between recurrent pregnancy loss [79]. This would save approxi-
specimen morphology and the likelihood of an abnormal mately $2,000 in expenses for testing that would otherwise
karyotype. Creasy studied the prevalence of chromosomal be done as part of a multiple miscarriage protocol. Given the
abnormalities and phenotype [35]. The results are summa- availability of therapy for many patients with nonchromo-
rized in Table 13.9. somal causes of pregnancy loss, the cost-benefit ratio may
Even though the degree of correlation between specimen actually be even better.
types and risk for chromosomal abnormalities is far from Although recurrent spontaneous abortion is often defined
ideal, some information regarding the likelihood of a karyo- as three consecutive losses, today, many couples find that
type abnormality can be gained from the embryonic pathol- three miscarriages are more than they are willing to accept
ogy. Absence of abnormalities in a pregnancy that has before looking for answers. There may indeed be justification
progressed to the fetal stage is a good predictor for a normal for initiating further evaluation after the second failed preg-
chromosomal complement. nancy. Coulam compared 214 couples with a history of two
Although morphology can help predict the likelihood of a or more consecutive abortions with 179 couples with a his-
chromosomal etiology for the loss, it cannot be expected to tory of three or more abortions [111]. Both groups showed
identify the particular karyotype abnormality involved. 6% of losses that were chromosomal, 1% that were anatomic,
However, even distinguishing probable chromosomal from and 5% that were hormonal. Sixty-five percent of the group
nonchromosomal losses is of considerable benefit to the with two losses and 66% of the group with three losses had
patient, as it can help in determining need for further studies immunologic causes. Twenty-three percent of the group with
and in predicting risk for recurrence. two losses and 22% of the group with three losses were unex-
There is a strong correlation between the chromosomal plained. The absence of any significant difference in preva-
constitutions of first and subsequent abortions. The patient lence between the two groups suggests that there is little to
with a chromosomally abnormal abortus is more likely to be gained by delaying evaluation until after the third preg-
experience abnormal karyotypes in subsequent losses, nancy loss.
whereas a patient with a normal karyotype in one loss is Tharapel and colleagues reviewed published surveys of
more likely to show normal karyotypes in any future preg- couples with two or more pregnancy losses (8,208 women
nancy losses [81, 82, 106]. and 7,834 men) and found an overall prevalence of major
Chromosome studies are especially useful for stillbirths chromosome abnormalities of 2.9% [112]. They go on to
suspected of having cytogenetic abnormalities, such as suggest that even with normal parental chromosomes, prena-
infants with congenital anomalies or intrauterine growth tal diagnosis should be offered because of the high incidence
restriction. There may also be increased risk in the presence of chromosomal abnormalities in spontaneous pregnancy
of fetal hydrops, maceration, or a history of prior losses [107, loss. Drugan and colleagues identified five anomalous
108]. Cytogenetic studies should probably be performed in fetuses, including one trisomy 18, two trisomy 21, one tri-
any case in which a pathophysiologic explanation for the somy 13, and one monosomy X fetus among 305 couples
demise is not identified [109]. A careful anatomic evaluation with recurrent pregnancy loss [113]. This 1.6% risk is greater
of both fetus and placenta is indicated in all stillbirths, as than the risk usually cited for amniocentesis. A control
are photographs and radiographic studies to document group of 979 patients revealed only three abnormalities
(0.3%), all sex chromosome aneuploidies. This would sug- improve chances for future success. Prenatal diagnosis can
gest an increased risk for nondisjunction among couples also be made available in those cases in which there is
experiencing repeated pregnancy failure. Their conclusion is increased risk for cytogenetic abnormalities or when addi-
that prenatal diagnosis is sufficiently safe and the risk for an tional reassurance of a normal fetal karyotype is needed. It is
abnormal result is sufficiently high to justify offering prenatal important to keep in mind that even with a history of a chro-
diagnosis to couples with a history of two losses. Although mosomally abnormal pregnancy, most couples have a good
this conclusion is based on a relatively small sample size and chance for a subsequent successful outcome.
not all obstetrical caregivers would agree, a discussion of
risks and benefits of prenatal diagnosis would appear to be
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108. Mueller RF, Sybert VP, Johnson J, Brown ZA, Chen W-J. Evans MI. Obstet Gynecol. 1990;75:381–4.
Evaluation of a protocol for post-mortem examination of still- 114. Munné S, Grifo J, Cohen J, Weier H-UG. Chromosome abnor-
births. N Eng J Med. 1983;309:586–90. malities in human arrested preimplantation embryos: a multiple-
109. Schauer GM, Kalousek DK, Magee JF. Genetic causes of still- probe FISH study. Am J Hum Genet. 1994;55:150–9.
birth. Semin Perinatol. 1992;16:341–51. 115. Zenzes MT, Casper RF. Cytogenetics of human oöcytes, zygotes,
110. Harger JH. Recurrent spontaneous abortion and pregnancy loss. and embryos after in vitro fertilization. Hum Genet. 1992;88:
In: Moore TR, Reiter RC, Rebar RW, Baker VV, editors. 367–75.
Chromosome Instability
14
Xiao-Xiang Zhang
Introduction The acquisition of chromosome abnormalities by target

cells is a central event that contributes to malignant transfor-
Two levels of genetic instability have recently been charac- mation and tumor development (see Chap. 16). This chapter
terized in human cancers: subtle sequence changes observed will focus on other forms of chromosome instability: fragile
at the nucleotide level and instability that is visible at the sites and chromosome breakage associated with chromo-
chromosomal level [1]. The high incidence of chromosome some instability syndromes.
instability reported in neoplastic processes has made this an
area of active investigation.
Chromosome instability describes a variety of chromo- Fragile Sites in Humans
some alterations, including numerical and structural chro-
mosomal rearrangements observed at an increased rate when Definition and Classification
compared with normal controls. Numerical changes can be
the consequence of abnormal segregation at the metaphase/ Chromosomal fragile sites are specific chromosome loci
anaphase transition. Dysregulation of genes involved in that usually appear as nonstaining gaps and breaks on meta-
chromosome condensation, sister chromatid cohesion, kine- phase preparations, either spontaneously or in response to
tochore structure and function, and centrosome/microtubule special agents or tissue culture conditions (Fig. 14.1). All
formation and dynamics have been implicated in the forma- fragile sites are part of the chromosome structure and are
tion of aneuploidy, hypodiploidy, and polyploidy, as have inherited as Mendelian codominant traits. Fragile site loci
cell cycle checkpoint genes. Chromosome breaks and telom- can be normal variants or can be associated with specific
ere dysfunction can result in various structural rearrange- genetic conditions/phenotypes. They are named by the
ments (deletions, duplications, inversions, insertions, and letters “fra” followed by designation for the specific chro-
translocations). Impairment of DNA repair, DNA replica- mosomes and bands where the fragile sites are located. For
tion, or DNA recombination is responsible for causing sister example, fra(10)(q25.2) describes a fragile site on chromo-
chromatid exchanges, fragile sites, chromatid/chromosome some 10 at band q25.2 [3]. The HUGO Gene Nomenclature
breaks, and mutagen sensitivity. Committee refers to this fragile site as FRA10B.
The most common forms of chromosome instability are Fragile sites are grouped into two classes, rare and com-
seen in cancers. Virtually all malignant human tumors con- mon, based on their frequency of occurrence and means of
tain chromosome rearrangements, and in many instances, induction. Common fragile sites are expressed in all individu-
these chromosomal changes were considered to have als at various rates. The most frequently observed common
occurred in the late stages of tumorigenesis. However, recent fragile sites occur, in decreasing order, at 3p14.2 (FRA3B),
evidence has suggested that chromosome instability was 16q23 (FRA16D), 6q26 (FRA6E), 7q32 (FRA7H), and Xp22
present in premalignant head and neck lesions and that high (FRAXB) [4]. The rare fragile sites are found only in some
levels of such instability were associated with subsequent families, with a population frequency of less than 5%. The
tumor progression [2]. fragile site at 16q22 (FRA16B) is seen most often, with an
occurrence of 1 in 20 in the German population, while 1p21.3
(FRA1M) is the rarest, having been reported only once.
X.-X. Zhang, M.D., Ph.D. (*)
Most fragile sites are not expressed spontaneously but
US Labs, a Labcorp Company, 201 Summit View Drive,
Suite 301, Brentwood, TN 37027, USA require induction using special chemical agents. Both the
e-mail: [email protected] rare and the common fragile sites are further subdivided
294 X.-X. Zhang
Fig. 14.1 An example of fragile sites on human chromosomes (arrows) in response to folate/thymidylate depletion using FUdR. See text for details
according to the culture conditions required for expression. classes of rare fragile sites that have been cloned thus far
While the majority of common fragile sites are induced by contain tandem repeat sequences (e.g., a CCG repeat in
aphidicolin, some are induced by 5-azacytidine or bro- FRAXA, a 33 bp AT rich in FRA16B, and an approximately
modeoxyuridine (BrdU). Most of the rare fragile sites are 42 bp variable AT-rich repeat in FRA10B). In contrast, no
observed when cells are grown in folic acid-deficient dinucleotide or trinucleotide repeat expansion has been
medium, but some are expressed when cells are grown in found at any of the common fragile sites that have been stud-
the presence of BrdU or distamycin. To date, 89 common ied, even though all fragile sites cloned are relatively AT
fragile sites and 30 rare fragile sites are described in the rich. Sequence analysis of FRA3B, FRA7G, and FRA7H
Human Genome Database (GBD) (http://www.gdb.org/). shows no striking molecular structure that explains the fra-
More than 150 fragile sites were recorded using the criteria gility in these regions. It has been proposed that delayed
formulated at a session known as the Chromosome DNA replication underlies expression of fragile sites and
Coordinating Meeting [5, 6]. that cytogenetic manifestation of these fragile sites is due to
Fragile sites have been found on every chromosome with incomplete DNA replication, which leads to a failure of
the exception of chromosome 21. Table 14.1 shows the chromatin compaction [11]. This becomes more obvious
classification and a list of the common and the rare fragile when DNA replication is perturbed by aphidicolin or folate
sites [7, 8]. It is interesting to note that the locations of many induction.
common fragile sites are highly conserved in man, gorilla,
chimpanzee, and orangutan, while none of the rare folate-
sensitive fragile sites have been identified in species other Clinical Significance
than humans [9, 10].
The physical basis of the cytogenetic expression of fragile The discovery of the fragile X syndrome (see Chap. 19) has
sites is not yet completely understood. However, advances in dramatically stimulated the search for other fragile sites that
the characterization of DNA sequences of fragile sites and might be associated with abnormal phenotypes. It has fre-
cell biology have shed some light on the mechanisms respon- quently been suggested that breakage and recombination at
sible for fragile site expression. All members of the three these sites may be mechanistically involved in constitutional
14 Chromosome Instability 295
Table 14.1 Common and rare fragile sites

Common fragile sites Rare fragile sites
Mode of induction Mode of induction
Aphidicolin inducible 1p36.1 3p14.2 7q22 12q24 Folate-sensitive 2q11.2
1p32 3q27 7q31.2 13q13.2 2q13
1p31.2 4p16.1 7q32.3 13q21.2 2q22.3
1p31 4p15.2 7q36 14q23 5q35
1p22 4q31.1 8q22.1 14q24.1 6p23
1p21.2 5p14 8q24.1 15q22 7p11.2
1q21 5q15 8q24.3 16q22.1 8q22.3
1q25.1 5q21 9q22.1 16q23.2 9p21
1q31 5q31.1 9q32 17q23.1 9q32
1q44 6p25.1 10q22.1 18q12.2 10q23.3
2p24.2 6p22.2 10q25.2 18q21.3 11q13.3
2p16.2 6q15 10q26.1 20p12.2 11q23.3
2p13 6q21 11p15.1 22q12.2 12q13.1
2q21.3 6q26 11p14.2 Xp22.31 12q24.13
2q31 7p22 11p13 Xq22.1 16p12.3
2q32.1 7p14.2 11q13 Xq27.2 16p13.11
2q33 7p13 11q14.2 19p13
2q37.3 7q11.2 11q23.3 20p11.23
3p24.2 7q21.2 12q21.3 22q13
Xq27.3
5-Azacytidine inducible 1q12 Distamycin A inducible 8q24.1
1q44 11p15.1
9q12 16q22.1
19q13 17p12
10q25.2
5-Bromodeoxyuridine (BrdU) inducible 4q12 6q13 13q21 BrdU requiring 12q24.2
5p13 9p21
5q15 10q21
Source: Data from references [6, 7]
rearrangements or the deletions observed in many tumors. progression. The theory that the common fragile sites might
The finding of a fragile site during the course of chromo- play a role in tumor development was initially proposed by
some analysis often raises questions regarding the potential Yunis et al. in 1984, soon after fragile sites were discovered
clinical significance and can create uncertainty regarding [15]. It has well been recognized that 50–70% of common
patient care. Currently, with the exceptions of FRAXA and fragile sites co-localize with oncogenes, tumor suppressor
FRAXE (two well-known causes of familial mental retarda- genes, and breakpoints in cancer rearrangements [16].
tion), and possibly FRA11B in relation to the breakpoint Subsequent experimental evidence has revealed that fragile
associated with Jacobsen syndrome, no other rare fragile sites appear to be preferential targets for viral integration
site has to date been shown to predispose to any heritable [17]. The observation of intrachromosomal amplification of
chromosome abnormality or malignancy [12]. In a study of the MET oncogene in a human gastric carcinoma via a
10,492 cases available from the literature, no statistical breakage-fusion-bridge within the FRA7G region further
association between fragile sites and constitutional break- supports the hypothesis that fragile sites play a key role in
points was noted [13]. The occurrence of folate-sensitive the amplification of some oncogenes during tumor progres-
autosomal rare fragile sites (ARFS) was compared in popu- sion [18, 19]. More direct evidence was reported by Egeli
lations of mentally retarded, mentally subnormal, and men- et al., who noted a significantly higher expression of fra(3)
tally normal children, and the frequencies did not differ (p14) in squamous cell lung cancer patients and their rela-
significantly [14]. tives than in healthy control subjects, and they suggested
On the other hand, compelling evidence has suggested that the high expression of fra(3)(p14) in these patients
that common fragile sites are highly unstable regions in the and their relatives may be a valid marker for genetic predis-
human genome, associated with cancer predisposition and position to lung cancer [20].
296 X.-X. Zhang
However, arguments downplaying the role of common Oram syndromes. Bone marrow failure leading to progres-
fragile sites in the tumorigenic process are based on the fact sive pancytopenia and predisposition to cancers, especially
that these sites are present in virtually everyone’s genome, AML, is the major cause of death in FA patients. Auerbach
and therefore it would be unreasonable to suggest that any suggested that the cellular defect in FA results in chromo-
one individual is at a particularly higher risk of developing a some instability, hypersensitivity to DNA damage, and
malignancy. Since there is no convincing evidence implicat- hypermutability for allele-loss mutations, predisposing to
ing common fragile sites in the cancer process, the following leukemia as a multistep process [22].
guidelines provided by Sutherland et al. can be used when The condition has a worldwide prevalence of 1–5 per mil-
dealing with patients who express fragile sites: lion and is found in all races and ethnic groups, with an
With the definite exceptions of FRAXA and FRAXE and possi- estimated carrier frequency of 0.3–1% [23]. The carrier
bly FRA11B, patients with any other fragile site, either rare or frequency in the Ashkenazi Jewish population is approxi-
common, can be strongly reassured the fragile site will not affect mately one in 90 [24]. Recent studies indicate that there are
their personal health or increase their risk of having chromoso- at least 13 genetically distinct complementation groups (A,
mally abnormal children. [21]
B, C, D1, D2, E, F, G, I, J, L, M, and N). The manner in
which these function has been identified [25]. The majority
of FA patients (60–80%) are assigned to group A, followed
Chromosome Instability Syndromes in frequency by groups C (8–14%) and G (~9%).
The gene for the FA-D1 subtype, FANCD1, is identical
The chromosome instability syndromes, formerly known as to the breast cancer susceptibility gene BRCA2. Patients
chromosome breakage syndromes, comprise a number of with heterozygous mutations of FANCD1/BRCA2 have an
rare but distinct clinical entities. The classic chromosome increased risk for breast/ovarian cancers while those with
instability syndromes are Fanconi anemia, ataxia telangi- homozygous mutations will suffer from the FA-D1 sub-
ectasia, Nijmegen syndrome, ICF syndrome, Robert syn- type of Fanconi anemia. All FA genes are recessive and
drome, Werner syndrome, and Bloom syndrome. They are are transmitted autosomally except FANCB, which is
all autosomal recessive, show increased frequency of chro- X-linked.
mosome changes (spontaneous or induced) and, with the Increasing evidence indicates that multiple FA proteins,
exception of Robert syndrome, are all associated with an including a ubiquitin ligase (FANCL), a monoubiquitinated
increased risk of development of malignancies. This higher protein (FANCD2), and DNA helicases (FANCJ and
incidence of neoplasia may also apply to family members of FANCD1/BRCA2), cooperate in a biochemical pathway
affected individuals. involved in cell cycle regulation and response to DNA dam-
These disorders were initially described as clinical syn- age (the FA pathway). The interaction of BRCA1 with the FA
dromes, independent of their mechanisms of action. However, protein pathway likely plays a critical role as a caretaker of
recent progress in molecular genetics and biochemistry indi- genomic integrity. Genetic defects of FA proteins result in a
cates that, despite their clinical characteristics, they essen- failure of recognition of interstrand DNA cross-links and
tially constitute disorders of DNA recombination. Although leave damaged DNA unrepaired. In a FANCA gene mutation
each has its own specific molecular defect related to abnor- analysis conducted on a panel of 90 patients, Wijker at al.
malities of DNA repair, cell cycle control, or apoptosis, the found no hot spots, and the mutations were scattered through-
common result is chromosomal instability leading to a neo- out the gene. Most mutations were predicted to result in the
plastic phenotype. absence of the FANCA protein [26].
The determination of the complementation group for
each patient has become increasingly important as more
Fanconi Anemia data on genotype–phenotype relationship has been col-
lected. Patients in the FA-C group showed a significantly
Fanconi anemia (FA) is a rare disorder characterized by poorer survival than those in groups A (FA-A) and group
diverse congenital anomalies and a predisposition to bone G (FA-G), and patients in the FA-D1 group have an
marrow failure and malignancy. FA patients present with a increased risk of developing medulloblastoma, Wilms
wide range of clinical heterogeneity, and many major organ tumor, and an early onset of acute leukemia. Certain geno-
systems can be affected. Approximately 50% of patients type–phenotype correlations have been noted; for example,
have radial ray anomalies ranging from bilateral absent FANCA-null patients tend to have more severe hematologi-
thumbs and radii to unilateral hypoplastic thumb or bifid cal manifestation and develop AML more often than non-
thumb. Malformations of the heart and kidney and anomalies null patients [27].
of the skeleton and limbs show considerable overlaps with The International Fanconi Anemia Registry (IFAR) was
some clinical syndromes, such as VATER, TAR, and Holt- established at the Rockefeller University in 1982 to study
this rare genetic disorder (database available online) [28]. In Ataxia Telangiectasia
a survey of the clinical features gathered from the IFAR, the
extreme variation of the phenotypes of FA makes the clinical Ataxia telangiectasia (A-T) is an autosomal recessive disor-
diagnosis difficult and unreliable. der associated with cerebellar degeneration, oculocutaneous
Fanconi anemia was the first disease in which spontane- telangiectasias, immunodeficiency, chromosome instability,
ous chromosome breakage was detected, both in vitro and radiosensitivity, and cancer predisposition. A-T patients
in vivo. Chromatid breaks and gaps are the most common present in early childhood with progressive cerebellar ataxia
spontaneous aberrations. Acentric and dicentric fragments, that can be misdiagnosed as ataxic cerebral palsy before the
rings, and endoreduplicated chromosomes are also seen in appearance of oculocutaneous telangiectases. Serum IgG2
the cells from FA patients, as are multiradial formations or IgA levels are diminished or absent in 80 and 60% of
(Fig. 14.2). A quantitative FISH analysis showed an accel- patients, respectively [30]. An elevated alpha-fetoprotein
erated telomere shortening in both arms of FA chromo- (APF) level is observed in a majority of A-T patients, who
somes; this may explain a tenfold increase in chromosome have a strong predisposition to develop lymphocytic leuke-
end fusions observed in FA cells [29]. Other cellular fea- mias and lymphomas. In general, lymphomas tend to be of
tures of FA include retardation of in vitro growth of FA B-cell origin, whereas leukemias tend to be T-cell type. Other
cells, delay during the G2 phase of the cell cycle, and solid tumors, including medulloblastomas and gliomas, are
hypersensitivity to cross-linking agents such as mitomycin also seen in A-T patients.
C (MMC) and diepoxybutane (DEB). Because the hetero- The incidence of A-T has been estimated at 1 in 89,000 in
geneity of the mutation spectrum and the frequency of the US Caucasian population and the A-T heterozygote fre-
intragenic deletions present a considerable challenge for quency is approximately 2.8% [31, 32].
the molecular diagnosis of FA, in vitro enhancement of The responsible gene, ATM (ataxia telangiectasia
chromosome breakage by DEB and MMC has been the mutated), encodes a large protein kinase with a phosphati-
gold standard for diagnosing FA. Currently, the best treat- dylinositol 3-kinase-like domain and was cloned in 1995.
ment is bone marrow transplantation. ATM is thought to play a central role in a signal transduction
Fig. 14.2 Metaphase from a

Fanconi anemia patient,
observed in a clastogen-
exposed lymphocyte culture.
Note the chromosome
breakage and radial
formations (Photo courtesy of
Dr. Susan Olson)
298 X.-X. Zhang
network that regulates cell cycle checkpoints, genetic recom- X-ray and radiomimetic substances, such as bleomycin, is
bination, apoptosis, and other cellular responses to DNA another characteristic cytogenetic hallmark. In a study that
damage. Heterozygous carriers of an ATM mutation have a utilized two recombination vectors, spontaneous intrachro-
6.1-fold relative risk of developing cancer, most commonly mosomal recombination rates were 30–200 times higher in
breast cancer. Such carriers may account for 5% of all cancer an A-T fibroblast cell line than in normal cells, but extra-
patients in the United States [33]. Although in vitro cell chromosomal recombination frequencies were near normal
fusion studies had suggested that A-T was genetically het- [36]. Therefore, the defects in ataxia telangiectasia seem to
erogeneous, all A-T patients studied to date have been found be related primarily to the processes of DNA recombination,
to harbor one of 200 different mutations in ATM [34]. and increased recombination may contribute to the high can-
However, a single mutation was observed in A-T patients of cer risk seen in A-T patients. Repair deficiencies after ion-
Jewish Moroccan or Tunisian origin [35]. Greater than 70% izing irradiation are secondary by-products of such
of mutations are predicted to lead to protein truncation. recombination defects. Nevertheless, treatment of malignan-
Approximately 90% of affected individuals showed no cies with conventional dosages of radiation can be fatal to
detectable ATM protein while about 10% of A-T patients A-T patients.
demonstrated trace amounts of ATM protein. The presence of early-onset ataxia along with oculocuta-
Elevated spontaneous chromosome breakage has been neous telangiectasias facilitates a clinical diagnosis of A-T,
observed in fibroblasts and peripheral lymphocytes from which can be problematic before the appearance of such
A-T patients, and tissue-specific chromosome aberrations telangiectasias. The large size of the ATM gene, together
are noted in A-T patient cells. For example, a high frequency with the diversity and broad distribution of ATM gene muta-
of balanced rearrangements involving chromosomes 7 and tions in A-T patients, greatly limits the utility of direct muta-
14 at loci for immunoglobulin and T-cell receptor genes tion analysis as a diagnostic tool, except where founder effect
(Fig. 14.3) is often seen in lymphocytes from A-T affected mutations are involved. Immunoblotting for intracellular
individuals. A greatly increased sensitivity of A-T cells to ATM protein depletion is to date the most sensitive and rather
Fig. 14.3 Sporadic (rows 1

and 2) and clonal (row 3)
rearrangements in ataxia
telangiectasia (R-banding).
Row 1, from left to right:
inv(7)(p14q35), t(7;7)
(p14;q35), t(14;14)(q11;q32),
inv(14)(q11q32). Row 2,
from left to right: t(7;14)
(p14;q11), t(7;14)(q35;q11),
t(7;14)(p14;q32), t(7;14)
(q35;q32). Row 3, from left to
right: inv(14)(q11q32),
t(X;14)(q28;q11) (note the
late replicating X on the left),
t(14;14)(q11;q32) (Courtesy
Alain Aurias and the Atlas of
Genetics and Cytogenetics in
Oncology and Hematology.
Modified from [36])
inexpensive test for establishing a diagnosis of A-T syn-

drome. A newly developed flow cytometric assay that mea-
sures the intranuclear phosphorylation of SMC (structural
maintenance of chromosomes) proteins claims to provide a
clear distinction between ATM mutation heterozygotes and
homozygotes [37].
Nijmegen Breakage Syndrome
Nijmegen breakage syndrome (NBS) is a rare disorder char-

acterized by progressive microcephaly, a “birdlike” face,
growth retardation, lack of secondary sex development in
females, recurrent sinopulmonary infections due to
immunodeficiency, and cancer predisposition, with the high-
est risk being for B-cell lymphoma. Because cells from NBS
patients share cytogenetic features with those from A-T Fig. 14.4 Chromosome 1 multiradial configuration from a patient with
patients, such as clonal rearrangements preferentially involv- ICF syndrome. Some stretching of the pericentromeric heterochromatin
ing chromosomes 7 and 14 in PHA-stimulated lymphocytes can also be seen (Photo courtesy of Dr. Jeffrey Sawyer)
and hypersensitivity to ionizing radiation, NBS was origi-
nally thought to be a variant of A-T [38]. Clinical features
differ, however. NBS patients have microcephaly, but neither hypertelorism, low-set ears, epicanthal folds, macroglossia,
cerebellar ataxia nor telangiectasias, and have normal serum recurrent respiratory infections, and variable immune
levels of AFP. Complementation studies and, moreover, the deficiency with a decreased level of IgA.
recent identification of the genes responsible for A-T (ATM) ICF syndrome is the only genetic disorder known to
and for NBS, NMN (NBS1, Nibrin, or p95 protein of the involve constitutive abnormalities of genomic methylation
NBS1/Mre11/Rad50 complex) have proven that A-T and patterns. Mutations in the DNA methyltransferase gene
NBS are related but separate entities [39, 40]. DNMT3B at 20q11.2 were identified in 75% of affected indi-
Several lines of evidence suggest that ATM and NMN viduals [44]. DNMT3B mainly affects de novo methylation
functionally interact in response to DNA damage induced by of the GC-rich classical satellite DNAs 2 and 3, which are
ionizing radiation (IR). Zhao et al. showed that NMN is phos- components of constitutive heterochromatin. In ICF patients,
phorylated by ATM in response to DNA damage. This there is constitutive hypomethylation of satellite 2 DNA,
involves S-phase checkpoint activation, formation of NBS1/ mostly located at the juxtacentromeric heterochromatin of
Mre11/Rad50 nuclear foci, and reversal of IR damage [41]. chromosomes 1 (1qh) and 16 (16qh), and of satellite 3 DNA,
This observation links ATM and NMN in a common signal- located on chromosome 9 (9qh).
ing pathway and provides an explanation for the phenotypic Cytogenetic analysis of peripheral blood lymphocytes
similarities between these two disorders. reveals multiradial configurations and a stretching of the
Diagnosis is based on molecular testing of NMN, the only pericentromeric heterochromatin of chromosome 1, 9, and
gene known to be associated with Nijmegen breakage syn- 16 (Fig. 14.4). An increase in formation of micronuclei is
drome. Disease-causing mutations are detected in almost also noted in ICF patients. Using FISH probes specific for
100% of NBS patients, and a 5-base pair truncating deletion alphoid satellite (centromere) DNA and classical satellite II
(657Del5) has been identified in 90% of patients. A German DNA (paracentric heterochromatin), Sumner et al. showed
group recently found a high carrier frequency (1/177) of that it is always the paracentromeric heterochromatin of
657Del5 mutations in three Slavic populations [42, 43]. chromosomes 1, 9, and 16 that becomes decondensed and
fused to form multiradial configurations [45]. The centro-
meric regions remain outside the regions of interchange.
ICF Syndrome These same juxtacentromeric heterochromatin regions are
subjected to persistent interphase self-associations and are
ICF syndrome (immunodeficiency, centromere instability, extruded into nuclear blebs or micronuclei. By an unknown
and facial anomalies) is a recessive disorder characterized by mechanism, the DNMT3B deficiency that causes ICF inter-
facial dysmorphism, immunoglobulin deficiency, and cen- feres with lymphogenesis (at a step after class switching) or
tromeric region instability involving chromosomes 1, 9, and lymphocyte activation. The stretched heterochromatic blocks
16. The most common clinical features of ICF syndrome are appear to be restricted to PHA-stimulated T-cells only.
300 X.-X. Zhang
When 35 individuals with ICF syndrome were screened

for DNMT3B gene mutations, only 20 affected patients
showed positive results, indicating genetic heterogeneity
[46]. The majority of mutations are missense mutations; oth-
ers include nonsense mutations and splice-site mutations. No
genotype–phenotype correlation was found.
Robert Syndrome (RS)
Robert syndrome (RS) is characterized by craniofacial

anomalies, limb defects, and pre- and postnatal growth retar-
dation. RS patients present with various degrees of limb mal-
formations, involving symmetric phocomelia or hypomelia.
Hypertelorism and cleft lip and palate are often seen in
affected individuals. Many of the malformation features are
similar to those observed in affected children whose mothers
took thalidomide during pregnancy; thus, RS is sometimes
called pseudothalidomide syndrome. RS is an extremely rare
disorder with only about 150 reported cases. It is an auto-
somal recessive condition, and parental consanguinity is Fig. 14.5 G-banded and C-banded (insert) image of cells from a
common. The carrier frequency for RS is unknown. patient with Robert syndrome, demonstrating premature centromere
Despite the heterogeneous clinical presentation, comple- separation (arrows) (Photo courtesy of Dr. Mazin Qumsiyeh)
mentation studies of cells derived from RS patients defined a
single complementation group [47]. Using a candidate gene
approach, Vega et al. found a variety of mutations in the leading to chromosome lag and aneuploidy. RS has been
ESCO2 gene at 8p21.1, including missense, nonsense, and designated as a human mitotic mutation syndrome that leads
frameshift mutations in 18 RS patients from 15 families of to secondary developmental defects.
different ethnic backgrounds [48]. N-acetyltransferase Clinical diagnostic criteria for RS were published by
ESCO2, also known as establishment of cohesion 1 homolog Vega et al. [50]. Cytogenetic analysis or a molecular assay
2 or ECO1 homolog 2, is an enzyme required for the estab- to identify ESCO2 mutations are required for confirmation
lishment of sister chromatid cohesion during the S phase of of the diagnosis [51]. Prenatal diagnosis requires an ultra-
mitosis. Mutations of ESCO2 leading to loss of ESCO2 sound examination in combination with cytogenetic testing
acetyltransferase activity is the only known cause for RS or prior identification of an ESCO2 mutation in the family.
thus far identified. No clear genotype–phenotype correlation Cytogenetic testing is uninformative for carrier status.
has been found.
Cytogenetic analysis using solid staining or C-banding
(see Chap. 4) to detect chromosome abnormalities involving Werner Syndrome
the heterochromatic regions has been used as a diagnostic
test for RS. Premature centromere separation (PCS; a phe- Werner syndrome (WS) is a human premature aging
nomenon of chromosome centromeres separating during syndrome manifested by scleroderma-like skin changes,
metaphase instead of anaphase) and heterochromatin repul- especially in the extremities, and cancer predisposition.
sion (HR; centromere splitting and puffing of heterochro- Individuals with WS generally show normal development
matic regions near centromeres), particularly of chromosomes until the end of the first decade. Lack of growth, graying and/
1, 9, and 16, are commonly seen in metaphases of 80% of or thinning of scalp hair, and scleroderma-like skin changes
RS patients (Fig. 14.5). Other cytogenetic abnormalities begin to manifest in the teens and 20s. Wizened and prema-
such as aneuploidy with random chromosome loss, micro- turely aged faces, described as “birdlike,” are often observed
nuclei, and abnormal nuclear morphology are also observed. in individuals with WS (Fig. 14.6).
Barbosa et al. demonstrated asynchronous replication of The most consistent feature of WS is bilateral cataracts.
homologous alpha-satellite DNA that was more evident in Variable features include diabetes mellitus, hypogonadism,
chromosomes 1, 9, and 16, in cells from RS patients [49]. osteoporosis and atherosclerosis, and an increased incidence
This asynchrony, in turn, prevents the establishment of of neoplasia. Malignant sarcomas, meningiomas, and carci-
proper cohesion between sister chromatid heterochromatin, nomas are seen in approximately 10% of WS patients; cancer
Fig. 14.6 A Werner syndrome patient at ages 15 and 48 years of age (Reprinted with permission from Epstein et al. et al. [52]. Photo courtesy of
Nancy Hanson)
is the leading cause of death in these patients. The prevalence [55]. WRN is a DNA helicase belonging to the RecQ family
of WS varies with the level of consanguinity, ranging from and is an exonuclease that participates in the pathways of
one in 20,000–40,000 in the Japanese population to an DNA repair, recombination, transcription, and replication.
estimated one in 200,000 in the US population. The carriers Loss of WRN function may promote genetic instability and
frequency is reported to be as high as one in 150–200 [53]. disease via recombination-initiated mitotic arrest, cell death,
The frequency of spontaneous chromosome damage in WS or gene rearrangement. Crabbe et al. proposed that lack of
is not as striking as in other chromosome instability syn- WRN helicase activity can result in dramatic telomere loss,
dromes. A variety of somatic chromosome rearrangements, which leads to chromosome fusion and breakage. They
including translocations, inversions, and deletions, were noted demonstrated that telomere elongation by telomerase
in lymphocytes and cultured skin fibroblasts from WS patients. significantly reduced the appearance of new chromosomal
Variegated translocation mosaicism (VTM), used to designate aberrations in cells lacking the WRN helicase, similar to
apparently balanced translocations, not numerical rearrange- complementation of Werner syndrome cells with the WRN
ments, has been observed in WS cells. In addition, skin helicase [56].
fibroblast lines established from WS patients have a dimin- Mutations of the WRN gene have been identified in approx-
ished in vitro life span. WS cells usually achieve only about 20 imately 90% of affected WS individuals, and these mutations
population doublings, in contrast with the approximately 60 are located at different sites across the coding region. All
doublings seen in normal control cells. Studies of cultured WRN mutations found to date either create stop codons or
cells from an obligate heterozygote revealed that these cells cause frameshifts that lead to premature termination and
exited the cell cycle at a faster rate than did normal cells. complete loss of function of the WRN gene product. No mis-
Wyllie et al. demonstrated that forced expression of telom- sense mutations have been identified. A splice-junction muta-
erase in WS fibroblasts confers extended cellular life span. tion is found in 50–60% of Japanese WS patients; no
Telomerase activity and telomere extension are sufficient to genotype–phenotype correlation has been observed [57].
prevent accelerated cell aging in WS fibroblast cultures [54]. Following the identification of mutations by sequencing anal-
The gene responsible for WS, WRN at 8p12, which con- ysis, a western blot has been developed to demonstrate the
tains a total of 35 exons, was identified by positional cloning absence of WS protein in the majority of affected patients.
302 X.-X. Zhang
Bloom Syndrome (BS) at the Holliday junction [60]. Gruber et al. demonstrated
that carriers of a BLM mutation are at increased risk for
Bloom syndrome (BS) is a rare genetic disorder character- colorectal cancer [61]. Mutation analysis indicates that the
ized clinically by severe pre- and postnatal growth restric- cause of most BS is the loss of enzymatic activity of the
tion, proportionately short stature, sun sensitivity, BLM gene product.
erythematous facial skin lesions, immunodeficiency, and Multiple BLM mutations have been identified. A specific
increased predisposition to cancer. Although BS occurs in 6-bp deletion/7-bp insertion at position 2,281 in exon 10 of
many ethnic groups and a single complementation group BLM, 2281del6ins7, often designated BLMAsh, was identified
exists among patients of diverse ethnic origin, Ashkenazi in 98% of affected individuals and in ~1% of unaffected
Jews have a significantly higher incidence, with a gene fre- individuals of Ashkenazi Jewish origin [62]. A PCR-based
quency estimated to be one in 110. mutation analysis is available for the Ashkenazi Jewish
Genomic instability is manifested by formation of quadri- population. Sequence analysis of the entire coding region of
radial configurations of symmetric shape with centromeres BLM is required to detect other mutations
in opposite arms, seen in approximately 1–2% in cultured The clinical diagnosis of BS is confirmed either by dem-
lymphocytes from BS patients (versus none in controls). onstrating a quadriradial formation in cultured lymphocytes
These rearrangements occur before mitosis and are a conse- or a highly increased rate of sister chromatid exchange in
quence of an equal exchange of chromatid segments near the cultured cells of any type. The diagnosis can also be
centromeres of two homologous chromosomes. confirmed by BLM mutation analysis.
The most characteristic and consistent cytogenetic feature
of BS is the greatly elevated (~10-fold) level of sister chro-
matid exchange (SCE; a reciprocal exchange of homologous Xeroderma Pigmentosum
segments between the two sister chromatids of a chromo-
some) in various cell types, including lymphocytes, Xeroderma pigmentosum (XP) is an autosomal recessive dis-
fibroblasts, and bone marrow cells in affected individuals order. Sensitivity to sunlight and the tendency to develop skin
[58] (Fig. 14.7). Increased SCE still represents the most dis- cancer at an early age are the key features of XP. The initial
tinctive cytogenetic diagnostic marker for BS. symptoms in most affected individuals are an abnormal reac-
BS arises from mutations in BLM at 15q26.1, a gene tion to sun exposure, including severe sunburn with blistering
encoding a protein with RecQ helicase function, and BLM and persistent erythema after minimal sun exposure. Freckling
is the only gene yet identified as causing BS. Hyper- in exposed areas occurs by two years of age in most patients.
recombination in BS is explained by a model in which BLM Neoplasms are predominantly basal cell or squamous cell
disrupts potentially recombinogenic molecules that arise at carcinomas and malignant melanomas. Approximately 90%
sites of stalled replication forks, promoting branch migration of squamous and basal cell carcinomas appear in the regions
Fig. 14.7 Sister chromatid exchange (SCE). (a) Two SCEs in a normal cell (arrows). (b) Multiple SCEs in a cell from a patient with Bloom
syndrome (Reprinted from Gardner and Sutherland [59]. By permission of Oxford University Press, Inc)
XP has been found in all races, with a frequency of approx-

imately 1 in one million in the United States and considerably
higher in Japan and North Africa. Consanguinity has been
reported in nearly 30% of cases. Seven complementation
groups (XPA to XPG) have been identified in one class of XP
patients with defective excision of pyrimidine dimers (excision-
deficient XP). A xeroderma pigmentosum variant (XPV) with
impaired replication of damaged DNA has also been identified.
Groups A and C are the most common forms worldwide, XPD
and XPF have intermediate frequency, group F has exclusively
been described in Japan. XPA, XPB, XPD, and XPG are asso-
ciated with neurological disorders, such as progressive mental
deterioration, abnormal motor activity, hearing loss, deafness,
and primary neuronal degeneration. The lowest level of DNA
repair is found in patients from group A. This may explain the
clinical severity involving both the skin and central nervous
system seen in these patients.
Elevated spontaneous chromosome breakage, a cytogenetic
hallmark for some chromosome instability syndromes, is not
seen in XP. However, an increased rate of sister chromatid
exchange and chromosome aberrations after exposure to ultra-
violet light and chemical carcinogens has been reported [65].
UV sensitivity in the form of deficient DNA repair is the
primary cellular feature of XP. Cells from XP patients lack
the ability to repair DNA damage by inserting new bases into
damaged DNA after UV irradiation. Colony-forming ability
after UV irradiation, as visualized under the microscope, can
Fig. 14.8 Above: Lesions of the face in an XP patient. Note multiple be used as an in vitro sensitivity test for XP. Nucleotide exci-
scars of carcinomas and an aged aspect of the skin. Below: multiple sion and repair (NER)-deficient XP fibroblasts are more sen-
basocellular carcinomas on the face of an XP patient. Thick arrow sitive than normal cells, and those from patients who have
points to a recent lesion and thin arrow to a scar of an old lesion
(Reprinted from Viguié [63]. Image courtesy of Daniel Wallach and
neurological defects generally exhibit the highest sensitivity.
used with permission of the Atlas of Genetics and Cytogenetics in Fibroblasts from patients with defects in XPV do not show a
Oncology and Haematology [63]) significant increase of UV sensitivity under standard test
conditions, but a dramatically increased sensitivity is seen
when XPV fibroblasts are incubated with caffeine after UV
of greatest sunlight exposure, such as the face, head, and exposure. Measurement of UV-induced unscheduled DNA
neck. The median age of onset of the first skin cancer is synthesis (UDS) is required for a definitive diagnosis of
8 years, nearly 50 years younger than that in the general pop- NER-deficient XP. Carrier detection and prenatal diagnosis
ulation in the United States. The rate of skin cancer in XP are possible if an unequivocal NER defect or the responsible
patients is nearly 2,000 times higher than in the general pop- mutation in the family has been characterized.
ulation under 20 years of age (Fig. 14.8). All XP genes have been cloned, and all reside on different
Ocular involvement is also common, and as a result, XP chromosomes. Some have the same names as the comple-
patients tend to develop photophobia. Abnormalities are mentation groups they are associated with (e.g., XPA and
restricted to sun-exposed anterior portions of the eye. XPC), while others have excision repair cross-complement-
Approximately 30% of XP patients also show progressive ing (ERCC2, 3, 4, and 5) or damage-specific DNA binding
neurologic degeneration including microcephaly, diminished protein names (e.g., DDB1). With the exception of XPV, the
deep tendon stretch reflexes, sensorineural deafness, and products of the XP genes are all involved in different steps of
cognitive impairment. the NER system, a major cellular defense against the carci-
Although the disorder is transmitted in an autosomal nogenic effects of UV exposure [66]. Cockayne syndrome
recessive manner, heterozygous carriers may be predisposed and the photosensitive form of trichothiodystrophy, two other
to skin cancers. Swift et al. reported that in 31 families of XP NER-deficiency syndromes, should be considered in any
patients, blood relatives had a significantly higher frequency differential diagnosis due to the common feature of extreme
of nonmelanoma skin cancer than their spouses [64]. sensitivity to sunlight shared by these disorders.
304 X.-X. Zhang
The diagnosis of XP is made clinically based on skin, eye, 19. Coquelle A, Pipiras E, Toledo F, Buttin G, Debatisse M. Expression
and neurologic manifestations. Sequencing analysis for XPA of fragile sites triggers intrachromosomal mammalian gene
amplification and sets boundaries to early amplicons. Cell.
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testing. Successful treatment for XP using a topical DNA common fragile sites and genetic predisposition to squamous cell
repair enzyme has been reported [67]. lung cancers. Cancer Genet Cytogenet. 1997;95(2):153–8.
21. Sutherland GR, Baker E. The clinical significance of fragile sites on
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Part IV
Cancer Cytogenetics
The Cytogenetics of Hematologic
Neoplasms 15
Aurelia Meloni-Ehrig
that errors during cell division were the basis for neoplastic
Introduction growth was most likely the determining factor that inspired
early researchers to take a better look at the genetics of the
The knowledge that cancer is a malignant form of uncon- cell itself. Thus, the need to have cell preparations good
trolled growth has existed for over a century. Several biologi- enough to be able to understand the mechanism of cell
cal, chemical, and physical agents have been implicated in division became of critical importance.
cancer causation. However, the mechanisms responsible for About 50 years after Boveri’s chromosome theory, the
this uninhibited proliferation, following the initial insult(s), first manuscripts on the chromosome makeup in normal
are still object of intense investigation. human cells and in genetic disorders started to appear, fol-
The first documented studies of cancer were performed lowed by those describing chromosome changes in neoplas-
over a century ago on domestic animals. At that time, the tic cells. A milestone of this investigation occurred in 1960
lack of both theoretical and technological knowledge with the publication of the first article by Nowell and
impaired the formulations of conclusions about cancer, other Hungerford on the association of chronic myelogenous leu-
than the visible presence of new growth, thus the term neo- kemia with a small size chromosome, known today as the
plasm (from the Greek neo = new and plasma = growth). In Philadelphia (Ph) chromosome, to honor the city where it
the early 1900s, the fundamental role of chromosomes in was discovered (see also Chap. 1) [4]. This finding stimu-
heredity and reproduction was already valued by a number of lated subsequent research on chromosome aberrations in
biologists. During that period, the most comprehensive view human neoplasms that still continues to augment our under-
of the role played by chromosomes in heredity was held by standing about cancer. This chapter will focus on the visibly
Boveri and Sutton, who independently theorized that it was recognizable chromosome abnormalities in human hemato-
necessary to have all chromosomes present in the cells for logic neoplasms and their implication in diagnosis, prognosis,
proper embryonic development to take place [1, 2]. This and therapeutic strategies.
innovative concept was later applied to the origin of tumor
cells by Boveri himself. Although he never experimented
with tumors, Boveri obviously sensed that tumors began Cytogenetic Methods for Diagnosis
from a single cell in which defects in the chromosome of Hematologic Neoplasms
makeup led cells to divide uncontrollably. He formulated his
theories in the book Zur Frage der Entstehung maligner Cytogenetics requires the presence of live cells or at least
Tumoren (On the Problem of Origin of Malignant Tumors), intact nuclei (for FISH studies; see Chap. 17). Although it is
published in 1914 [3]. This book is probably the most impor- understood that human cancer cells divide spontaneously
tant early contribution on the genetics of cancer, as it offered and that culturing might not be a necessary step, it is also true
some of the concepts still applicable today, specifically that that neoplastic cells are regulated by different growth cycles,
chromosome imbalances, mitotic disturbance, and monoclo- and therefore longer times in culture, as well as mitogen
nality are all attributes found in cancer cells. The thought stimulation (in the case of mature lymphoid neoplasms),
may be beneficial [5–8]. Cytogenetics starts with proper
sample collection, which in the case of hematologic neoplasms
A. Meloni-Ehrig, Ph.D., D.Sc. (*)
includes bone marrow aspirate, peripheral blood, as well as
Department of Cytogenetics, Ameripath Central Florida, 8150
Chancellor Drive, Suite 110 Orlando, FL, USA various body fluids and solid tissues in which infiltration
e-mail: [email protected] by the neoplastic hematologic cells has occurred [5, 9].
310 A. Meloni-Ehrig
Fig. 15.1 G-banded karyogram of a normal

bone marrow cell
Collection of samples should be performed aseptically, and in various tissue preparations. Their invention was seen as a
the case of solid tissues, samples should be placed in a room potential competitor to conventional cytogenetics, due to their
temperature medium, preferably enriched with growth higher resolution. Nevertheless, several years after the intro-
factors and antibiotics. Longer transit times (>48 h) might duction of these sophisticated technologies, conventional
affect the viability of the neoplastic cells and should be cytogenetic analysis is still the best method for the diagnosis
avoided when possible [5]. Analyzable chromosome prepa- of most hematologic neoplasms since it has the advantage
rations are obtained by first exposing the cells to mitotic of an overall examination of all chromosomes, compared to
inhibitors and subsequently treating them with hypotonic the more focused detection of abnormalities with the other
solution and fixation [10, 11]. Chromosome preparations are molecular genetic methods. Undisputed, in fact, is the ability
then subjected to banding techniques, the most widespread of conventional cytogenetics to identify related and distinct
of which is the trypsin-Giemsa banding method [12, 13]. See clonal populations, which is challenging for FISH and practi-
Fig. 15.1; see also Chap. 4. The terms used in cancer cytoge- cally impossible for array CGH [24, 25]. Furthermore, the
netics are listed in Table 15.1, and the karyotypes are presence of abnormalities acquired during clonal evolution,
described according to An International System for Human an important indicator of disease progression, might be
Cytogenetic Nomenclature (the most recent version appeared missed during a targeted FISH analysis [26–29].
in 2009; see also Chap. 3) [14].
Chromosome Abnormalities in Hematologic

Importance of Conventional Cytogenetics in Neoplasms
the Diagnosis and Prognosis of Hematologic
Neoplasms Cytogenetics began in 1956, when Tijo and Levan, and soon
after them Ford and Hamerton declared that normal human
There is no question that the development of sophisticated cells contained 46 chromosomes and not 48, as previously
techniques such as fluorescence in situ hybridization (FISH), believed (see Chap. 1) [30, 31]. From that point on, experi-
multicolor karyotyping (M-FISH, SKY), and, to some extent, mental work on cell cultures and banding was geared to the
array comparative genomic hybridization (array CGH) has improvement of chromosome spreading and morphology and
enhanced the knowledge of chromosome abnormalities in was presented in subsequent publications [4, 32]. It was the
hematologic neoplasms [15–23] (see also Chaps. 17 and 18). detection of the Philadelphia chromosome by Nowell and
These techniques have immensely contributed to the discov- Hungerford, however, that definitively established that chro-
ery of significant cryptic rearrangements as well as to the mosome abnormalities in leukemia are acquired and as such
detection of such rearrangements in nondividing cells of they are present exclusively in the neoplastic cells [4]. But it
15 The Cytogenetics of Hematologic Neoplasms 311
Table 15.1 Glossary of cytogenetics terminology used in this chapter

Acentric fragment A chromosome fragment lacking a centromere and therefore incapable of attaching to the spindle. Acentric
chromosomes are distributed randomly among daughter cells
Aneuploidy Deviation of the chromosome number that is characteristic for a particular species caused by either gain or loss
of one or more chromosomes
Autosome Any chromosome other than the sex chromosomes
Banding Alternating intrachromosomal light and dark segments along the length of chromosomes
Breakpoint Specific band on a chromosome containing a break in the DNA as the result of a chromosome rearrangement
Centromere An area of chromosomal constriction that holds the two chromatids together and is needed for spindle site
attachment. Based on the position of the centromere, chromosomes are classified as metacentric (middle
position), submetacentric (above the middle), and acrocentric (extremely small short arm consisting of satellites
and stalks)
Chromosome Arrangement of nuclear genetic material into formations containing a centromere and two chromosome arms.
The normal chromosome number in human somatic cells is 46, whereas in germ cells it is 23
Chromosome rearrangement Structural aberration in which chromosomes are broken and rejoined. These rearrangements can occur on a
single chromosome or involve multiple chromosomes
Clonal evolution A stepwise evolution characterized by the acquisition of new cytogenetic abnormalities
Cytogenetics The examination of chromosomes
Deletion Loss of a chromosome segment. Deletions can either be terminal or interstitial
Dicentric A chromosome containing two centromeres
Diploid Normal chromosome complement (two copies of each autosome and two sex chromosomes) in somatic cells
Double minute Cytogenetic visualization of gene amplification. So called because of their appearance as two adjacent dots.
Each double minute is thought to contain hundreds of copies of a particular oncogene
Duplication Two copies of the same segment present on a single chromosome
Haploid Half (i.e., 23 chromosomes) of the normal human chromosome complement in somatic cells. This is the number
of chromosomes present in normal germ cells
Homogeneously staining Cytogenetic visualization of gene amplification. Multiple copies of a particular oncogene are inserted into one
region of more chromosome region giving the appearance of a uniform staining
Hybrid gene Fusion of two different genes as a result of a structural chromosomal rearrangement. A hybrid gene leads to a
hybrid protein with abnormal function
Hyperdiploid Gain of one or more chromosomes
Hypodiploid Loss of one or more chromosomes
Idiogram Diagrammatic representation of a partial or complete karyogram
Insertion Balanced or unbalanced relocation of chromosomal material into a different or the same chromosome
Inversion Structural rearrangement affecting a single chromosome. This is generated by a 180° rotation of a segment
included between 2 breaks along a single chromosome. Inversions can be paracentric (breaks involving a single
arm) or pericentric (breaks involving both arms)
ISCN Suggested guidelines of An International System of Human Cytogenetic Nomenclature used for the description
of karyotypes
Isochromosome Structural rearrangement affecting a single chromosome generated by the misdivision of the centromere in
transverse plane resulting in loss of one arm and duplication of the other
Karyogram Arrangement of metaphase chromosomes according to size, position of centromere, and banding patterns
Karyotype Description of the chromosome complement according to ISCN guidelines
Locus Location of a particular gene on a chromosome
Marker chromosome Chromosome whose origin cannot be identified using standard banding methods
Metaphase Arrangement of chromosomes in one plane at the equator of the cell. This phase of mitosis is characterized by
the disappearance of the nuclear membrane and appearance of the spindle with subsequent attachment of the
centromeres to the spindle
Monosomy The absence of one member of a homologous pair of chromosomes
Oncogene Gene that promotes cell growth and development. One abnormal allele is sufficient to cause uncontrolled
growth and lead to tumor formation
Polyploid A cell containing a multiple of the haploid chromosome complement
Pseudodiploid Approximate diploid number of chromosomes, often accompanied by structural rearrangements
Recurrent abnormality Structural rearrangement or numerical abnormality detected in multiple patients with the same or similar
disease
Ring chromosome A circular formation of a chromosome originating from two breaks on opposite arms and reunion of the broken
ends
(continued)
312 A. Meloni-Ehrig

Sex chromosomes The X and the Y chromosomes. With some exceptions, XX is observed in females and XY in males
Translocation Chromosome abnormality resulting from a break in two or more chromosomes and exchange of the material
distal to the breaks. In a balanced translocation, there is exchange but no loss of DNA, whereas in an unbal-
anced translocation there is gain or loss of DNA. With unbalanced translocations, abnormal chromosomes are
referred to as derivatives if the exchanged material is known. The term add is used if the origin of the exchanged
material cannot be identified
Trisomy Three copies of a chromosome
Tumor suppressor gene Locus that inhibits tumor growth when at least one allele is functional. Loss of both alleles is associated with
tumor growth
was not until the middle 1970s that reports of cytogenetic Myeloid Neoplasms
abnormalities in cancer started to increasingly populate the
scientific literature [33–35]. Today, a complete list of these The classification of myeloid neoplasms has recently been
abnormalities can be found in Mitelman Database of modified [67]. This reclassification more than ever before
Chromosome Aberrations in Cancer [36]. It is immediately takes into account the genetic and cytogenetic changes asso-
evident from consulting Mitelman’s database that the most ciated with these neoplasms. Consequently, neoplasms with
common rearrangements in hematologic neoplasms are bal- similar morphologic and genetic features have been grouped
anced translocations [37–39]. In the majority of cases, trans- together. The myeloid neoplasms include the myelodysplas-
locations represent the sole abnormality, whereas in other tic syndromes (MDSs), myeloproliferative neoplasms
cases, they are identified during disease progression [40–44]. (MPNs), MDS/MPN, and acute myeloid leukemias. These
The significance of a primary translocation versus a later- are described in more detail in the following sections.
appearing abnormality differs, the latter usually suggestive of
a more aggressive clinical course. Similarly, the significance
of the same translocation in de novo and treatment-related Myelodysplastic Syndromes
hematologic neoplasms differs, with the latter, again, carry-
ing a worse prognostic outcome and, in some cases, a greater The term myelodysplastic syndrome (MDS) refers to a
resistance to therapy [45]. Balanced translocations are often fairly heterogeneous group of hematopoietic stem cell
the sole abnormality in the majority of acute and chronic neoplasms characterized by a series of similar features
myeloid leukemias and in a large number of acute and mature such as dysplastic cellular morphology, defect in cellular
lymphoid neoplasms [46, 47]. It is interesting to note that the maturation, and increased risk of transformation into
product of a translocation in leukemia is almost always a acute myeloid leukemia (AML) via a multistep process
hybrid protein with abnormal function, whereas in lymphoma [68, 69]. MDS is rare in children as it makes up approxi-
no hybrid protein is produced [48–50]. In lymphoma, the mately 5% of the pediatric hematologic neoplasms. MDS
relocation of an oncogene to a site under the control of an occurs mainly in adults with a median age of 70 years, and
immunoglobulin promoter often leads to overproduction of a although there is a risk for developing AML, about 50%
protein with oncogenic activity [51–53]. Translocations of deaths occur as a result of unrelated causes, such as
appear to be less frequent in myelodysplastic syndromes and bleeding or infection [70].
classical myeloproliferative neoplasms where partial or full There are two main types of MDS: primary or de novo
unbalances, leading to loss of tumor suppressor genes and/or MDS, and secondary or therapy-related MDS. Although
gain of oncogenes, dominate [54–60]. Apart from balanced secondary MDS occurs as a result of treatment with radiation
translocations, practically every abnormality known today and/or alkylating agents or treatment with DNA topoi-
has been observed in hematologic neoplasms, including ring somerase inhibitors for an unrelated malignancy, the initial
chromosomes, double minutes (dmin), and homogeneously insults leading to the development of primary MDS are still
staining regions (hsr), which for some time were considered being debated. Some of the possible triggers include
to be present exclusively in solid tumors [61–66]. The exposure to radiation, tobacco, and benzene.
specificity and recurrence of chromosome abnormalities in
hematologic neoplasms have gained significance to the point Classification of MDS
that the latest version of the World Health Organization Cytogenetic studies, which are routinely performed in
(WHO) guidelines focuses intensively on the genetic and patients with these neoplasms, are useful since chromosome
cytogenetic features of hematologic neoplasms as predictors abnormalities provide both diagnostic and prognostic infor-
of diagnostic and prognostic outcome [67]. mation [57, 58, 70]. Table 15.2 describes the subdivision of
Table 15.2 Subdivision of MDS neoplasms according to the 2008 Table 15.3 Recurrent chromosome abnormalities in primary MDS
WHO classification and percent of chromosome abnormalities in each and t-MDS
category
Abnormality Primary MDS t-MDS Fig. 15.2
Neoplasm Marrow blasts (%) Cytogenetics (%) +1/+1q + a
RCUD <1 25–50 der(1;7)(q10;p10) + ++ b
RARS <5 5–20 del(3p) + c
RCMD <5 50 3q21.3 rearrangements + d
RAEB-1 5–9 50–70 3q26.2 rearrangements + d
RAEB-2 10–19 50–70 −5/del(5q) + ++ e
MDS-U <5 50 +6 + –
MDS with isolated <5 100 del(6p) + ++ f
del(5q) −7/del(7q) + ++ g
Childhood MDS 5–10 5–10 t(7;12)(q36.3;p13.2) + h
t-MDS <5 90 +8 ++ + –
Abbreviations: RCUD refractory cytopenia with unilineage dysplasia, del(9q) + i
RARS refractory anemia with ring sideroblasts, RCMD refractory cytope- +10 + –
nia with multilineage dysplasia, RAEB-1 refractory anemia with excess
+11 + ++ –
blasts-1, RAEB-2 refractory anemia with excess blasts-2, MDS-U
myelodysplastic syndrome—unclassifiable, t-MDS therapy-related MDS del(11q) + j
11p15.4 rearrangements + k
11q23 rearrangements + l
MDS neoplasms according to the 2008 World Health 12p13 rearrangements + ++ m
Organization classification [67]. Chromosome abnormalities +13 +
have been observed in approximately 50% of patients with del(13q) + n
de novo MDS and in as many as 90% of patients with thera- +14 + –
py-related MDS. There appears to be a correlation between +15 + –
the frequency of chromosomal abnormalities and the sever- del(15q) + o
del(16q) + p
ity of disease, and this is evident in this Table [57, 69]. About
t(16;21)(q24.3;q22.3) + q
25% of patients with low-grade MDS, such as refractory
del(17p) + ++ r
anemia and refractory anemia with ring sideroblasts, have an
dic(17;20)(p11.2;q11.2) + ++ s
abnormal karyotype, compared with 50–70% of patients
i(17)(q10) + t
with refractory anemia with excess blasts (RAEB-1 and +19 + –
RAEB-2). The karyotypes observed in MDS are variable as del(20q) + u
they present with single or complex chromosome rearrange- ider(20)(q10)del(20q) + v
ments [56–58]. −21 + –
i(21)(q10) + w
idic(X)(q13) + x
Chromosome Abnormalities in MDS + indicates the presence of an abnormality and ++ indicates increased
frequency
The most frequent chromosome abnormalities are complete
or partial loss of chromosomes 5 and/or 7, deletions on
the long arm of chromosome 20, and gain of chromosome 8 MDS with Deletion of 5q
[36, 37, 71–75] (see Table 15.3 and Fig. 15.2). In general, The significance of del(5q) in MDS has to take into account
aggressive neoplasms are characterized by more complex not only the presence of this abnormality but also the
karyotypes than those seen in low-grade MDS. Furthermore, associated morphologic picture [72, 75, 80]. The size of
as a general rule, dosage aberrations appear to be more rep- the deleted portion of the long arm of chromosome 5 is
resented in primary MDS, whereas balanced translocations highly variable. The critical deleted region is approximately
are encountered more frequently in secondary MDS [56] 1.5 Mb in size and is located at 5q31.2, where the EGR1
(see Table 15.3). Among therapy-related MDSs, complex gene is located [75].
karyotypes with loss/deletion of chromosomes 5 and/or 7 del(5q) can be associated with the so-called 5q− syn-
together with deletions of 6p, 12p, and/or 16q are typical of drome. In this hematologic syndrome, patients present
alkylating agent-induced MDS, whereas balanced transloca- with refractory macrocytic anemia and demonstrate
tions involving 11q23 (MLL) and 21q22.3 (RUNX1) are hypolobulated micromegakaryocytic hyperplasia in the
associated with preceding therapy with DNA topoisomerase marrow [73, 80]. A female predominance has been noted
II inhibitors [76–79]. (sex ratio: 1M/3F). The clinical course is said to be relatively
314 A. Meloni-Ehrig
Fig. 15.2 Partial karyograms of recurrent abnormalities in MDS (Refer to Table 15.3 for additional information on the various rearrangements
illustrated in this figure)
indolent, with a very low-risk of developing acute leuke- with the most favorable prognostic outcome that is typi-
mia. In the International Prognostic Scoring System cally seen in patients with 5q− syndrome. Deletion 5q and/
(IPSS), del(5q) MDS patients are placed in the most favor- or complete loss of chromosome 5 in the context of a com-
able prognostic category [81, 82]. About 15% of patients plex karyotype is frequently seen in high-grade as well as
do not fit into this category but still have a del(5q) as the therapy-related MDS [63, 65]. Here, deletions of 5q might
sole abnormality [83, 84]. These cases do not appear to be derived from unbalanced translocations with a variety
have the same favorable prognostic outcome, demonstrat- of chromosome regions. The most common of these is a
ing the importance of the specific deletion for the progno- dic(5;17)(q11.2;p11.2), a result of which is loss of TP53 at
sis and response to therapy [58, 85]. Similarly, del(5q) 17p13.1, a marker of poor prognostic outcome in numer-
together with other abnormalities is no longer associated ous neoplasms [86].
MDS with Deletion of 7q or Monosomy 7 karyotype, can be the result of various rearrangements, includ-
Deletion of 7q/monosomy 7 has been viewed as a marker of ing simple deletions, unbalanced translocations, formation of
poor prognostic outcome [56, 57, 81]. However, the 2007 prog- an isochromosome, and monosomy 17 [86, 107]. Deletion of
nostic score criteria places patients with this rearrangement in 17p is recurrent in myeloid disorders, mainly refractory ane-
an intermediate risk [82]. As a sole abnormality, del(7q) occurs mia with excess of blasts (RAEB-1 and RAEB-2) and AML.
in approximately 1% of cases [87]. Three regions are most About 30% of AML and MDS cases with 17p deletion are
frequently deleted: 7q22, 7q31.1, and 7q31.3 [88, 89]. Some therapy related [108]. Deletion of 17p has been found to
studies indicate that retention of band 7q31 may be found in correlate with a particular form of morphological dysgranu-
patients with longer survival, suggesting that 7q31 might be the lopoiesis, sometimes associated with TP53 mutation [109].
location of a tumor suppression gene [89, 90]. More often, The clinical significance of sex chromosome loss in the
del(7q) or −7 occurs as part of a complex karyotype (approxi- bone marrow of patients with hematologic neoplasms is still
mately 5–10% of cases), characterized by recurrent abnormali- questionable [110, 111]. Loss of the Y chromosome is
ties that include one or more of the following: rearrangements observed in approximately 10% of MDS cases, but since it is
of chromosome 3, −5/del(5q), del(6p), +8, +9, del(9q), del(11q), seen also in males of increasing age without evidence of a
del(12p), del(17p), +19, del(20q), +21 [56]. Monosomy 7 as a hematologic neoplasm, it is generally interpreted to represent
sole abnormality is seen in pediatric patients with all MDS sub- an age-related phenomenon of no clinical significance [112].
types as well as in juvenile myelomonocytic leukemia (JMML) However, it is interesting to note that some elderly males with
[91]. Loss of chromosome 7 is also seen in siblings with the MDS and loss of the Y chromosome show the Y chromosome
so-called −7 syndrome and a predisposition to develop juvenile in their marrow cells when they achieve complete hemato-
MDS [92, 93]. Literature shows that the lost chromosome 7 can logic remission. Loss of an X chromosome in the bone mar-
come from either parent, suggesting that some other genetic row of female patients is less frequent than loss of the Y
defect that predisposes these children to lose one chromosome chromosome in males, and tends to be viewed as being asso-
7 is at the origin of this phenomenon [94, 95]. ciated with a hematologic neoplasm rather than as an age-
related phenomenon [113].
MDS with Trisomy 8 An interesting association is loss of the Y chromosome
Gain of one copy of chromosome 8 is recurrent in all myeloid together with gain of chromosome 15, which is also charac-
neoplasms. In MDS, it is found in over 10% of patients teristically seen in males with increasing age [114] (Fig. 15.3).
[96–98]. According to both the old and new prognostic scor- The significance of trisomy 15 with or without the loss of the
ing systems for MDS, trisomy 8 is associated an intermediate Y chromosome is not fully understood. In some cases, par-
risk when detected as the sole abnormality [97]. The presence ticularly when only a few abnormal metaphase cells are pres-
of additional abnormalities generally worsens the prognostic ent, this finding is thought to be a transient phenomenon by
outcome [96]. Trisomy 8 is often present as an additional some but not all authors [115–117].
abnormality, particularly in addition to del(5q). In about 2% Apparently balanced translocations have been reported in
of cases, four copies of chromosome 8 (tetrasomy 8) might be MDS, but they appear to be less common than the unbalanced
seen. These patients are given a high prognostic risk [99]. rearrangements. Chromosomes 1, 2, 3, 5, 6, 7, 13, 15, 17, 18, 19,
and 20 appear to be more frequently involved [118]. Table 15.3
MDS with Other Chromosome Abnormalities shows some of the most well-characterized translocations. From
Rearrangements of chromosome 3, specifically bands 3q21.3 this Table, it is apparent that balanced translocations have been
and/or 3q26.2, occur in about 5% of cases [100]. They have found in both de novo and therapy-related MDS.
been observed in de novo as well as therapy-related MDS, in Due to the variety of chromosome abnormalities reported
AML, and in accelerated phase or blast crisis CML [101, 102]. in MDS, it is understandable that, at present, the best genetic
The most common rearrangements include inv(3)(q21.3q26.2), test at diagnosis is conventional cytogenetics [119]. FISH is
t(3;3)(q21.3;q26.2), and del(3)(q21.3q26.2) [103]. Generally, unquestionably useful when a limited number or no meta-
these patients present with trilinear dysplasia in their bone phase cells are available or as a follow-up tool for a patient
marrow with dysmegakaryopoiesis. These rearrangements are with a known cytogenetic abnormality, but adds little to a
associated with an adverse prognostic risk in both MDS and normal conventional chromosome study based on the analy-
AML. This adverse prognosis probably correlates to the highly sis of 20 metaphase cells [120].
increased MECOM (EVI1, at 3q26.2) expression, detectable in
the vast majority of these patients [104, 105]. Some patients
with the 3q21.3q26.2 rearrangement do not have detectable Prognostic Significance of Chromosome
MECOM expression, suggesting that the poor prognosis in Abnormalities in MDS
these patients may be independent of such expression [106].
Another recurrent abnormality in MDS is del(17p). This The 1997 International Prognostic Scoring System (IPSS;
deletion, which often is observed in the context of a complex Table 15.4), which was constructed with data gathered from
316 A. Meloni-Ehrig
Fig. 15.3 Karyogram showing the simultaneous gain of chromosome 15 and loss of the Y chromosome (arrows). The significance of trisomy 15,
particularly when present in few cells, is not clear
Table 15.4 International Prognostic Scoring System of common elements in the marrow and mostly affect adult individuals
abnormalities in MDS [81] [121]. These neoplasms are known by different names,
Abnormalities Risk Median survival (mo.) depending on the lineage affected. The classic MPNs include
Normal, isolated − Y, isolated Favorable 42 chronic myelogenous leukemia (CML), polycythemia vera
del(5q), isolated del(20q) (PV), primary myelofibrosis (PMF), and essential thrombo-
Complex with ³3 abnormalities, Unfavorable 8 cythemia (ET) [122]. Other hematologic disorders included in
−7/del(7q)
the MPN category are chronic eosinophilic leukemia (CEL),
Other abnormalities Intermediate 28
systemic mastocytosis, chronic neutrophilic leukemia (CNL),
and the unclassifiable MPNs [67, 123, 124]. Except for CML,
patients with de novo MDS, was revised in 2007 to also include which is characterized by the presence of the t(9;22)
patients who received established treatments for MDS [81, 82]. (q34;q11.2)—the Philadelphia (Ph) chromosome transloca-
This new prognostic scoring system named IPSS-IMRAW tion—the classic MPN exhibits similar cytogenetic abnormal-
(International MDS Risk Analysis Workshop) is a combined ities, such as gain of 1q, +8, +9, del(13q), and/or del(20q) [59,
effort by European and American institutions and lists 22 groups 71, 125, 126]. Two or more of these abnormalities might be
of chromosome abnormalities compared with only 7 listed in present in the same karyotype (Table 15.6).
the 1997 IPSS (Table 15.5). These guidelines are still a work in
progress, and experts from around the world are intensively Chronic Myelogenous Leukemia
working on a more updated and satisfactory version. Chronic myelogenous leukemia (CML) is a stem cell
neoplasm that can occur at any age but is most frequent in
the 5th and 6th decades of life [127, 128]. It is characterized
Myeloproliferative Neoplasms by high white blood cell count with increased levels of gran-
ulocytes and megakaryocytes, often in the presence of
Myeloproliferative neoplasms (MPNs) are stem cell disorders eosinophilia and basophilia. CML is characterized by the
characterized by proliferation of one or more myeloid cellular t(9;22)(q34;q11.2), which leads to the formation of a chimeric
Table 15.5 Revised prognostic scoring system of common chromosome derived from different breakpoints in the BCR gene are
abnormalities in MDS based on the combined German-Austrian, known: P210BCR-ABL1, P190BCR-ABL1, and P230BCR-ABL1. The
Spanish MDS Registry, and IMRAW cohorts (IPSS-IMRAW) [82]
P210BCR-ABL1 is found in the majority of patients with CML
Abnormalities Risk Median survival (mo.) and in 30% of patients with Ph-positive acute lymphoblastic
Normal, −Y, isolated del(5q), Favorable 51 leukemia (ALL); the P190BCR-ABL1 is found in about 20% of
del(11q),del(12p), del(20q),
t(11;V)(q23;V), +21, any 2 adults and 80% of children with ALL, in Ph-positive AML,
abnormalities including and rarely in CML; and the rare P230BCR-ABL1 is found only in
del(5q) cases of neutrophilic-chronic myeloid leukemia (CML-N),
+1q, t(3q21.3;V), Intermediate-1 29 which has been described as a CML variant associated with
t(3q26.2;V),+8, t(7q;V), +19, a more benign clinical course than classic CML [133, 134].
−21, any other single
abnormality, any 2 abnor- There are three main clinical phases of CML: chronic,
malities not including 5q or accelerated, and blast crisis [135]. The chronic phase of
7q CML is characterized by mild or no symptoms and less than
−X, −7 or del(7q), any 2 Intermediate-2 15.6 5% blasts. At this stage, the only abnormality is the t(9;22).
abnormalities with −7 or
About 6% of cases have a variant translocation due to the
del(7q), complex with 3
abnormalities involvement of one or more additional chromosomes,
Complex with >3 Unfavorable 5.9 whereas in approximately 3% of cases the translocation can-
abnormalities not be identified by routine cytogenetics mostly due to cryp-
V variable translocation partners tic insertions of ABL1 sequences from chromosome 9 into
the BCR region on chromosome 22 or vice versa [136, 137].
Table 15.6 Classification of myeloproliferative neoplasms according These variants and cryptic rearrangements generally have
to the WHO, including the most common chromosome abnormalities the same prognostic outcome of the standard t(9;22), but
associated with them some are associated with a more aggressive course. This may
Abnormalities during be due to the fact the variant translocations might be the
Neoplasm Frequent abnormalities progression result of one, two, or more events or they might lead to a
CML t(9;22)(q34;q11.2) +8, i(17q), +der(22)t(9;22) deletion of either BCR or ABL1 sequences adjacent to the
PV +8, +9, del(20q) −5/del(5q), −7/del(7q), translocation breakpoints. Fluorescence in situ hybridization
del(17p)
(FISH) has revealed small deletions adjacent to the ABL1
PMF +8, 13q−, del(20q) +1q, −5/del(5q), −7/
del(7q), del(17p)
and BCR breakpoints in approximately 16 and 8% of cases,
ET +1q, +8, +9, del(20q) +1q, −5/del(5q), −7/del(7q) respectively [138] (see also Chap. 17).
SM 4q12 rearrangements (KIT −7/del(7q), +8, +9, Conventional cytogenetic analysis can sometimes reveal
mutations) del(11q), del(20q) abnormalities in addition to the t(9;22). It is important to
CNL +8, +9, del(11q), del(20q), del(12p) note, however, that an additional balanced rearrangement in
+21 all metaphase cells in chronic phase CML (or any neoplasm,
CEL, NOS No specific abnormalities Unknown for that matter) might be constitutional in origin. This should
MPN, U No recurrent abnormalities Unknown be investigated and removed from the equation when deter-
Abbreviations: CML chronic myelogenous leukemia, PV polycythemia mined to be the case. When the abnormality in addition to
vera, PMF primary myelofibrosis, ET essential thrombocythemia, SM
the t(9;22) is obviously (or proven to be) acquired, it is indic-
systemic mastocytosis, CNL chronic neutrophilic leukemia, CEL, NOS
chronic eosinophilic leukemia, not otherwise specified, MPN, U myelo- ative of clonal evolution. At the clinical level, such clonal
proliferative neoplasm, unclassified progression is associated with the accelerated phase or blast
crisis, both characterized by an increase in the number of
blasts and worsening of clinical symptoms [139]. The most
transcript between the ABL1 and BCR genes at 9q34 and recurrent chromosome abnormalities (about 90% of cases) in
22q11.2, respectively [129, 130] (Fig. 15.4). The derivative these phases are an additional Ph chromosome, +8, i(17)
chromosome 22 is also known as the Philadelphia (Ph) (q10), and/or +19 [140] (Fig. 15.5). Other abnormalities,
chromosome and is the first abnormality to have been associ- such as −Y, −7, del(7q), t(8;21)(q22;q22.3), t(15;17)
ated with a specific malignant neoplasm (see also Chap. 1). (q24.1;q21.2), inv(16)(p13.1q22.1), as well as 3q21.3,
The Ph chromosome was described in 1960 by Nowell 3q26.2, and 11q23 rearrangements, have been reported but
and Hungerford and is named after the city in which it was only in a small number of cases.
discovered [4].
The BCR-ABL1 rearrangement is also the first reported Polycythemia Vera
example of a “hybrid” gene leading to the production of an Polycythemia vera (PV) is a myeloproliferative neoplasm of
abnormal tyrosine kinase [131, 132]. Three fusion proteins adults (50–60 years of age) characterized by a proliferation
318 A. Meloni-Ehrig
Fig. 15.4 Karyogram of a patient with CML in chronic phase. The abnormal chromosomes involved in the t(9;22)(q34;q11.2) are indicated with
arrows. The derivative 22 is the Philadelphia (Ph) chromosome
Fig. 15.5 Karyogram of a patient with CML in blast crisis. This karyogram contains the three most common additional abnormalities observed
in the progressive phases of CML, specifically +8, i(17q), and + Ph
Fig. 15.6 Karyogram of a patient with

polycythemia vera. The three most common
abnormalities are present in this karyogram,
specifically +8, +9, and del(20q)
of red blood cells, which in some patients leads to bleeding megakaryocytes and immature granulocytes and associated
and thrombosis [141]. At the chromosome level, patients are anemia. Affected patients are generally in their 5th and 6th
BCR-ABL1 fusion-negative, and most, if not all, cases have a decade of life [154, 155]. Approximately 50% of patients
mutation at codon 617 in the Janus kinase 2 gene (JAK2, with PMF have the JAK2 V617F mutation, but unlike PV, no
located at 9p24.1) that results in a substitution of phenylala- mutations of JAK2 other than V617F have been found. A
nine for valine (V617F) [60, 142]. Mutations in exons 12 and small number of patients have mutations of other genes, par-
13 have also been described in patients negative for JAK2 ticularly MPL [156]. At diagnosis, about 40–50% of cases
V617F mutations [143, 144]. Other mutations involving the show chromosome abnormalities, the most common of
MPL, TET2, and CBL genes have been found in some of which are del(13q), del(20q), and gain of chromosome 8
these patients [143, 144]. These mutations are receiving [157, 158]. Additional abnormalities are detected during
increasing attention, particularly in the area of possible tar- disease progression, including del(5q), del(7q), gain of 1q, and
geted therapy using tyrosine kinase inhibitors. About 20% of del(17p) [159].
cases have an abnormal karyotype at diagnosis, mostly char-
acterized by +8, +9/+9p, and/or del(20q) [98, 145] (Fig. 15.6). Essential Thrombocythemia
Furthermore, gain of 9p is usually the result of a derivative Essential thrombocythemia (ET) is associated with an increased
chromosome, the most common of which is a der(9;18) number of platelets and megakaryocytes, plus fibrosis in the
(p10;q10) [146–148]. Gain of chromosome 9 or 9p is marrow. Patients are generally asymptomatic, with about 50%
assumed to represent a gain-of-function mechanism with presenting with circulation problems such as bleeding and
respect to JAK2 [149]. Less frequently gain of 1q, or partial thrombosis [160]. Similar to the other classic MPN, JAK2 muta-
trisomy 1q, might be seen. This gain is often the result of tions are also detected in these patients. Approximately 50%
unbalanced translocations involving chromosome 1 and vari- have the characteristic JAK2 V617F mutation found in PV and
ous chromosome regions [150]. The detection of chromo- MPF, whereas another 4–5% of patients have mutations of MPL
some abnormalities in PV increases as the disease progresses [161]. Only 10% of cases have chromosome abnormalities,
to MDS or AML [151]. The most common abnormalities which are similar to those seen in PV and PMF. Specifically, +8,
during disease progression are del(5q), del(7q), and/or +9, del(13q), and del(20q) are the most common, followed by
del(17p) [152, 153]. gain of 1q, del(5q), and del(7q) [162]. As in other MPNs, karyo-
typic abnormalities are more frequent during disease progres-
Primary Myelofibrosis sion to MDS or AML. Because ET is often a diagnosis of
Primary myelofibrosis (PMF), also known as idiopathic exclusion, some clinicians prefer to definitively rule out CML
myelofibrosis and agnogenic myeloid metaplasia, is charac- by testing for t(9;22) or a BCR-ABL1 rearrangement in these
terized by marrow fibrosis with an increased number of patients when the karyotype is normal.
320 A. Meloni-Ehrig
Chronic Eosinophilic Leukemia, Not Otherwise Some cases, particularly those associated with hypereosino-
Specified (NOS) philia, present with the same FIP1L1-PDGFRA fusion
Chronic eosinophilic leukemia, not otherwise specified (CEL, and other rearrangements involving PDGFRA observed in
NOS), is characterized by hypereosinophilia and represents a CEL [168]. Other detectable chromosome abnormalities
rare MPN [163]. The diagnosis is usually achieved by the are similar to those reported for other MPNs and leukemias,
exclusion of conditions that might be causing the abnormal specifically +8, +9, del(7q), del(11q), del(20q), t(8;21), and
increase of eosinophils in the marrow and blood. Two entities inv(16)/t(16;16). The association of mastocytosis with core
exist: CEL, not otherwise specified, and CEL with rearrange- binding factors AML, specifically those leukemias with
ments involving the platelet-derived growth factor receptors t(8;21) and inv(16)/t(16;16), makes it necessary to investigate
(PDGFRA and PDGFRB) [123, 155]. Pertinent literature these patients for KIT mutations [169].
indicates that CEL should be distinguished from idiopathic
hypereosinophilia by the presence of leukemic blasts. No Chronic Neutrophilic Leukemia
specific abnormalities have been reported in CEL, NOS. Chronic neutrophilic leukemia (CNL), as the name implies,
Among the CELs with PDGFR rearrangements, the most is characterized by an increase in mature neutrophils [170].
common abnormality is deletion of CHIC2 located at 4q12, Patients often present with splenomegaly, but no fibrosis is
which leads to a FIP1L1-PDGFRA fusion [164]. See later present in the marrow. Approximately 20% of cases have an
section: “Myeloid and Lymphoid Neoplasms Associated with abnormal karyotype. The abnormalities observed so far
PDGFRA, PDGFRB, and FGFR1.” include +8, +9, del(11q), del(20q), +21, and less frequently
del(12p) [171, 172].
Systemic Mastocytosis Some CNL patients present with a t(9;22)(q34;q11.2) as
Patients with systemic mastocytosis (SM) present with prolif- seen in typical CML but with a p230 BCR-ABL1 transcript
eration of mast cells in the bone marrow and/or other organs [173]. According to the WHO 2008 classification, these
[165]. Most patients are characterized by symptoms such as cases should be considered CML with a variant BCR-ABL1
hepatomegaly, osteoporosis, and ascites, among others. This transcript and not CNL.
is a very complex disease, as it comprises several distinct
entities and is also found in association with neoplasms such Myeloid and Lymphoid Neoplasms Associated with
as MPN and leukemia [165]. The disease course can vary PDGFRA, PDGFRB, and FGFR1
from indolent to aggressive. A large number of cases have This is a rare group of stem cell myeloid and lymphoid neo-
rearrangements involving chromosome 4, most likely due to plasms that have in common the presence of eosinophilia
the fact that this disease is often associated with mutations in and the involvement of genes that code for a tyrosine kinase
KIT located at 4q12 [166]. The most common KIT mutation, [174]. In the WHO 2008 classification, these neoplasms are
which results in substitution of valine for asparagine, occurs grouped together under the name, “myeloid and lymphoid
at amino acid position 816 and is thus known as D816V. This neoplasms with eosinophilia and abnormalities of PDGFRA,
mutation leads to relative resistance to the tyrosine kinase PDGFRB, or FGFR1” [67]. Various translocations involv-
inhibitor imatinib mesylate (Gleevec®) and therefore pro- ing the PDGFRA (4q12), PDGFRB (5q33.1), and FGFR1
vides relevant information for treatment selection [167]. (8p12) genes have been reported (Fig. 15.7). It is essential to
Fig. 15.7 Partial karyograms showing some of the most common t(5;12)(q33;p13.2) fuses PDGFRB with ETV6 (b), whereas t(8;13)
translocations involving PDGFRA, PDGFRB, and FGFR1. In this par- (p12;q12) leads to fusion of FGFR1 and FLT3 (c)
ticular figure, t(4;12)(q12;p13.2) fuses PDGFRA with ETV6 (a), and
clarify that although some earlier publications position the Table 15.7 Rearrangements involving genes that code for tyrosine
FGFR1 gene locus at 8p11, the present chromosome loca- kinases and neoplasms associated with them
tion following more precise mapping is at 8p12 [175]. The Abnormality Gene fusionsa Neoplasms
most common translocation observed in these neoplasms is t(1;12)(q25;p13.2) ABL2-ETV6 AML
t(5;12)(q33.1;p13.2) leading to a PDGFRB-ETV6 fusion t(2;13)(p16;q12.2) SPTBN1-FLT3 aCML
[176, 177]. Some of the rearrangements are cryptic at the t(5;9)(q33.3;q22) ITK-SYK T-Cell lymphoma
chromosome level. Since the presence of translocations t(8;9)(p22;p24.1) PCM1-JAK2 aCML, AML,
CEL, ALL
involving PDGFRA and PDGFRB is associated with respon-
t(9;12)(p24.1;p13.2) JAK2-ETV6 aCML, ALL
siveness to tyrosine kinase inhibitors, it is important, when
t(9;12)(q34;p13.2) or ABL1-ETV6 aCML, AML, ALL
one of these particular MPNs is suspected, to perform appro- ins(12;9)(p13.2;q34q34)
priate molecular studies to investigate whether any are pres- t(9;12)(q22;p13.2) SYK-ETV6 MDS
ent. Some translocations involving 4q12, 5q33.1, or 8p12, t(9;22)(p24.1;q11.2) JAK2-BCR aCML
but not resulting in a rearrangement of the PDGFRA, t(12;13)(p13.2;q12.2) ETV6-FLT3 MPN, AML, ALL
PDGFRB, and FGFR1 genes, respectively, have been also t(12;15)(p13.2;q25.3) ETV6-NTRK3 AML
reported. In these cases, as well, the final interpretation Abbreviations: AML acute myeloid leukemia, aCML atypical chronic
should be dependent on the presence or absence of the myeloid leukemia, CEL chronic eosinophilic leukemia, ALL acute lympho-
molecular rearrangement. The rearrangement involving blastic leukemia, MDS myelodysplastic syndrome, MPN myeloprolifera-
tive neoplasm
PDGFRA and FIP1L1 at 4q12 is cryptic with conventional a
Genes that code for tyrosine kinases are in bold
cytogenetics and can be detected only by FISH or by
RT-PCR. However, FISH appears to be superior as it can
provide information about other rearrangements involving Myelodysplastic/Myeloproliferative Neoplasms
the 4q12 region [178]. Rearrangements involving PDGFRB,
located at 5q33.1, include various translocations, the most This group includes neoplasms with morphologic features
common of which is t(5;12)(q33.1;p13.2), which fuses the that can be seen in both MDS and MPN [183]. Generally, the
PDGFRB and the ETV6 genes [179]. FISH is useful and bone marrow is hypercellular, but there is also some degree
should be performed on these patients since the presence of of dysplasia. The number of blasts is always below 20%. The
these rearrangements requires a specific alternative treat- neoplasms included here are chronic myelomonocytic leuke-
ment. See Chap. 17, Fig. 17.12a, b and discussion on tyrosine mia (CMML), atypical chronic myeloid leukemia (aCML),
kinases that follows. juvenile myelomonocytic leukemia (JMML), and myelodys-
plastic syndrome/myeloproliferative neoplasm, unclassifiable
Myeloproliferative Neoplasms, Unclassifiable (MDS/MPN, U). Table 15.8 presents some clinical and cyto-
This category includes stem cell neoplasms that do not have genetic data for each of these neoplasms. The workup of the
the morphologic characteristics typically seen in any particu- diagnosis includes the absence of BCR-ABL1 fusion and of
lar MPN [67]. They might have overlapping features seen in rearrangements of PDGFRA, PDGFRB, and FGFR1. On the
various MPNs but nothing specific enough to be classifiable other hand, mutations involving transcription factors such as
as a specific MPN. Genetically, no rearrangements of CEBPA, NPM1, or WT1 are frequent in these neoplasms, and
PDGFRA, PDGFRB, or FGFR1 are present, and no recur- one or more of these mutations might be present at the same
rent chromosome abnormalities have been associated with time. Other significant gene mutations involve TET2, RUNX1,
these neoplasms. ASXL1, and CBL [184]. The prognosis associated with MDS/
MPN is considered, in most cases, unfavorable since these
Myeloid Neoplasms with Translocations Involving patients rapidly progress to acute leukemia and are generally
Genes Coding For Tyrosine Kinases resistant to chemotherapy with associated short survivals
A number of myeloid neoplasms exhibit translocations after transformation [183].
involving genes that code for tyrosine kinases other than
PDGFRA, PDGFRB, or FGFR1. These neoplasms are not at Chronic Myelomonocytic Leukemia
this time included in a specific group but deserve some con- Chronic myelomonocytic leukemia (CMML) is an MPN
sideration, particularly in view of the increasing interest in characterized by persistent monocytosis and variable degree
these genes for therapeutic advancements. See Table 15.7 for of dysplasia [185]. The cases that were described previously
a list of these translocations and associated neoplasms. The as having a t(5;12)(q33.1;p13.2) leading to a PDGFRB-ETV6
majority of neoplasms where these translocations have been fusion are now included in the group of neoplasms with rear-
observed fall into the category of atypical CML (aCML), and rangements of PDGFRA, PDGFRB, and FGFR1 [67, 177,
the rest have been observed in other myeloid or lymphoid 186]. Although no specific abnormality has been associated
neoplasms [16, 180–182]. with CMML, recurrent chromosome abnormalities, such as
322 A. Meloni-Ehrig
Table 15.8 MDS/MPN according to WHO 2008 and most common specific/recurrent abnormality associated with a particular
chromosome abnormalities in order of frequency subtype of AML is sufficient to make the diagnosis. The
Percent of classification of these hematologic neoplasms has been
Neoplasm blasts (%) Recurrent chromosome abnormalities revised by the WHO to account for the various genetic and
CMML <20 −7/del(7q), +8, 12p rearrangements, cytogenetic changes that characterize this neoplasm [67].
i(17q), del(5q)
See Table 15.9 and Fig. 15.8. Although AML more fre-
aCML <5 +8, del(20q), involvement of chromo-
somes 12, 13, 14, and 17 quently affects adults in their 6th decade of life, it has been
JMML <5 −7/del(7q), del(5q) described in children and young adults as well [198]. Among
MDS/MPN,U <20 del(5q) (infrequent) the myeloid neoplasms, this is the group that accounts for the
Abbreviations: CMML chronic myelomonocytic leukemia, aCML atyp- majority of specific abnormalities and for a large number of
ical chronic myeloid leukemia, JMML juvenile myelomonocytic leuke- balanced rearrangements, most of which are translocations
mia, MDS/MPN, U: myelodysplastic syndrome/myeloproliferative [39, 199–201].
neoplasm, unclassified
Acute Myeloid Leukemia with Recurrent Genetic
−7/del(7q), gain of chromosome 8, and less commonly Abnormalities
del(5q), 12p rearrangements, and i(17)(q10), have been The AMLs included in this group are characterized by the
observed [187–189]. See Table 15.8. presence of well-established genetic abnormalities, the
most common of which are t(8;21)(q22;q22.3), inv(16)
Atypical Chronic Myeloid Leukemia (p13.1q22.1) or (16;16)(p13.1;q22.1), t(15;17)(q24.1;q21.2),
Atypical chronic myeloid leukemia (aCML) is an interesting t(9;11)(p22;q23), t(6;9)(p23;q34.1), inv(3)(q21.3q26.2) or
neoplasm that presents with features seen in classic CML as t(3;3)(q21.3;q26.2), and t(1;22)(p13.3;q13.1). These translo-
well as with myelodysplastic characteristics [190]. Although cations/inversions belong to the so-called class 2 mutations,
this neoplasm has many similarities with classic CML, it which have the ability to arrest differentiation of the lineage
lacks the typical t(9;22)(q34;q11.2). Chromosome abnor- affected by the rearrangement [202]. This results in prolif-
malities are detected in the majority of cases and are similar eration of only a particular subset of myeloid cells. As such,
to the ones described for CMML, except for losses involving these chromosome abnormalities have been associated with
chromosomes 6 and 7 and i(17)(q10), which seem to be particular subtypes of AML.
confined to CMML. Thus, gain of chromosome 8 and rear-
rangements resulting in deletions of 12p are the most fre- AML with t(8;21)(q22;q22.3)
quent aberrations [191, 192]. Furthermore, the t(8;9) This represents one of the core binding factor (CBF) myeloid
(p22;p24) (leading to a PCM1-JAK2 fusion) that was previ- leukemias and affects approximately 8–10% of AML
ously associated with aCML is no longer associated with this patients, mainly adults [203]. The t(8;21) leads to a RUNX1-
neoplasm but most likely belongs with chronic neutrophilic RUNXT1 (formerly AML1-ETO) fusion and is generally
leukemia (CNL). In fact, neoplasms with JAK2 mutations associated with a favorable prognostic outcome [46, 47].
should not be considered as aCML [193]. This particular AML is also known as AML with maturation
and as subtype M2 according to the French-American-British
Juvenile Myelomonocytic Leukemia (FAB) classification [204, 205]. Less commonly, t(8;21) can
As the name implies, juvenile myelomonocytic leukemia be also seen in AMML (FAB M4) and in therapy-related
(JMML) is an MPN of childhood, characterized by an abnor- MDS/AML [206]. Variant translocations, usually affecting a
mal proliferation of myelocytes and monocytes in the bone third chromosome, have been reported in 3% of cases [26,
marrow [190]. As with the other MPNs in this category, the 207–212]. The presence of additional abnormalities is com-
final diagnosis is based on the exclusion of the BCR-ABL1 mon (about 70% of cases). The most frequent additional
fusion [67]. The most common abnormality is −7/del(7q) abnormality is loss of a sex chromosome (the Y in males),
and less frequently del(5q) [55, 194, 195]. followed by del(9q), del(7q), +8, and/or +21. Although most
of these additional abnormalities do not appear to affect the
favorable prognostic outcome associated with t(8;21), gain
Acute Myeloid Leukemia of chromosome 6 is also seen, and some reports indicate a
less favorable disease course when trisomy 6 is part of the
Acute myeloid leukemia (AML) is defined by the presence karyotype [26, 213].
of myeloblasts in the bone marrow, peripheral blood, and Regardless of the presence or lack of additional abnor-
other tissues [196, 197]. At least 20% blasts should be present malities, patients exhibit a good response to chemotherapy
in the marrow. However, <20% blasts and presence of a together with a high rate of complete remission and disease-free
Table 15.9 Acute myeloid leukemia (AML) classification and associated chromosome abnormalities according to the World Health Organization
[67] (See Fig. 15.8)
Common additional abnormalities
Neoplasm Frequency (%) Chromosome abnormality (typical and variants) (in order of frequency)
AML with recurrent genetic
abnormalities
AML with t(8;21) 5–10 t(8;21)(q22;q22.3) −X or − Y, del(9q), del(7q), +8
AML with inv(16) or t(16;16) 5–8 inv(16)(p13.1q22.1) or t(16;16)(p13.1;q22.1) +22, +8, del(7q)
AML with t(15;17) 5–8 t(15;17)(q24.1;q21.2) +8, del(7q), del(9q)
AML with t(9;11) 9–12 (pediatric) t(9;11)(p22;q23) −X or − Y, +8
2 (adult)
AML with t(6;9) 1–2 t(6;9)(p23;q34.1) +8, +13, +21
AML with inv(3) or t(3;3) 1–2 inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2) −7, del(5)
AML (megakaryoblastic) with <1 t(1;22)(p13;q13) del(5q), del(7q), +21
t(1;22)
AML with NPM1 mutation 25–30 Normal or no specific abnormality No specific abnormality
AML with FLT3 mutation 20–40 Normal or no specific abnormality No specific abnormality
AML with CEBPA mutation 5–15 Normal or no specific abnormality No specific abnormality
AML with KIT mutation t(8;21) or inv(16)/t(16;16) Same are for t(8;21) and
inv(16)/t(16;16)
AML with WT1 mutation 20–25 Normal or no specific abnormality No specific abnormality
AML with myelodysplastic- 25–35 del(5q), del(7q), +8, del(20q), del(9q) −X or − Y, +1q,
related changes
Therapy-related myeloid 10–20 Complex with del(5q), del(7q), +8, 3q21.3 or del(6p), del(12p), del(17p), del(9q),
neoplasms 3q26.2 rearrangements, 11q23 rearrangements del(20q), +21
AML, not otherwise specified
AML with minimal <5 Complex with del(5q), del(7q), +8, MLL rearrange- del(17p), del(12p)
differentiation ments, RUNX1 rearrangements
AML without maturation 5–10 +8, del(9q) No recurrent abnormality
AML with maturation 8–10 +8 No recurrent abnormality
AML (myelomonocytic) 5–10 +8 No recurrent abnormality
AML (monoblastic/monocytic) <5 t(8;16)(p12;p13.3) No recurrent abnormality
AML (erythroid)
Pure erythroid leukemia <5 Complex with −5/del(5q), −7/del(7q), +8, del(20q)a del(6p), del(12p), del(17p)
Erythroleukemia (erythroid/ <5 Complex with t(3;3)/inv(3), −5/del(5q), −7/del(7q), del(6p), del(12p), del(17p)
myeloid) +8, del(20q)a
AML (megakaryoblastic) <5 Children: t(1;22)(p13;q13), +21 Adults: Complex del(6p), del(12p), del(17p)
with −5/del(5q), −7/del(7q), t(3;3)/inv(3), +8,
del(20q)a
AML (basophilic) <1 No recurrent abnormality No recurrent abnormality
AML (panmyelosis with Rare Complex with −5/del(5q), −7/del(7q), +8, del(20q)a del(6p), del(12p), del(17p)
myelofibrosis)
Myeloid sarcoma Abnormalities similar to AML with recurrent Similar to AML with recurrent
genetic abnormalities genetic abnormalities
Myeloid proliferation related to Down syndrome
Down syndrome patients
Transient abnormal 10 newborns Additional copies of chromosome 21 (in addition +8
myelopoiesis to constitutional trisomy 21)
Myeloid leukemia associated 1–2 children Additional copies of chromosome 21 (in addition −7, +8
with Down syndrome (<5 years of age) to constitutional trisomy 21)
Blastic plasmacytoid dendritic Rare del(4q), del(5q), del(12p), del(13q), del(6q), No recurrent abnormalities
cell neoplasms del(15q), del(9p), del(9q)
survival. However, the favorable prognostic outcome is AML with inv(16)(p13.1q22.1) or t(16;16)(p13.1;q22.1)
without exception altered by the presence of KIT mutations The characteristic of this AML is the presence of myelo-
[214, 215]. monocytic blasts and atypical eosinophils. Also known as
324 A. Meloni-Ehrig
Fig. 15.8 Partial karyograms showing recurrent (or specific) rearrangements in AML. These translocations/inversions define particular AML
subtypes in the WHO classification
AML M4EO according to the FAB classification, this of this translocation leads to a fusion between PML at
leukemia makes up 7–10% of AML cases and is generally 15q24.1 and RARA at 17q21.2 [48]. The PML-RARA fusion
associated with a favorable prognostic outcome [203]. is associated with a favorable prognosis and response to
However, patients have a higher risk of central nervous treatment with all-trans retinoic acid (ATRA) [220].
system (CNS) involvement at diagnosis or at relapse than Translocations with additional rearrangements involving
patients with other types of AML. Adults are more fre- either chromosome 15 or 17 or complex translocations
quently affected than children. The hallmark of this AML involving a third chromosome occur in approximately 5% of
is the inv(16)(p13.1q22.1) or, less commonly, the t(16;16) cases [222, 223]. In these cases, it is important to determine
(p13.1;q22.1). Either abnormality leads to the fusion of that the PML-RARA fusion is intact. RT-PCR can easily be
MYH11 at 16p13.1 with CBFB at 16q22.1 [216]. The used to verify this as well as determine the size of transcript,
identification of these rearrangements by conventional which could negatively influence the prognostic outcome
cytogenetics might be challenging, particularly when the [224]. Other variants involving 17q21.2 and not 15q24.1
chromosome morphology is not optimal. In those cases, exist but are rare. The most known of these variants are
FISH or RT-PCR can be helpful [217]. These rearrange- t(5;17)(q35.1;q21.2) leading to a fusion of NPM1 and RARA
ments have been reported occasionally in tMDS and tAML and t(11;17)(q23.2;q21.2) leading to a fusion of ZBTB16
[218]. Chromosome abnormalities in addition to inv(16) (PLZF) and RARA [225]. The t(5;17) seems to respond to
or t(16;16) are detected in approximately 30% of cases ATRA, whereas t(11;17) does not. The presence of t(15;17)
[219]. The most common is +22, which is considered a and variants in therapy-related neoplasms is infrequent, but it
clue by many cytogeneticists, particularly when the pres- has been reported [226]. These cases show dysplastic fea-
ence of inv(16) or t(16;16) is not obvious. Other additional tures and often are associated with additional chromosomal
chromosome abnormalities include +8, del(7q), and/or and molecular changes.
+21. Although this leukemia has been associated with Additional abnormalities have also been observed in
complete remission and improved long-term survival, de novo APL, of which +8, del(9q), and del(7q) are the most
molecular testing for KIT mutations is necessary, as these frequent. The presence of chromosome abnormalities in
are associated with adverse prognosis and necessitate addition to t(15;17) does not appear to affect the prognosis
more aggressive therapy [214]. associated with this neoplasm [227].
Acute Promyelocytic Leukemia with t(15;17) AML with t(9;11)(p22;q23) and Other Translocations
(q24.1;q21.2) Involving MLL
The vast body of research of the past 30 years has contrib- This translocation leads to fusion of MLLT3 at 9p22 with MLL
uted to the successful management of acute promyelocytic at 11q23 and is found in AML with a monocytic or myelo-
leukemia (APL) [42, 220]. Originally considered one of the monocytic phenotype, lack of CD34 expression, and frequent
most aggressive leukemias, it is now a model for targeted RAS mutations [228]. This is the most common translocation
therapy [221]. Due to the high risk of early death and the involving MLL [67]. Among the approximately 85 known
potential for high cure rate, it is essential to immediately MLL translocations, t(9;11) is thought to be associated with a
identify this leukemia. The t(15;17) is the specific abnormal- better prognostic outcome [229]. However, large-scale retro-
ity that characterizes this subtype of AML [42]. The formation spective studies could not confirm this earlier result [230].
Fig. 15.9 Partial karyograms illustrating common MLL (11q23) translocations observed in AML. Some of these translocations have been observed
also in therapy-related MDS/AML
With conventional cytogenetics, MLL translocations are usu- ticularly together with gains of chromosomes 8, 13, and/or
ally present in all or almost all metaphase cells analyzed. 21 [236].
Additional abnormalities can be seen, and the most common
are loss of a sex chromosome (−Y in males) and +8 [231]. AML with inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2)
Another frequent MLL translocation is t(11;19) with variant These two abnormalities have in common the involvement
breakpoints on chromosome 19. Specifically, the breakpoint at of two genes associated with an unfavorable prognostic
19p13.1 (ELL) is seen mainly in adults, whereas the break- outcome, RPN1 at 3q21.3 and MECOM (EVI1) at 3q26.2
point at 19p13.3 (MLLT1) is typical of childhood AML [232]. [103]. There are several other balanced and unbalanced rear-
t(6;11)(q27;q23) which involves the MLLT4 and MLL genes, rangements involving these two regions, including 1p36,
respectively, is at times difficult to identify and often has been 2p15, 3p12, 3p24, 3q23, 5q31.2, 5q34, 7q21, 8q24, 11p15,
erroneously identified as del(11q) [233]. 12p13, 12q21, 17q22, 18q11, and 21q22.3 [104–106]. Some
Two recurring translocations involving chromosomes 10 of these rearrangements are also common in therapy-related
and 11 have been observed. The most common involves the MDS/AML. Rearrangements of 3q21.3 and 3q26.2, particu-
MLLT10 gene at 10p12.3. The fusion of this gene with MLL larly t(3;21)(q26.2;q22.3), are also seen as additional abnor-
is often the result of an inverted insertion of a variant seg- malities during progression of CML to accelerated phase and
ment of chromosome 11 containing the 3¢ portion of MLL blast crisis. The most common additional abnormalities that
into the short arm of chromosome 10 rather than a reciprocal accompany cases with rearrangements of 3q21.3 and 3q26.2
translocation [234]. The other is t(10;11)(q21.3;q23), which are −7 and, less frequently, del(5q) [237].
fuses TET1 with MLL.
Due to the cryptic nature of some of these translocations, Megakaryoblastic AML with t(1;22)(p13.3;q13.1)
it is always good practice to perform FISH to look for Acute megakaryoblastic leukemia (AMKL; FAB M7) is a
an MLL rearrangement when the karyotype appears to be clonal stem cell neoplasm that makes up about 3–15% of all
normal. See also Chap. 17, Fig. 17.8. AML cases [238]. This leukemia is seen mostly in children,
In addition to de novo AML, MLL translocations have with the median age at presentation between 1 and 8 years
also been reported in therapy-related MDS/AML [79] [238, 239]. The incidence of developing this subtype of
(Fig. 15.9). AML is much higher in children with Down syndrome (DS)
than in children without DS [240]. Interestingly, DS children
AML with t(6;9)(p23;q34.1) generally have a more favorable prognosis compared to
This somewhat rare translocation results in the fusion of patients without constitutional +21 [240].
DEK at 6p23 with NUP214 at 9q34.1. t(6;9) is probably the Three entities of AMKL have been described. The first
abnormality most frequently associated with basophilia and subtype is observed in Down syndrome (DS) children and is
had been seen in both pediatric and adult patients [235]. In characterized by mutation of GATA1 and also by t(1;22)
most of the cases, this abnormality is present as the only (p13.3;q13.1), leading to a fusion of RBM15 at 1p13.3 with
change, but it can also be seen in a complex karyotype, par- MKL1 at 22q13.1 [241]. GATA1 mutations are rare in non-DS
326 A. Meloni-Ehrig
AMKL [242]. The few non-DS cases with GATA1 mutation mutations should be provided for patients characterized by
are characterized by the presence of an acquired trisomy 21 one of these chromosome abnormalities.
in the karyotype [242]. This is intriguing and raises the pos-
sibility that GATA1 mutation might be dependent on the Acute Myeloid Leukemia with Myelodysplasia-Related
presence of an additional copy of chromosomes 21. The sec- Changes
ond subtype is observed in about 20% of infants with Down As the name implies, these are myeloid leukemias character-
syndrome and transient myeloproliferative disease (TMD) ized by abnormalities typically seen in MDS, specifically −5/
who subsequently develop AMKL [240]. GATA1 is likely to del(5q), −7/del(7q), and +8, as well as translocations involv-
play a critical role in the etiology of TMD and mutation of ing 3q21.3, 3q26.2, and 11q23. The majority of these AML
this gene represents a very early event in the development of have complex karyotypes similar to what has been reported
AMKL. The karyotype of these DS patients typically con- in high-grade MDS and tMDS/tAML. However, according
tains additional copies of chromosome 21 (four or more cop- to the WHO, this group should not include patients that have
ies of chromosome 21 can be seen), as well as gain of a prior history of cytotoxic or radiation therapy [246].
chromosome 8. The third subtype of AMKL is found in
infants that show the t(1;22) but do not have Down syndrome Myeloid Sarcoma
[241, 242]. Detection of the t(1;22) is diagnostic in this Myeloid sarcoma or granulocytic sarcoma is the name
group. The prognosis associated with the t(1;22) used to be given to a myeloid leukemic process that forms a mass at an
considered unfavorable but is now considered intermediate anatomical site outside of the bone marrow; the term
since these patients are responsive to AML therapy and extramedullary myeloid tumor is therefore also used to
exhibit long clinical remission times. describe this leukemia [247]. Other terms used include
In adults, this leukemia is often secondary in nature, either granulocytic sarcoma and chloroma. This leukemia occurs
posttreatment or during leukemic transformation [239]. at any age and affects males more than females. It can arise
Approximately 50% of adult patients have chromosome de novo, represent a relapse of a known leukemia, or occur
abnormalities at diagnosis. The most common rearrangements as a transformation of a chronic myeloproliferative neo-
involve the regions 3q21.3 and 3q26.2. In addition, frequently, plasm or myelodysplastic syndrome.
the abnormal karyotype includes −5/del(5q), −7/del(7q), and Several cytogenetic abnormalities have been observed in
+8 [238]. t(1;22) has not been observed in adults. myeloid sarcoma. The most common are −7, +8, del(5q),
del(20q), +4, +11, del(16q), inv(16)/t(16;16), MLL rear-
Acute Myeloid Leukemia with Gene Mutations rangements, and t(8;21)(q22;q22.3) [248–250]. The prog-
This new category of AML is characterized by a normal nosis is variable as it is influenced by several factors
karyotype and recurrent gene mutations involving genes including but not limited to age, morphology, and cytoge-
such as nucleophosmin (NPM1) located at 5q35.1, fms-like netic abnormality.
tyrosine kinase 3 (FLT3) located at 13q12.2, CCAAT/
enhancer-binding protein-a(alpha) (CEBPA) located at Blastic Plasmacytoid Dendritic Cell Neoplasm
19q13.1, and mixed lineage leukemia (MLL) located at Blastic plasmacytoid dendritic cell neoplasm is a very aggres-
11q23 [243, 244]. Specifically, 45–60% patients have muta- sive leukemia derived from the plasmacytoid monocytes and
tions involving NPM1, 20–35% patients show mutations of usually involves the skin, bone marrow, and peripheral blood.
FLT3, 10–20% have CEBPA mutations, and 5–25% have This neoplasm is best known as blastic natural killer lym-
MLL tandem duplications. Some of these mutations are not phoma [251]. The median survival is around 12 months.
mutually exclusive; most notably, NPM1 and FLT3 might be Patients are typically in their sixth decade of life at presenta-
present at the same time. The prognosis is variable and tion. Around 20% of cases transform into acute myeloid leu-
depends on which gene is mutated. Patients with NPM1 or kemia, preferentially acute myelomonocytic leukemia. The
biallelic CEBPA mutation alone have a favorable prognosis, majority of cases have an abnormal karyotype that is usually
whereas the presence of FLT3 or MLL mutations is associ- complex. The most common abnormalities include del(5q),
ated with an unfavorable prognostic outcome [245]. del(12p), del(13q), del(6q), del(15q), del(4q), del(9p), and
Therefore, patients with both FLT3 and NPM1 mutations del(9q) [252].
have an adverse prognosis. Other less frequent mutations
observed in AML with normal or abnormal karyotype involve Acute Leukemia of Ambiguous Lineage
KIT, WT1, KRAS, and NRAS. Of note, KIT is also associated This group of neoplasms includes acute undifferentiated leu-
with abnormalities involving the core binding factor genes, kemia (AUL) and mixed phenotype acute leukemia (MPAL)
such as t(8;21)(q22;q22.3) and inv(16)(p13.1q22.1)/t(16;16) that have not differentiated into a particular lineage or
(p13.1;q22.1) [214]. Since KIT mutations affect the clinical expressed cell surface markers of more than one lineage,
course, a suggestion to investigate for the presence of KIT respectively [253]. In other words, AUL blasts express nei-
ther lymphoid nor myeloid markers, whereas MPAL blasts Acute Lymphoid Neoplasms
express markers of different lineages. Although no specific
or recurrent abnormalities are observed in AUL, MPAL is Acute B-cell Lymphoblastic Leukemia/Lymphoma
characterized by two recurrent abnormalities, t(9;22) This neoplasm is defined as leukemia when it involves the bone
(q34;q11.2) and 11q32 (MLL) rearrangements [254]. The marrow and peripheral blood and as lymphoma when it pres-
prognosis associated with these leukemias is poor. However, ents as a lesion without evidence of bone marrow and periph-
patients characterized by the BCR-ABL1 fusion are expected eral blood involvement [256]. There is often extramedullary
to have a better course due to response to imatinib. involvement, particularly of the central nervous system,
lymph nodes, spleen, liver, and testis in cases of B-ALL and of
skin, soft tissue, bone, and lymph nodes in cases of LBL.
Lymphoid Neoplasms A large percentage of ALL cases, especially those involving
children, are classified as precursor B-cell ALL (pre-B-ALL).
This group of hematologic neoplasms includes immature Several factors impact the prognosis. Approximately 85%
and mature neoplasms of B-cell, T-cell, and natural killer of B-ALL patients are children [257]. In general, older age
(NK) cell subtypes. Neoplasms of B-cell origin are more fre- (³10 years) and high WBC are factors associated with high-
quent than those of T-cell origin [255]. Immature B-cell neo- risk B-ALL, compared with younger age and low WBC,
plasms include precursor B-cell lymphoblastic leukemia/ which are associated with low-risk disease [256].
lymphoma (pre-B-ALL/LBL) and precursor T-cell lympho- Chromosome abnormalities have been reported in the major-
blastic leukemia/lymphoma (pre-T-ALL/LBL) [256]. The ity of cases and are useful for prognostic stratification [258,
yearly incidence of these immature neoplasms is estimated 259]; conventional metaphase cytogenetics is still considered
to be 1–4.75/100,000 individuals worldwide. They are by far the basic method for the detection of these abnormalities.
more common in children than adults. Approximately 85% Pediatric cases with t(9;22)(q34;q11.2), 11q23 (MLL) rear-
are of B-cell origin and present as ALL, whereas precur- rangements, t(1;19)(q23.3;p13.3), and hypodiploidy (£45
sor T-cell lymphoblastic neoplasms present mostly as lym- chromosomes) are known to have an unfavorable prognosis,
phoma and affect mainly adolescent males. whereas t(12;21)(p13.2;q22.3) and hyperdiploidy (>50
Disorders of mature cells make up 90% of all lymphoid chromosomes) are associated with a favorable prognostic
neoplasms [255]. These lymphomas are more frequent in outcome particularly if trisomies 4 and 10 are present in the
developed countries with 33 cases/100,000 individuals diag- latter [258] (see Table 15.10). Cytogenetic and FISH analyses
nosed each year. are indicated for proper risk stratification.
The majority of lymphoid neoplasms (both precursor and Children and young adults (generally up to 21 years of
mature types) are characterized by recurrent chromosome age) enrolled in the Children’s Oncology Group (COG)
abnormalities. Some of the most common subtypes are dis- program are required to have their bone marrow or informa-
cussed as follows. tive peripheral blood sample analyzed by conventional cyto-
genetics and FISH [260]. The latter is mainly geared toward
Table 15.10 Recurrent chromosome abnormalities and involved genes in B-ALL

Cytogenetic abnormality Gene(s) involved Common additional abnormalities Prognosis % of patients
t(9;22)(q34;q11.2) ABL1-BCR +der(22)t(9;22), –7 High risk 2.5% children
25% adults
t(12;21)(p13.2;q22.3) ETV6-RUNX1 del(6q), del(11q), 12p rearrangements, Low risk 30% children
del(16q), +21 Absent in adults
Hyperdiploidy (³50 Dosage Rare structural rearrangements Low risk 25% children
chromosomes) 5% adults
Hypodiploidy (£45 Dosage Few structural rearrangements High risk 2% children and teenagers
chromosomes)
t(1;19)(q23.3;p13.3) PBX1-TCF3 dup(1q), del(6q), +8, i(9q), i(17q), +21 High risk Children: 25% pre-B-ALL
and 5% B-ALL
Adults: 3% pre-B-ALL
del(9)(p21.3) CDKN2A del(6q), del(12p) Undetermined 10% children and adults
RUNX1 amplification RUNX1 Generally none High risk 5% children
2% adults
11q23 rearrangements, including MLL Generally none High risk 80% infants
partial deletions and duplications 10% children and adults
328 A. Meloni-Ehrig
the detection of prognostic markers such as BCR-ABL1 Chromosome abnormalities in addition to the Ph chromosome
and ETV6-RUNX1 fusions, MLL rearrangements (including are seen in greater than 60% of patients and are similar to those
partial deletions/duplications), as well as trisomies 4 and 10. observed in CML during progression to accelerated phase or
Some of these recurrent abnormalities are discussed at length blast crisis, specifically +8 and one extra copy of the Ph chro-
as follows. Other FISH probes are available to detect and/or mosome [266]. However, i(17)(q10) is seen primarily in CML,
clarify less common, atypical, or prognostically less infor- while −7, +X, and del(9p) are seen primarily in B-ALL [267]
mative chromosome abnormalities. See also Chap. 17. (Fig. 15.10). While additional abnormalities are associated
with disease progression in CML, they do not appear to modify
The Philadelphia Chromosome the disease’s course in B-ALL. However, patients with loss of
The Philadelphia (Ph) chromosome derived from the t(9;22) chromosome 7 seem to have a much worse prognosis than
(q34;q11.2) occurs in approximately 2.5% of children and patients without this abnormality, probably due to its associa-
approximately 25% of adults with B-ALL [261]. At the tion with resistance to therapy [268, 269].
molecular level, the breakpoints in B-ALL and CML differ,
and this variation leads to the production of p190 and p210 MLL Rearrangements
fusion proteins, respectively. Approximately 20% of Rearrangements involving MLL at 11q23 have been reported
Ph-positive B-ALL patients, however, have been found to in infants, children, and adults with B-ALL [228, 268, 269].
generate both the p190 and p210 fusion transcripts, possibly They have been observed in approximately 80% of infants
as a result of alternative splicing or missplicing events in the (<1 year old) and 5–10% of children and adults with B-ALL.
BCR gene [262, 263]. Alternatives to the typical transloca- In children, the cells have a pre-B immunophenotype that
tion include insertions of ABL1 into the BCR locus and vice express myeloid antigens and are CD19+/CD10− by flow
versa to form the BCR-ABL1 fusion [264, 265]. Some of cytometry. The CD10− immunophenotype, high WBC, and
these variants and most of the insertions will not result in a young age are helpful clues suggestive of the presence of an
classic Ph chromosome with conventional chromosome MLL rearrangement.
analysis. However, the presence of the gene fusion will be The most frequent MLL translocations include t(4;11)
revealed by FISH and/or PCR testing. (q21.3;q23) leading to MLL-AFF1 fusion and t(11;19)
Fig. 15.10 Karyogram of a patient with B-ALL. The t(9;22)(q34;q11.2) is rarely the sole abnormality in B-ALL. In this case, there is also loss
of one copy of chromosome 7
Fig. 15.11 Partial karyograms illustrating some of the most recurrent MLL (11q23) translocations in B-ALL. These are presented in order of
frequency. The majority of patients with MLL rearrangements are infants
(q23;p13.3) leading to MLL-MLLT1 fusion [15, 270, 271]. sion has been reported in approximately 5% of pediatric
Occasionally, t(9;11)(p22;q23) involving MLLT3 and MLL B-ALL cases [274].
or other less frequent translocations might be seen
(Fig. 15.11). MLL rearrangements are associated with an Hypodiploidy
unfavorable prognostic outcome in both children (particu- Hypodiploidy is associated with an unfavorable prognosis.
larly infants) and adults, and bone marrow transplant is still Fortunately, only 2% of pediatric patients with B-ALL and
the treatment of choice. rare cases of adult patients are found to have a hypodiploid
MLL rearrangements have also been reported in T-ALL chromosome complement [278–280].
[272]. Even though these rearrangements are uncommon, Three separate groups have been observed. The most
recent studies indicate that MLL rearrangements are one of common is the near-haploid karyotype, with a chromo-
the early leukemogenic hits in T-ALL [273]. some count ranging from 26 to 29 [281]. The loss and
retention of chromosomes in this group is not random. In
t(1;19)(q23.3;p13.3) fact, invariably, the karyotypes with 26 chromosomes
Approximately 5% of children with pre-B-ALL and 5% of retain two copies of chromosomes 14, 21, and the sex
children with B-ALL have a t(1;19)(q23.3;p13.3), which chromosomes, with a single copy of all other chromo-
leads to a fusion of TCF3 located at 19p13,3 with PBX1 at somes. The chromosomes that are preferentially lost
1q23.3 [274]. This translocation is rare (3% of cases) in include chromosomes 3, 7, 15, and 17 [281]. Therefore,
adults, who also present with a pre-B immunophenotype. the investigation of hypodiploidy by FISH should target
The majority of patients (75% of cases) have an unbalanced regions on the preferentially lost chromosomes. The sec-
form of the translocation, der(19)t(1;19)(q23.3;p13.3), ond and third groups include karyotypes with a chromo-
whereas 25% have the balanced t(1;19). Whether the unbal- some count ranging from 30 to 39 and 40 to 44, respectively.
anced form in pediatric B-ALL patients is associated with a Generally, a lower number of chromosomes correspond to
better prognostic outcome than the balanced t(1;19) is still a worse prognosis. A peculiarity of hypodiploid karyo-
controversial [275]. A variant form of t(1;19) is t(17;19) types is their tendency to double the chromosome comple-
(q22;p13.3), which leads to a fusion between the HLF and ment via endoreduplication. This is not a culture-induced
TCF3 genes located at 17q22 and 19p13.3, respectively, (in vitro) doubling, but rather it occurs in vivo, as demon-
and has been observed in approximately 1% of pediatric strated by DNA index studies of uncultured specimens
B-ALL patients. This variant translocation is associated [282] (Fig. 15.12a, b). These studies have shown the pres-
with poor prognostic outcome [276]. Both t(1;19) and ence of three distinct populations: a hypodiploid comple-
t(17;19) are easily identifiable with conventional cytoge- ment with DNA index below 1, a diploid complement with
netics. However, since cells with these abnormalities are DNA index equal to 1, and a hyperdiploid complement
characterized by low mitotic activity in culture, their detec- with DNA index above 1 [283].
tion might require the analysis of more than the 20 meta- From the DNA index, it is possible to estimate the num-
phase cells typically examined. Probes that target both ber of chromosomes present in the karyotype by using the
TCF3 (break-apart probe) and the actual TCT3-PBX1 fusion simple mathematical formula 46 × DNA index = number of
are available for initial detection and monitoring [277]. chromosomes in the karyotype. The reason(s) that cells
These FISH studies have also proven useful to detect cryp- with a hypodiploid complement endoreduplicate is not
tic rearrangements such as inv(19)(p13.3q13.4), which clear. Some authors have suggested that hypodiploid cells
leads to a fusion of the TCF3 and TFPT genes. This inver- are unstable and doubling their complement gives them the
330 A. Meloni-Ehrig
Fig. 15.12 Karyograms of a patient with B-ALL showing (a) a of the most basic doubling, from which it is easy to suspect the presence
near-haploid karyotype and (b) its doubling version. Note the reten- of the hypodiploid counterpart
tion of chromosomes 14, 21, and the sex chromosomes. This is the one
ability to survive longer. This might be the reason why, A relatively young age (2–10 years) is associated with a favor-
with conventional cytogenetics, more cells with the dou- able prognosis; children also tend to present with favorable
bled complement than with the hypodiploid complement features such as low WBC and a pre-B immunophenotype.
are observed. It is imperative to distinguish a doubling from Modal chromosome numbers between 51 and 55 are thought
a true hyperdiploid clone since they are associated with dif- to be associated with a relatively less favorable prognosis than
ferent prognostic outcomes. Molecular testing, such as those from 56 to 68 chromosomes [259]. The better prognosis
microsatellite markers and SNP array, could be useful to of the latter seems to correlate with the presence of trisomies 4
distinguish true hyperdiploid from an endoreduplicated and 10. The most common gains involve chromosomes 4, 6, 8,
hypodiploid cell population. 10, 14, 17, 18, 19, and 21 (Fig. 15.13). Gain of chromosome
21 (often tetrasomy 21) is the most common numerical abnor-
Hyperdiploidy mality in B-ALL, present in more than 95% of cases with
True hyperdiploidy (51–68 chromosomes) occurs in approxi- hyperdiploidy. This is followed by gains of chromosomes 6
mately 25% of children and 5% of adults with B-ALL [282]. (85% of cases, especially in adults). X and 14 (80% of cases),
Fig. 15.13 Hyperdiploid karyogram of a patient with B-ALL. Chromosomes X, 4, 10, 14, 17, 18, and 21 are usually overrepresented. The best
prognosis is associated with the presence of trisomies 4 and 10, as seen here
17 and 18 (70% of cases), and 10 (55% of cases). For reasons After the first detection of the ETV6-RUNX1 fusion by
that are not completely clear, the prognostic outcome of adult FISH, a large number of studies demonstrated that the
B-ALL patients with hyperdiploidy is not as favorable as t(12;21) is rarely the only abnormality present. Additional
in children. abnormalities include del(6q), del(11q), rearrangements
High hyperdiploidy is sometimes associated with the pres- of 12p, and del(16q), and often these abnormalities pro-
ence of poor prognostic markers such as t(9;22) and t(1;19) vide a clue that a t(12;21) might be present [287, 288]
[284]. Another hyperdiploid group with 47–50 chromosomes (Fig. 15.14).
has been described in approximately 10–15% of children and Variant ETV6-RUNX1 fusion patterns can be seen, as
2–5% of adults with B-ALL [259]. Trisomy 21 is again the demonstrated by FISH studies. The most common of these
most common numerical abnormality. Furthermore, patients variants is loss of the native ETV6 allele, generally subse-
in this group often also exhibit structural chromosome quent to a translocation or other rearrangement involving
rearrangements [285]. 12p [289]. Molecular studies have demonstrated that fusion
with ETV6 converts RUNX1 from an activator to a repressor
t(12;21)(p13.2;q22.3) of transcription [290]. Molecular studies have also demon-
Prior to the discovery of the cryptic t(12;21), which fuses strated that the presence of t(12;21) occurs early and is most
ETV6 at 12p13.2 with RUNX1 at 21q22.3, at least 30% of likely present in utero. Using PCR, researchers confirmed
B-ALL cases were thought to have a normal or prognosti- the presence of t(12;21) in cord blood and perinatally
cally informative karyotype [286]. This translocation is most obtained blood samples (Guthrie cards) of patients who later
often seen in children between 2 and 12 years old, with a developed t(12;21)-associated ALL. Furthermore, these
peak at 3–5 years of age. These children have disease that is studies demonstrated the presence of both the typical and
characterized by a long duration of first remission and excel- several variant ETV6-RUNX1 fusion patterns at levels higher
lent cure rates. t(12;21) is rare in adults with an incidence of than what is seen in overt B-ALL [291, 292]. This implies
2–5% of cases. that the t(12;21)-carrying cells present in those early samples
332 A. Meloni-Ehrig
Fig. 15.14 Karyogram of a patient with B-ALL and cryptic t(12;21). Even if the translocation is cryptic at the conventional cytogenetic level,
additional abnormalities, in this case deletions of 6q and 11q (arrows), often serve as a clue
most likely represent clones predisposed to leukemia devel- cytogenetics. Therefore, FISH testing targeting the
opment and that the acquisition of more genetic aberrations CDKN2A gene located at 9p21.3 is very helpful to demon-
is needed for one of these clones to become fully malignant. strate the deletion [296]. Homozygous deletions of this
The usefulness of ETV6-RUNX1 FISH studies goes gene, frequently unsuspected, are also confirmed by FISH.
beyond the ability to merely detect the fusion. Two additional Although the CDKN2A gene is thought to play a key role,
abnormalities in particular can be detected with the ETV6/ other genes located at 9p, such as MLLT3, PAX5, MTAP,
RUNX1 FISH probes—deletions of ETV6 and amplification IFN, JAK2, and PTPLAD2, may play important roles as
of RUNX1. Specifically, a small group of B-ALL patients well [296]. In adults with B-ALL, the presence of del(9p)
with a median age of 9 years have been found to have appears to be associated with improved outcome, whereas
amplification of RUNX1, defined as multiple copies of this in children the same deletion is associated with poor out-
gene clustered in a marker chromosome. In the majority of come [297]. Besides pure deletions, other unbalanced
the cases, this amplification is actually concentrated on an rearrangements include dic(9;20)(p13.2;q11.2), dic(9;12)
abnormal chromosome 21 (iAMP21) [293]. This abnormal- (p13.2;p12.2), and i(9)(q10), all of which are associated
ity is associated with an unfavorable prognosis characterized with an excellent prognostic outcome [298].
by high risk of relapse and a decreased event-free and overall This being a textbook dedicated to cytogenetics, it is
survival at 5 years. important to mention some of the “tricks of the trade.” One
of these involves the apparent loss of chromosome 20 in
Rearrangements of 9p some of the karyotypes of patients with B-ALL. FISH has
Unbalanced rearrangements of the short arm of chromo- shown that the apparent loss of chromosome 20 is actually
some 9, usually leading to loss of 9p, have been observed the result of a dic(9;20)(p13.2;q11.2). This abnormality,
in various neoplasms and are particularly frequent in ALL which results in loss of most of 9p and 20q, might be difficult
of both B- and T-lineages [294, 295]. In B-ALL, these to detect in a sample with poor chromosome morphology,
deletions have been observed in approximately 10% of and the apparent loss of chromosome 20 can provide a
patients and often are not easily detected with conventional helpful hint [299] (Fig. 15.15).
Fig. 15.15 Karyogram of a patient with B-ALL showing apparent loss of chromosome 20. Upon careful review, it is possible to recognize that
the right chromosome 9 is in reality a dic(9;20)(p13.2;q11.2)
Rearrangements of 14q32.3 (IGH@) region, via juxtaposition of this gene to the IGH@ enhancer.
Most of the IGH@ rearrangements observed in B-ALL are This deregulation can also arise via a cryptic interstitial dele-
balanced translocations. The most common are t(8;14) tion within the pseudoautosomal region, that is, del(X)
(q11.2;q32.3), inv(14)(q11.2q32.3), t(14;14)(q11.2;q32.3), (p22.33p22.33)/del(Y)(p11.32p11.32) via juxtaposition of
t(14;19)(q32.3;q13.1), and t(14;20)(q32.3;q13.1) [300–302]. CRLF2 to the P2RY8 promoter also located in the X/Y
These translocations appear to be more frequent in adults pseudoautosomal region. The deregulation of CRLF2 is
(approximately 10% of cases) than in children (approxi- associated with activating mutations in JAK2 [309].
mately 2% of cases) with B-ALL. Therefore, performing FISH with an IGH@ probe is a valu-
These recurrent IGH@ translocations have in common able option, particularly in Down syndrome patients with a
the deregulated expression of unmutated CEBP genes karyotype lacking acquired chromosome abnormalities.
(CCAAT enhancer-binding protein transcription factors)
[303]. A rare t(5;14)(q31.1;q32.3), which leads to an IL3- Acute T-Cell Lymphoblastic Leukemia
IGH@ fusion, has also been observed in B-ALL and is often Approximately 15% of children and 25% of adults diagnosed
associated with eosinophilia [304, 305]. Other reported yearly with ALL have T-cell ALL/LBL [67]. There appears to
IGH@ translocations include t(6;14)(p22.3;q32.3) and be a prevalence of adolescent males (age range 12–19 years),
t(9;14)(p13.2;q32.3), leading to a fusion of IGH@ with ID4 but this is by no means exclusive [310]. Patients with this
and PAX5, respectively [306, 307]. leukemia often present with mediastinal mass, CNS involve-
Rarely, deletions involving IGH@ have been reported. ment, and leukocytosis [256].
Based on the small number of cases reported so far with this The cytogenetic abnormalities most often found in T-ALL
abnormality, the prognosis is unknown [308]. involve the T-cell receptors, specifically TRA@ and TRD@
Two cryptic translocations, t(X;14)(p22.3;q32.3) and at 14q11.2, TRB@ at 7q35, and TRG@ at 7p14 [311]. TRD@
t(Y;14)(p11.3;q32.3), have recently been described in is contained within the TRA@ locus, and thus TRA@ is com-
B-ALL, especially in patients with Down syndrome. These monly used as reference.
translocations lead to overexpression of the cytokine recep- Several translocations involving these genes have been
tor gene, CRLF2, located in the X/Y pseudoautosomal reported; some are cryptic with conventional cytogenetics [312].
334 A. Meloni-Ehrig
Table 15.11 Recurrent chromosome abnormalities and involved genes in T-ALL

Abnormality Genes % Children % Adults
t(1;7)(p32;q34) TAL1-TRB@ 5
t(1;14)(p32;q11.2) TAL1-TRA@ 10
t(4;11)(q22.3;p15.4) RAP1GDS1- NUP98 2–5
t(5;14)(q35.1;q32.2) (cryptic) TLX3-BCL11B 20 10–20
del(6q) Unknown 10–20 5–10
inv(7)(p15.2q34) or t(7;7) HOXA10-TRB@ 1–2 2
t(7;9)(q34;q31.2) TRB@-TAL2 Rare
t(7;9)(q34;34.3) TRB@-NOTCH1 Rare
t(7;10)(q34;q24.3) TRB@-TLX1 7 30
t(7;11)(q34;p13) TRB@-LMO2 5–10
t(7;12)(q34;p13.3) TRB@-CCND2 3–5
t(7;14)(q34;q32.1) TRB@-TCL1A Rare
t(7;19)(q34;p13.2) TRB@-LYL1 Rare
t(8;14)(q24.2;q11.2) MYC-TRA@ 2
del(9)(p21.3) (homozygous/hemizygous) CDK2NA 30 3
80 by FISH 8–10
t(9;12)(p24.1;p13.1) JAK2-ETV6 Rare
t(10;14)(q24.3;q11.2) TLX1-TRD@ 5–10
t(11;14)(p13;q11.2) LM02-TRA@ 5–10
t(11;14)(p15.4;q11.2) LMO1-TRA@ Rare
t(12;14)(p13.3;q11.2) CCND2-TRA@ 2–5
12p rearrangements ETV6, others 10–15 5
inv(14)(q11.2q32.1) or t(14;14)(q11.2;q32.1) TRA@-TCL1A Rare Rare
Except for the Y chromosome, virtually every chromosome has chromosome 9 homologs. This deletion is less frequent
been involved with one or more of these T-cell receptors. The (8–10%) in adults with T-ALL.
most common translocations involve chromosomes 1, 7, 9, 10, A relatively new abnormality, discovered by chance while
11, 12, and 14 [313] (see Table 15.11). investigating the frequency of BCR-ABL1 fusion in T-ALL,
A rare but recurrent abnormality seen in T-ALL is is the amplification of ABL1 [318, 319]. The rearrangement
inv(14)(q11.2q32.1) or t(14;14)(q11.2;q32.1). Either of is a cryptic episomal NUP214-ABL1 translocation and occurs
these rearrangements generally leads to overexpression of in approximately 6% of patients, most of whom are children.
TCL1A/TCL1B at 14q32.1 via relocation to the TRA@/ FISH with the BCR-ABL1 probes and RT-PCR can both
TRD@ locus at 14q11.2. TCL1A and TCL1B have approxi- detect T-ALL patients with ABL1 amplification. The quick
mately 65% homology, and therefore overexpression of one identification of this rearrangement is fundamental in the
affects the other [314]. Other genes in the vicinity of clinic because this T-ALL subset is Gleevec® sensitive but
TCL1A/TCL1B, such as BCL11B at 4q32.3, can also be may become resistant due to the development of additional
involved, and thus overexpressed, as a result of this inver- mutations (9p21.3 deletions often accompany this amplification)
sion [315]. The prognosis associated with this abnormality [320]. ABL1 quantitative RT-PCR may be easily applied to
is unknown. monitor minimal residual disease.
Another frequent abnormality is deletion of 9p21.3 lead-
ing to loss of CDKN2A. This gene encodes the p14ARF
protein, which binds to and inactivates HDM-2. HDM-2 in Mature B-Cell Neoplasms
turn targets the TP53 tumor suppressor protein for degrada-
tion [316]. Therefore, deletions of 9p21.3 result in reduc- Culturing of Mature B-Cell Neoplasms for
tion in the amount of p14ARF, loss of inhibition of HDM-2, Cytogenetic Analysis
and subsequent inhibition of TP53 protein production. This The detection of chromosome abnormalities in mature B-cell
deletion can be seen in about 30% of cases by conventional lymphomas by conventional cytogenetics is dependent upon
cytogenetics and about 80% of cases with FISH [317]. This the culturing method used. Historically, the utilization of
indicates that the majority of deletions involving 9p are B-cell mitogens has proven effective in promoting the growth
cryptic. Approximately 50% of these deletions affect both of the abnormal clonal population in culture [321, 322].
Table 15.12 Neoplasms stimulated with DSP-30/IL-2 and associated Most NHL cases are of B-cell origin and are characterized
cytogenetic abnormality rates. A. Meloni-Ehrig, personal data by rearrangements involving the immunoglobulin genes:
Number of Abnormal Normal IGH@ at 14q32.3, IGK@ at 2p12, and IGL@ at 22q11.2
Diagnosisa cases cases cases % abnormal [321]. Translocations can be simple or complex and at times
v-CLL 14 14 0 100 have partial deletions or partial duplications of the genes
HCL 13 13 0 100 involved in the translocation. By far, the majority of translo-
b-MCL 8 8 0 100
cations involve IGH@ [36, 37].
B-PLL 2 2 0 100
The most common associations between chromo-
sMZBCL 29 28 1 97
some anomalies and specific lymphomas include t(14;18)
DLBCL 36 32 4 89
CLL 430 367 63 85
(q32.3;q21.3) and follicular lymphoma (FL), t(8;14)
LPL 10 8 2 80 (q24.2;q32.3) and Burkitt lymphoma (BL), t(11;14)
MCL 19 15 4 78 (q13;q32.3) and mantle cell lymphoma (MCL), and
BL 6 4 2 66 t(11;18)(q21;q21.3) and mucosa-associated lymphoid
MALT 14 9 5 64 tissue (MALT) lymphoma [37].
HL 3 1 2 33
FL 35 9 26 26 Follicular Lymphoma
NHLb 34 1 33 3 Follicular lymphoma (FL) affects 1 in 24,000 individuals per
LPDb 12 0 12 0 year in the USA [322]. The majority of patients have an
Totals 665 511 154 77 indolent disease, and few develop a more aggressive form
Abbreviations: BL Burkitt lymphoma, CLL chronic lymphocytic leukemia, [326]. Approximately 85–90% of patients with FL and
v-CLL variant type, DLBCL diffuse large B-cell lymphoma, FL follicu- 25–30% of patients with diffuse large B-cell lymphoma
lar lymphoma, HCL hairy cell leukemia, HL Hodgkin lymphoma, LPD
lymphoproliferative disorder, MCL mantle cell lymphoma, b-MCL
(DLBCL) exhibit the t(14;18)(q32.3;q21.3), which results in
mantle cell lymphoma, blastoid type, MALT mucosa-associated fusion of BCL2 at 18q21.3 and IGH@ at14q32.3 [327]
lymphoid tissue, sMZBCL marginal zone B-cell lymphoma, splenic (Fig. 15.16a). This translocation, which is one of the most
type, NHL non-Hodgkin lymphoma, B-PLL B-cell prolymphocytic leu- common abnormalities in NHL, repositions the BCL2 onco-
kemia, LPL lymphoplasmacytic lymphoma
a
Defined according to immunophenotypic and/or morphologic analyses
gene so that it is under the control of IGH@ promoter, lead-
b
No specific subtype ing to overexpression of BCL2 and therefore overproduction
of BCL2, one of the proteins involved in regulation of apop-
The recent introduction of CpG-oligodeoxynucleotides tosis [52]. Variant translocations, such as t(2;18)(p12;q21.3)
(CpG-ODNs) has increased the detection of clonal abnor- and t(18;22)(q21.3;q11.2) involving IGK@ at 2p12 or IGL@
malities. CpG-ODNs are made of short single-stranded at 22q11.2, respectively, have been described in both FL and
DNA, are known to activate cells of the immune system in a DLBCL [328]. These translocations also lead to the overex-
sequence-dependent manner, and are also known to promote pression of BCL2.
proliferation of chronic lymphocytic leukemia (CLL) cells Numerous additional chromosome abnormalities are
[323]. One of these CpG-ODNs is DSP-30. In combination identified by conventional cytogenetics. In addition to
with interleukin-2 (IL-2), DSP-30 has proven to be effective t(14;18), certain numerical abnormalities, speci fi cally
in increasing the detection of chromosome abnormalities trisomies 2, 7, and/or 8, are associated with a more favor-
with conventional cytogenetics when compared to other tra- able course of disease when compared with patients
ditional well-established B-cell mitogens, not only in CLL with structural abnormalities, specifically del(1p), del(1q),
but also in other mature B-cell lymphoid neoplasms [8, 324, del(6q), +der(18), or del(22q), or gain of an X chromosome
325]. Table 15.12 shows the author’s experience using the or chromosome12, which are associated with an unfavor-
DSP-30/IL-2 cocktail. Table 15.13 lists recurrent chromo- able outcome [29, 329]. Progression of FL to DLBCL
some abnormalities in B-cell neoplasms. occurs in 60–80% of cases and is accompanied by accu-
mulation of secondary abnormalities, including +7,
Non-Hodgkin Lymphoma del(10q), del(6q), and/or +der(18) [330] (Fig. 15.16b).
Non-Hodgkin lymphoma (NHL) comprises a heterogeneous The lifespan of the FL cells is very short; therefore,
group of disorders characterized by localized proliferation of longer transit times and longer culture times should be
lymphocytes. The WHO recognizes that genetic anomalies avoided whenever possible. B-cell mitogens do not appear
represent one of the most reliable criteria for classification of to promote the mitotic activity of the clonal population
malignant lymphomas [67]. Some tend to be confined to a par- and more success is obtained with overnight cultures. In
ticular lymphoma—for example, t(14;18)(q32.3;q21.3) in fol- contrast to what is seen in adults, neither the t(14;18) or
licular lymphoma—whereas others are nonspecific and can be variant translocations nor overexpression of BCL2 is
seen in a variety of lymphomas, such as del(6q) and del(14q). observed in children with FL, who are found to show
336 A. Meloni-Ehrig
Table 15.13 Mature B-Cell lymphoid neoplasms and associated recurrent chromosome abnormalities, according to the World Health
Organization
Mature B-cell neoplasm Primary chromosome abnormalities Chromosome abnormalities during progression
CLL/SLL del(6q), del(11q), del(13q), +12, del(17p), t(2;14), t(14;19), del(14q),+18, t(8;14) or variants, 13q rearrangements
t(14;18),
PLL del(6q). del(11q), +12, del(13q), del(17p) MYC translocations
MCL t(11;14) and other CCND1 variants; t(6;14) and CCND3 Gains of chromosomes 3, 8, and 15q; losses of 1p,
variants; t(12;14) and CCND2 variants 8p, 9p, 11q, and 13q.
Splenic MZBCL del(7q); +3, +12, +18 del(17p)
Nodal MZBCL +12, +18, 3q27 rearrangements del(17p)
MZBCL (MALT type) t(11;18) or t(14;18); +3 with or without +18; t(1;14) and del(17p)
variants; (3;14)
HCL +5, del(6q), del(7q), +12, del(17p) Variable
LPL del(6q), +4, +3, +7 del(17p)
FL t(14;18) and variants Gains of chromosomes X, 2, 7, 8, and 12; del(1p),
del(1q), del(6q), del(10q), +der(18), del(22q)
DLBCL t(14;18), 3q27 rearrangements 1q and 14q rearrangements, del(6q), del(10q),
del(11q), del(13q), del(17p), +X, +7, +12, +18
BL t(8;14) and variants Gain of 1q
PCM High risk: hypodiploidy, 1p/1q rearrangements, del(13q), del(4q), del(6q), del(16q), del(20q), MYC
t(4;14), t(14;16), t(14;20), del(17p) translocations
Standard risk: hyperdiploidy with gain of odd number
chromosomes (+5, +9, +11, +15), without high-risk
markers; t(11;14)
PBL Similar to high-risk PCM: hyperdiploidy with gain of odd MYC translocations
number chromosomes (+5, +9, +11, +15) and high-risk
markers [rearrangements of chromosome 1, del(13q),
del(17p)]
Unclassifiable—DLBCL/ t(8;14) and variants, t(14;18) and/or 3q27 rearrangements, Same as DLBCL
Burkitt and/or t(11;14)
Unclassifiable—DLBCL/ 3q27 rearrangements, del(17p) Variable
Hodgkin
HL Hyperdiploidy, del(1p), del(6q), del(7q), del(13q), del(16q), Variable
del(17p), gain of 2p, 9p, +12, rearrangements of 3q27
Abbreviations: CLL chronic lymphocytic leukemia, SLL small lymphocytic lymphoma, PLL prolymphocytic leukemia, MCL mantle cell
lymphoma, MZBCL marginal zone B-cell lymphoma, MALT mucosa-associated lymphoid tissue, HCL hairy cell leukemia, LPL lymphoplasma-
cytic lymphoma, FL follicular lymphoma, DLBCL diffuse large B-cell lymphoma, BL Burkitt lymphoma, PCM plasma cell myeloma, PBL
plasmablastic lymphoma, HL Hodgkin lymphoma
variable rearrangements of 14q32.3 (IGH@) and 3q27 overexpression of MYC. In the majority of BL cases, the
(BCL6) [331]. reciprocal translocation involving MYC and one of the immu-
noglobulin genes is the sole cytogenetic abnormality.
Burkitt Lymphoma
This lymphoma is named for Dr. Denis Burkitt, who first Diffuse Large B-Cell Lymphoma
described it in 1958 [332]. Burkitt lymphoma (BL) has an Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous
incidence of approximately 1,200 cases per year in the USA group of neoplasms characterized by a diffuse infiltrate of
and affects children as well as adults [333]. large lymphoid cells. Some of these lymphomas are clearly
Three immunoglobulin gene translocations, all affecting defined, while the classification of others remains challenging
MYC at 8q24.2, are seen in BL [334] (Fig. 15.17). The most [67, 335]. In general, these lymphomas affect individuals of
common of these, t(8;14)(q24.2;q32.3), is detected in about increasing age, and their presence in children and young
75–80% of patients. Two variant translocations, t(8;22) adults is uncommon.
(q24.2;q11.2) and t(2;8)(p12;q24.2), are seen in ten and 5% In addition to being heterogeneous at the cellular level,
of patients, respectively. All three translocations involve these neoplasms are also cytogenetically diverse. Among the
MYC and one of the three immunoglobulin genes (IGH@ at recurrent abnormalities is t(14;18)(q32.3;q21.3), which
14q32.3, IGK@ at 2p12, or IGL@ at 22q11.2) and lead to involves IGH@ and BCL2. This translocation is observed in
Fig. 15.16 (a) Karyogram of a patient with follicular lymphoma B-cell lymphoma. Typical abnormalities during progression are the
showing t(14;18)(q32.3;q21.3) as the sole abnormality and (b) together gain of chromosomes X, 7, and the der(18), as seen in this case
with other rearrangements in a patient in progression to diffuse large
25–30% of DLBCL cases [336]. t(14;18) in DLBCL is usu- prognostic outcome [337]. Translocations involving 3q27
ally present in the context of a complex karyotype. Additional (BCL6) have been detected in approximately 35% of patients
abnormalities include rearrangements of 1q and 3q, del(6q), with DLBCL. More than 30 different partner genes have
+7, +8, del(10q), del(11q), +12, del(13q), rearrangements of been translocated with this locus, the most recurrent of which
14q and 17p, +der(18)t(14;18), and + X (Fig. 15.16b). It is include 2p12, 3q29, 4p13, 6p21.2, 6p22, 7p12, 8q24.2,
believed that the more complex the karyotype the worse the 11q23, 13q14, 14q32.3, 15q22, 16p13, 17q11.2, 18p11.2,
338 A. Meloni-Ehrig
Fig. 15.17 Partial karyograms illustrating the three translocations and IGL@ loci, located at 2p12, 14q32.3, and 22q11.2, respectively,
involving MYC (at 8q24.2) in Burkitt lymphoma. In these transloca- leads to its overexpression
tions, the relocation of MYC to the immunoglobulins IGK@, IGH@,
and 22q11.2 [338]. These translocations juxtapose different presence of rearrangements involving MYC, BCL2, and/or
promoters derived from partner chromosomes to the BCL6 BCL6 is strongly suggested. FISH is very helpful in these
coding domain causing overexpression of this oncogene. cases, as rearrangements are often complex and difficult
Since BCL6 functions as a transcriptional repressor of genes to sort out.
containing its binding sites, its overexpression leads to tran-
scription deregulation [339]. Many 3q27 rearrangements are B-Cell Lymphoma, Unclassifiable, with Features
not detectable with conventional cytogenetics. Therefore, Intermediate Between Diffuse Large B-Cell Lymphoma
FISH or PCR are useful to provide evidence of them. The and Hodgkin Lymphoma
prognostic significance of BCL6 rearrangements is not clear, This second category of unclassifiable lymphomas includes
and different outcomes have been reported [340]. DLBCL with features of classic Hodgkin lymphoma (HL)
Other recurrent abnormalities observed in DLBCL include [342]. The diagnosis of these cases is difficult. However, a
partial or complete gain of chromosome 3, specifically 3q; large B-cell lymphoma with a strong expression of CD15, or
loss of chromosome 6; and gain of chromosome 18 and a classic HL with a strong and diffuse expression of CD20
t(14;15)(q32.2;q11.2), which leads to fusion of the BCL8 at and CD79, would support inclusion in this category. Similarly
15q11.2 with IGH@ at 14q32.3. It is thought that among these to what has been observed with DLBCL/BL, the prognosis
abnormalities, only gain of chromosome 3 is associated with of this neoplasm is worse than that of DLBCL or HL. The
an adverse prognosis [341]. genetics of this entity is not well established. However, ear-
lier reports have implicated rearrangements of 3q27 (BCL6)
B-Cell Lymphoma, Unclassifiable, with Features and 17p13.1 (TP53) [346].
Intermediate Between Diffuse Large B-Cell Lymphoma
and Burkitt Lymphoma Mantle Cell Lymphoma
This is a new lymphoma category described by the WHO in Mantle cell lymphoma (MCL) is an aggressive lymphoma
2008 [67]. As its name implies, this lymphoma does not meet that affects primarily adult male patients (median age
the criteria for either classical BL or DLBCL [342]. This neo- 65 years) and represents 5–10% of all lymphoma cases in the
plasm occurs in fewer than 2% of lymphoma patients and is USA [347]. Typical immunophenotypic findings are positiv-
associated with aggressive histology and poor prognosis [343]. ity for CCND1, CD20, CD19, and CD5 and negativity for
These cases have translocations involving MYC with CD10, CD23, and BCL16.
either immunoglobulin or non-immunoglobulin genes; there- With conventional cytogenetics, about 70% of MCL patients
fore, t(8;14), t(2;8), and t(8;22) have been observed. Among exhibit a t(11;14)(q13;q32.3) (Fig. 15.19). This translocation
the translocations involving MYC and non-immunoglobulin relocates the CCND1 gene at 11q13, a gene involved in cell
genes, the most common is t(8;9)(q24.2;p13) [344]. cycle control, to the IGH@ locus on chromosome 14. Similar
The differentiator between typical BL and these to what occurs with other immunoglobulin gene translocations,
unclassifiable lymphomas is the simultaneous presence of t(11;14) leads to overexpression of CCND1 [348].
rearrangements involving MYC, BCL2, and/or BCL6 The use of B-cell mitogens such as DSP-30/IL-2 has
[345]. As such, this entity is also known as double-hit or proven useful to promote the growth of the abnormal lym-
triple-hit lymphoma (Fig. 15.18). FISH to investigate the phocytes in culture [8]. The best method, however, to detect
Fig. 15.18 Karyogram of a patient with the aggressive “double-hit” lymphoma. In this particular case, the complex karyogram shows the simulta-
neous involvement of MYC and BCL2 rearrangements in the form of t(8;14;18)(q24.2;q32.3;q21.3). Arrows point to the abnormal chromosomes
Fig. 15.19 Karyogram of a patient with mantle cell lymphoma at diagnosis. In these cases, t(11;14)(q13;q32.3) is usually the sole abnormality
340 A. Meloni-Ehrig
Fig. 15.20 Partial karyograms illustrating translocations observed less t(6;14)(p21;q32.3) leads to fusion of CCND3 with IGH@. Other com-
frequently in mantle cell lymphoma. The translocations (11;22) binations of these key genes are possible but much less frequent than
(q13;q11.2) and (2;11)(p12;q13) fuse CCND1 with IGL@ and IGK@, those illustrated here
respectively. The t(2;12) (p12;p13) fuses CCND2 with IGK@, and
the presence of t(11;14) is FISH, with a detection rate of up The t(11;14) has been previously associated with other
to 95% [349]. Various archival samples, including touch B-cell lymphomas, including atypical CLL, sMZBCL, and
preparations and non-decalcified paraffin-embedded tissue, B-PLL, and with PCM [356]. However, presently, most MCL
can be used for FISH testing. and some PCM cases are thought to be characterized by the
Variant translocations involving CCND1 and IGK@ t(11;14). The others are now considered variants or leukemic
[t(2;11)(p12;q13)], or CCND1 and IGL@ [t(11;22) variants of MCL [67].
(q13;q11.2)], have been observed in a limited number of t(11;14) is not limited to MCL as it is also found in other
cases, but their detection is equally important for the diagno- B-cell neoplasms, including CLL, sMZBCL, B-PLL, and
sis of MCL [350, 351] (Fig. 15.20). An IGH@-CCND1 FISH PCM [356]. See following sections.
study showing three signals for CCND1 should be evaluated
further if MCL is a concern since the third CCND1 signal Marginal Zone B-Cell Lymphoma
could be the result of a splitting of this region due to a trans- Marginal zone B-cell lymphoma (MZBCL) comprises a
location involving an immunoglobulin gene other than group of indolent NHL arising from the marginal zone of
IGH@. lymphoid tissues and accounts for approximately 10% of
About 5% of patients have been found to be CCND1- NHL [357]. According to the 2008 WHO classification, this
negative because they lack CCND1 expression by IHC and lymphoma is subdivided into three subtypes: splenic (sMZ-
do not exhibit t(11;14)/CCND1-IGH@ by either conven- BCL), nodal, and MALT or extranodal [67]. The splenic and
tional cytogenetics or FISH [351, 352]. Some of these cases nodal subtypes are rare (1% of NHL), whereas MALT is
involve other cyclin genes such as CCND2 at 12p13 and relatively frequent (8% of all NHL).
CCND3 at 6p21, specifically the cryptic t(12;14)(p13;q32;3)
and the t(6;14)(p21;q32.3), respectively (Fig. 15.20). MCL Mucosa-Associated Lymphoid Tissue (MALT) Lymphoma
rarely displays variant translocations involving CCND2 or This particular subtype of MZBCL has received much atten-
CCND3 with either IGK@ or IGL@ [353]. tion particularly because of its association between low-
Furthermore, a novel cryptic t(2;14)(p24;q32.3) grade gastric MALT lymphoma and Helicobacter pylori
involving MYCN and IGH@ has been observed in two infection [358]. There is also evidence that suggests that
patients with blastoid MCL [354]. The blastoid variant of chronic antigenic stimulation in autoimmune diseases, such
MCL, as the term implies, is associated with a very as Hashimoto thyroiditis, contributes to an increased risk of
aggressive clinical course and shorter survival than typi- developing MALT lymphoma [357].
cal MCL [355]. One of the specific aberrations occurring in 50% of MALT
Both CCND1-positive and CCND1-negative patients lymphoma cases is t(11;18)(q21.3;q21.3), which leads to a
might have the same secondary chromosome abnormali- fusion between BIRC3 (API2) at 11q21.3 and MALT1 at
ties, which include partial or complete gain of chromo- 18q21.3 [359]. When present, this translocation is usually
somes 3 and 8, gain of 15q, and losses of 1p, 8p, 9p, 11q, the only chromosome abnormality. Interestingly, it is also the
and 13q. Highly complex chromosome complements, only recurrent translocation in NHL that does not involve an
particularly those with loss of 9p, 17p, and gain of 3q and immunoglobulin gene. The BIRC3-MALT1 fusion is easily
8q, have been described in blastoid variant of MCL [355] identified using a dual-color BIRC3-MALT1 FISH probe or
(Fig. 15.21). the “break-apart” strategy of the dual-color MALT1 probe
Fig. 15.21 Complex karyogram showing various abnormalities in addition to t(11;14)(q13;q32.3). Recurrent abnormalities in these karyograms
include losses of 1p, 8p, 9p, 13q, and 17p, and gain of 3q. Similar karyotypes have been described in blastoid variant of mantle cell lymphoma
(see Chap. 17). The other specific MALT1 translocation is Splenic Marginal Zone B-Cell Lymphoma
t(14;18)(q32.3;q21.3), which relocates the MALT1 gene Splenic marginal zone B-cell lymphoma (sMZBCL) is a rare
from 18q21.3 to the IGH@ locus at 14q32.3. This transloca- neoplasm that typically affects individuals aged greater than
tion has been observed in approximately 2% of MALT lym- 60 years. It represents 1–2% of non-Hodgkin lymphomas.
phoma cases. As the name implies, the primary site of involvement is the
In contrast to BIRC3-MALT1-positive cases, patients spleen, but bone marrow and peripheral blood might be
with IGH@-MALT1 fusion have disease outside the gastro- involved as well [363].
intestinal tract, usually presenting with ocular, skin, liver, Immunophenotypically, this lymphoma is positive for
or salivary gland tumors [360]. With conventional cytoge- CD19, CD20, CD22, and CD79b. The majority of cases also
netics, this translocation is indistinguishable from the lack expression of CD5, CD10, and CD23 [363].
t(14;18) with involvement of BCL2 seen in follicular lym- Cytogenetically, they are characterized by recurrent numeri-
phoma. Therefore, FISH studies are useful to distinguish cal and structural abnormalities [364]. One of the most com-
the two translocations. The combination of the BIRC3- mon structural abnormalities is deletion of 7q (30–40% of
MALT1 dual fusion and IGH@ break-apart probes is most cases). The deletion appears to be concentrated to bands
practical to detect the presence of either of these 7q22 ~ q32 and can be detected with conventional as well as
rearrangements. molecular cytogenetics [365]. Other recurrent chromosome
Two other translocations seen in approximately 5% of the abnormalities include partial or complete trisomy 3, particu-
cases include t(1;14)(p22.3;q32.3) and its variant t(1;2) larly involving the long arm (30–50% of cases), and partial
(p22.3;p12), which relocate BCL10 from 1p22.3 to the or complete trisomy 12 (20–30% of cases) [366, 367]. In
IGH@ locus at 14q32.3 or the IGK@ locus at 2p12, respec- addition to these abnormalities, the karyotype of some
tively [361]. aggressive cases often includes a deletion of 17p which leads
All four translocations described previously are known to to loss of TP53 [368].
activate the nuclear factor (NF)-kB activation pathway [362].
Another translocation seen in rare cases of MALT lymphoma Nodal Marginal Zone B-Cell Lymphoma
is t(3;14)(p13;q32.2), which leads to fusion of the FOXP1 Nodal marginal zone B-cell lymphoma (MZBCL) is a rare
gene on chromosome 3p14.1 with IGH@ resulting in over- low-grade neoplasm comprising 1–2% of all NHL in adults.
expression of FOXP1F [361]. This lymphoma contains a heterogeneous group of cells that
342 A. Meloni-Ehrig
Fig. 15.22 Partial karyograms illustrating recurrent abnormalities observed in CLL, in order of frequency
express CD19, CD20, and CD79a, with variable expression noticeably over the years [377]. According to the WHO, the
of CD138 depending on the degree of plasma cell differen- current incidence rate is estimated to be 2–6 cases per
tiation. Like other MZBCLs, it is negative for CD5, CD10, 100,000 individuals per year, increasing to up to 12.8 cases
and CD23 [369]. Cytogenetically, it is not well defined, but per 100,000 for individuals at age 65 and older [67].
partial or complete trisomy 12 appears to be more common The most important risk factor for the development of
than in sMZBCL with a frequency of 40–50% [369]. CLL is family history. In fact, among patients with newly
diagnosed CLL, 8–10% have one or more blood relatives
Lymphoplasmacytic Lymphoma with this neoplasm [378]. The prognosis of familial and
Lymphoplasmacytic lymphoma (LPL) is an indolent neo- nonfamilial cases is highly variable, with survival varying
plasm characterized by the expansion of small B-lymphocytes from months to several years, and is highly dependent on the
with variable plasmacytoid differentiation that are negative presence of recurrent chromosome abnormalities
for CD5, CD10, and CD23 [370]. LPL represents 5% of all (specifically del(6)(q23.3)/MYB, del(13q)(q14.3), +12,
NHL affecting adult individuals and often is associated with del(11)(q22.1)/ATM, and del(17)(p13.1)/TP53) [379–381]
Waldenström macroglobulinemia (WM). There is also an (Fig. 15.22; see also Chap. 17, Fig. 17.10), and also gene
association between this lymphoma and hepatitis C virus mutations. Other less frequent abnormalities have been
infection [371]. reported in CLL and are discussed at the end of this section.
Although most cases of LPL appear to be sporadic, familial Morphologically, the worst prognosis is associated with the
cases have been reported. A familial history of at least one presence of prolymphocytes and the development of Richter
first-degree relative with either LPL or another B-cell disorder syndrome (RS). RS corresponds to progression of CLL to a
has been observed in approximately 20% of patients [372]. high-grade non-Hodgkin lymphoma, prolymphocytic leuke-
Chromosome abnormalities have been observed in this mia, Hodgkin disease, or acute leukemia [382].
neoplasm. However, it is important to specify that these rear-
rangements tend to occur in bone marrow-based LPL and are Deletion of 13q
not typically seen in nodal LPL [67]. The most common From the cytogenetic point of view, the best prognosis is
rearrangement is deletion of 6q, which is found approxi- related to the presence of del(13q) as a sole abnormality,
mately 50% of the cases, followed by gain of chromosome 4 with a survival of more than 130 months [383]. The deleted
(20%), and other less frequent abnormalities such as del(17p) portion of chromosome 13 can vary in size, but it always
and gains of chromosomes 3 and 7 [373–375]. The clinical involves band 13q14.3 (Fig. 15.22). In a significant propor-
or pathogenic significance of chromosome abnormalities in tion of cases, del(13q) involves both the RB1 gene and the
LPL is unclear. D13S25/D13S319 loci, while in others only the latter are
deleted. Because of the minimal size of the deletion, this
Chronic Lymphocytic Leukemia/Small abnormality is often cryptic at the chromosome level and can
Lymphocytic Lymphoma only be detected by FISH [383]. In fact, the frequency of
Chronic lymphocytic leukemia (CLL)/small lymphocytic del(13q) with conventional cytogenetics is 10–15%, but with
lymphoma (SLL) is a B-cell neoplasm that leads to prolifera- FISH it can be detected in over 70% of cases [384]. The
tion of mature, normal-appearing lymphocytes in the periph- occurrence of this abnormality surpasses that of trisomy 12,
eral blood, bone marrow, spleen, and lymph nodes. These previously believed to be the most common chromosome
lymphocytes are typically positive for CD5, CD19, CD20, abnormality in CLL.
and CD23 and negative for CD10 [ 376 ] . The term SLL FISH has also demonstrated the existence of biallelic 13q
is used to indicate the aleukemic counterpart that is deletions [385]. Although patients with deletions of one or
morphologically and genetically comparable to CLL but both copies of chromosome 13 seem to have similar progno-
without significant lymphocytosis and mostly nodal involve- sis, it is believed that the presence of a high number of cells
ment [376]. Although CLL accounts for 30% of all adult leu- with del(13q) is characterized by a more aggressive clinical
kemias, the apparent incidence of this neoplasm has fluctuated course, equivalent to that seen in the “intermediate”-risk
FISH category [385]. Furthermore, del(13)(q14.3) leads to Deletion of 17p

deletion or reduced expression of two microRNAs, miR15a Patients with del(17p) and associated loss of TP53 at
and miR16, which have been identified as regulators of BCL2 17p13.1 are characterized by a poor response to chemo-
mRNA [384]. Therefore, lack or reduced expression of these therapy (e.g., alkylating agents) and short survival
two miRs is considered to be the basis of the consistent (24–32 months after diagnosis) [394]. This deletion is seen
overexpression of BCL2 in CLL exhibiting del(13)(q14.3). in approximately 5% of cases with conventional cytogenet-
ics (Fig. 15.22) and in about 7–20% by FISH (see also
Trisomy 12 Chap. 17, Fig. 17.10) [386].
After the discovery of the high incidence of del(13q) in CLL, The majority of abnormalities leading to del(17p) are
gain of chromosome 12 has settled in as the second most unbalanced translocations. Generally, the loss of 17p is
common abnormality [386] (Fig. 15.22). Patients with this present in the context of a complex karyotype. However, a
abnormality often present with atypical lymphocyte mor- few cases with i(17)(q10) as the only change have been
phology and intermediate risk, with a survival of about described [395].
115 months [387]. Factors that worsen the prognostic out- Mutations of TP53 are found in 8–10% of patients with
come include advanced age, male gender, elevated CD38 untreated CLL. Some reports indicate that the clinical behav-
expression, and unmutated IGHV@. At least 50% of patients ior of cases with TP53 mutation only is very similar to cases
with trisomy 12 have been found to carry unmutated IGVH with deletion of one allele and mutation of the remaining
and to progress to Richter syndrome [388]. By conventional allele. However, the overall survival after treatment was
cytogenetics, this abnormality is detected in 10–15% of all significantly improved in patients with del(17p) but without
cases. With the use of FISH, its detection has increased to TP53 mutation [396].
30% [389]. In approximately 30% of cases, gain of chromo-
some 12 is associated with additional abnormalities, the most Deletion of 6q
common of which is del(13q) (12% of cases). Less frequent del(6q) occurs in approximately 6–7% of patients with CLL
is the association of trisomy 12 with del(11q) and del(17p), [397]. The deletion is associated with atypical lymphocyte
believed to occur mostly as clonal evolution [389]. The pres- morphology, higher white blood cell count, splenomegaly,
ence of trisomy 12 together with del(14q) or t(14;18) has also and short survival compared to patients with normal karyo-
been reported [390, 391] (Fig. 15.23). These patients appear type or 13q deletion [397]. Thus, this abnormality is consid-
to have somewhat a more aggressive disease due to the fre- ered an intermediate marker in CLL.
quent association of this cytogenetic entity with unmutated Deletion of 6q is rarely the sole abnormality. More often,
IGHV@ (see later section on “IGH@ Rearrangements”). it is present in the context of a complex karyotype. At least
two regions of minimal deletion have been observed, one
Deletion of 11q involving bands 6q21–q23.3, which includes MYB, and the
CLL patients with del(11q) have a progressive disease course other involving bands 6q25–q27 [398, 399]. The majority of
[386]. Survival in this group is about 80 months [387]. The deletions were found to occur in the 6q21–q23.3 region
detection frequency of this deletion is 5–10% by conven- (Fig. 15.22). However, not all of these deletions involve
tional cytogenetics (Fig. 15.22) and 20–25% by FISH [392]. MYB. Therefore, it is important to point out that since CLL is
The deleted region at 11q22.3 includes the ataxia-telangi- characterized by variable 6q deletions, probes targeting
ectasia-mutated (ATM) gene and is also the site of MIR34B. solely the MYB locus will not detect all 6q deletions.
Approximately 10% of all CLL patients and about 30% of
those with del(11q) have an ATM mutation on the homolo- IGH@ Abnormalities
gous chromosome 11. Furthermore, in up to 12% of patients It is important to distinguish the IGH@ rearrangements
with ATM deletions, these are large enough to also result in that occur in CLL from those that are seen in other neo-
loss of MLL at 11q23 [392]. plasms. t(11;14)(q13.q32.3) had been reported in CLL, but
ATM is an important checkpoint gene involved in cell these cases are now believed to belong to a different entity,
damage control. The ATM/TP53 interaction has been shown and therefore the terminology atypical MCL is used to refer
to have an important impact on cell proliferation. Therefore, to these neoplasms [68, 400, 401]. Other IGH@ transloca-
deletion of ATM removes this checkpoint and blocks the tions that are observed in CLL include t(2;14)(p16.1;q32.3),
activation of TP53. As a result, even if TP53 is present, t(14;19)(q32.3;q13), and t(14;18)(q32.3;q21.3), and their
there is no attempt at repairing damaged cells [393]. variants (Figs. 15.23 and 15.24). These translocations,
Additional abnormalities are present in approximately respectively, relocate BCL11A, BCL3, and BCL2 to the
60–70% of patients with del(11q) and often include those IGH@ locus (or less commonly to one of the immuno-
that are recurrent in CLL, such as del(13q), +12, del(6q), globulin light chain loci) leading to their upregulation
and del(17p) [392]. [391, 402, 403].
344 A. Meloni-Ehrig
Fig. 15.23 Karyogram showing the simultaneous presence of trisomy 12 and t(14;18)(q32.3;q21.3) in a patient with atypical CLL
Fig. 15.24 Karyogram showing del(14)(q24) and t(14;19)(q32.3;q13) in a patient with CLL. Arrows indicate the chromosomes involved in the
t(14;19). The arrowhead indicates the deletion of 14q
Deletions of IGH@ are apparently more frequent but While patients with unmutated IGHV@ follow an unfavor-
are often cryptic; some reports have indicated that IGH@ able course with rapid progression and short survival, those
deletions, whether cryptic or not, are present in over 30% with mutated IGHV@ often show slow progression and long
of CLL cases [404]. FISH studies have demonstrated that survival [411].
up to 80% of IGH@ deletions involve the 5¢ portion of the Zeta-chain (TCR)-associated protein kinase 70 kDa
gene. Loss of one complete copy of IGH@ is indicative of (ZAP70) is a molecule involved in T-cell receptor signaling.
a larger deletion, generally involving band 14q24.1 It is coded for by ZAP70 on the proximal long arm of chro-
(Fig. 15.24). Detecting these deletions, either by conven- mosome 2, and is abnormally expressed in some CLL
tional cytogenetics or with FISH, is important since in patients. The presence of ZAP70 in at least 20% of leukemic
over 60% of cases del(14)(q24.1) is found in association cells is associated with unmutated IGHV@ [411]. The clini-
with unmutated IGHV@ [405]. Furthermore, in approxi- cal reliability of ZAP70 analysis has been questioned by
mately 50% of cases it is found associated with gain of some experts [413]. However, prognostic determinations can
chromosome 12 [390, 406]. Given the unfavorable prog- be made based on the combinatorial status of these markers,
nosis associated to deletion of IGH@, it is good practice where ZAP70 can provide useful information. In general,
to perform FISH to investigate the status of this gene in CLL patients with unmutated IGHV@ and/or CD38 expres-
patients with CLL. sion, but without expression of ZAP70, will not require ther-
apy by current criteria for many years after diagnosis [414].
Non-IGH@ Reciprocal Translocations Due to genetic variability of CLL, the combination of
Balanced reciprocal translocations, particularly those that do conventional cytogenetics, FISH, mutation analysis of
not involve IGH@, are rare in CLL [407]. Occasionally, IGHV@, and expression studies of ZAP70 and CD38 pro-
t(8;14)(q24.2;q11.2) involving MYC and IGH@ (or less fre- vides the best prognostic tool for the diagnosis and manage-
quently another immunoglobulin gene) is observed as an ment of patients with CLL.
additional abnormality in some CLL cases [408]. The recent
introduction of new mitogenic stimulants such as CpG- B-Cell Prolymphocytic Leukemia
oligodeoxynucleotides (CpG-ODNs) has increased the B-cell prolymphocytic leukemia (B-PLL) is a rare chronic
detection of these rearrangements with conventional cytoge- lymphoproliferative neoplasm accounting for only 1% of all
netics [325]. Interestingly, several of these apparently bal- chronic leukemias of lymphoid origin. This neoplasm is
anced translocations are found to involve chromosome 13. characterized by the presence of >55% prolymphocytes in
FISH has demonstrated, however, that the majority of these the peripheral blood. The bone marrow and/or spleen are
translocations are not balanced but often have a cryptic dele- often involved [415].
tion of 13q14.3, suggesting that they are most likely second- Patients present with severe leukocytosis and prominent
ary to the deletion [409]. Very few of these—for example, splenomegaly. Lymphocytes show the immunophenotypic
t(6;13)(p21;q14.1) or t(10;13)(q24;q14)—are recurrent [409, expression of B-cell markers such as CD19, CD20, CD22,
410]. The reason that these translocations are being detected CD79a, and FMC7. There is weak expression of CD5 and
only now with the use of CpG-ODNs is not clear. What is absent expression of CD10 and CD23. Approximately 50%
clear is that the broader use of ODNs is likely to increase the of the cases express CD38 and/or ZAP70 [415].
number of cases with these apparently balanced transloca- B-PLL has overlapping features with other mature neo-
tions, and a review of these cases might ultimately help plasms such as CLL and MCL. In fact, patients are diag-
elucidate their incidence and significance in the prognosis of nosed as having either de novo PLL or PLL that progressed
patients with CLL. from CLL or MCL [415, 416]. This overlapping is also evi-
dent in the cytogenetic findings. Approximately 25% of
Prognostic Markers patients were reported to have the t(11;14) that is the hall-
A set of genetic markers has significant prognostic value in mark of MCL; PLL cases with t(11;14) are now considered a
CLL. The most well-recognized are CD38, IGHV@ (IgVH), leukemic variant of MCL (Fig. 15.25). Other abnormalities
and ZAP70 [411]. include gain of chromosome 12 and deletions of 6q, 11q,
CD38 is a 45-kDa transmembrane glycoprotein that acts 13q, and 17p, abnormalities that are often seen in CLL, and
as both as an enzyme and a receptor [412]. CLL patients with thus these cases are thought to have progressed from CLL.
expression of CD38 in over 30% of cells tend to have unmu- An additional abnormality seen in some cases of PLL is
tated IGHV@, resistance to chemotherapy, and shorter over- the t(8;14) characteristic of Burkitt lymphoma. Less fre-
all survival compared with patients with low expression of quently, a variant of the t(8;14), either t(2;8) or t(8;22), might
CD38 [324, 411, 412]. be seen [417]. Rearrangements of chromosome 17 leading to
IGHV@ mutation status is a good parameter for assess- loss of 17p13.1 (TP53) have been reported in approximately
ing the prognosis of CLL patients at presentation [324]. 50% of PLL cases [418].
346 A. Meloni-Ehrig
Fig. 15.25 Karyogram with t(11;14) in the context of a complex karyotype in a patient diagnosed with B-cell PLL. These cases are now
considered to be a leukemic variant of MCL
Hairy Cell Leukemia (Typical and Variant) malities involve chromosomes 1, 6, 14, and 19 [419, 421,
Hairy cell leukemia (HCL) is an indolent mature B-cell lym- 423]. HCLv often shows a more complex karyotype than that
phoproliferative neoplasm that affects adult individuals aged seen in typical HCL and tends more frequently to exhibit
50 years and over and accounts for 2% of all B-cell lympho- gain of chromosome 12 and rearrangements of chromosome
mas [419]. The sites involved include primarily the bone 17 leading to loss of TP53 [423, 424] (Fig. 15.26).
marrow and spleen [419, 420].
The circulating peripheral blood B-lymphocytes are Plasma Cell Myeloma
small to medium in size and have a characteristic morphol- Plasma cell myeloma (PCM), also known as multiple
ogy with prominent cytoplasmic projections termed hairs, myeloma, is a neoplasm that affects the terminally differenti-
thus the name “hairy cell” used to refer to this entity [420]. ated plasma cells in the bone marrow and accounts for
Flow cytometric analysis shows expression of CD20 (bright), approximately 12% of hematologic neoplasms. Generally,
CD22, CD25, CD103, and CD11c. CD5 and CD10 are often patients are in their seventh decade of life and present with
negative [419, 420]. an excess of plasma cells in the bone marrow, monoclonal
The variant counterpart of HCL (HCLv) shares many proteins in the blood and urine, osteolytic bone lesions, and
similarities with typical HCL, except that patients present multiorgan dysfunction [425].
with leukocytosis and the hairs are less evident in the affected Conventional cytogenetics and FISH have been crucial
blood lymphocytes [421]. in the characterization of prognostically significant mark-
There are no specific chromosome abnormalities in either ers in PCM. However, due to the short life and low prolif-
typical or variant HCL. However, conventional cytogenetics erative rate of plasma cells, it is challenging to consistently
has demonstrated the recurrent gain of chromosome 5, obtain abnormal metaphases for analysis; the detection of
specifically the region 5q13-q31, and deletion of chromo- chromosome abnormalities by conventional cytogenetics is
some 7, specifically the region 7q22-q36. Of importance is believed to be between 25 and 40% [426]. This detection
the discovery of the BRAF V600E mutation in HCL. rate has been somewhat increased by the use of specific
Interestingly, the BRAF gene is located on 7q34, a chromo- mitogens, the most widely used of which is interleukin-4
some region often implicated in HCL. This mutation results (IL-4) [427].
in the production of an aberrant protein that is most likely FISH with a subset of probes targeting common abnor-
suitable for targeted therapy [422]. Other less frequent abnormalities in PCM has certainly improved the detection of
Fig. 15.26 Karyogram of a patient with hairy cell leukemia. Some of the recurrent abnormalities include gain of chromosome 5 and rearrange-
ments leading to deletion of 17p, as seen in this case. Here, the derivative (13;17) leads to the net loss of 17p
chromosome abnormalities of prognostic significance in this are t(4;14)(p16.3;q32.3), t(14;16)(q32.3;q23.1), and t(14;20)
neoplasm [428]. More recently, the use of plasma-cell- (q32.3;q12) which are associated with an unfavorable prog-
enriched fractions obtained with the use of CD138-coated nosis [430].
immunomagnetic beads has provided a concentrated number An interesting characteristic of PCM with hypodiploidy is
of plasma cells for easy identification of chromosome abnor- the tendency to double the abnormal chromosome comple-
malities by FISH analysis [429]. See also Chap. 17. ment, similar to what is seen in hypodiploid acute lympho-
PCM is characterized by distinct karyotypic entities, each blastic leukemia cases. Karyotypes with 70–90 chromosomes
with an associated prognostic outcome [430]. and a double content of structural rearrangements, including
the relative losses of chromosomes 13 and 17, most likely rep-
Hypodiploidy resent the doubling of a hypodiploid clone [431, 432]
Hypodiploidy (<46 chromosomes) generally includes the (Fig. 15.27a, b). The original hypodiploid clone has a very low
loss of chromosome 13, speci fi cally 13q14.3, and/or mitotic proliferation in culture compared to the clone with the
chromosome 17, specifically 17p13.1 (TP53), both of near-tetraploid (doubled) karyotype. Both carry the same
which are associated with unfavorable prognosis [430, adverse prognosis.
431]. As such, patients with these karyotypes are placed in
a high-risk category. In the majority of cases, the hypodip- Hyperdiploidy
loid chromosome complement includes structural abnor- Another group of PCM patients is characterized by hyper-
malities, involving, in particular, chromosomes 1, 4, 6, 14, diploidy and few or no structural abnormalities. Gains are
16, and 20. Specifically, loss of 1p and/or gain of 1q, losses nonrandom and often involve chromosomes 3, 5, 7, 9, 11, 15,
of 4q and 6q, loss and/or rearrangements of 14q and 16q, 19, and 21 [430] (Fig. 15.28). Patients with the presence of
and partial or complete loss of chromosome 20 are most these additional chromosomes are placed in a standard-risk
commonly seen [431]. category, as long as there is no deletion of 13q or 17p [432].
Translocations involving chromosome 14, specifically The most common translocation in this group involves
14q32.3 (IGH@), are seen in approximately 85% of the IGH@; it is t(11:14)(q13;q32.3) and is present in approxi-
cases. The most common IGH@ translocations in this group mately 25% of cases.
348 A. Meloni-Ehrig
Fig. 15.27 (a) Hypodiploid karyogram in a patient with high-risk often undergo reduplication and give origin to a doubling version. The
plasma cell myeloma showing, among other abnormalities, the simulta- prognosis associated with these karyograms is unfavorable
neous loss of chromosomes 13, 14, and 17. (b) The hypodiploid cells
Patients with this translocation are placed in a separate FISH can be useful in the differentiation of these clonally
prognostic category because of their specific treatment and different populations.
their improved prognostic outcome compared to patients
with other IGH@ translocations [430]. Deletion of 13q/Loss of Chromosome 13
The prognostic relevance of hyperdiploid karyotypes An interstitial deletion of 13q, involving either 13q14.2
might be difficult to ascertain when structural abnormalities (RB1) or 13q14.3 (D13S319), is one of the most common
are present. For the sake of proper prognostic stratification of abnormalities in PCM and has been detected by FISH in
PCM patients, every effort should be made to differentiate over 50% of cases [426, 427, 433] (see also Chap. 17, Fig.
true hyperdiploidy from the doubling of a hypodiploid clone. 17.11c). The region of deletion in these cases can be very
Fig. 15.28 True hyperdiploidy in plasma cell myeloma. These karyograms are characterized by gain of odd-numbered chromosomes (+3, +5, +7,
+9, +11, +15, +19, and +21)
small and in some cases has been found to involve either TP53, is considered a marker of adverse prognostic out-
D13S319 or RB1, but not both. In these cases, therefore, the come associated with aggressive disease, short survival,
deletion is cryptic and so is not detected with conventional and resistance to treatment. It is believed that in PCM,
cytogenetics; only 10–20% of PCM cases exhibit del(13q) deletions of 17p13.1 occur as secondary events during dis-
with chromosome analysis. ease progression. In fact, this deletion has been observed
In other instances, when a sufficient number of FISH in both hypodiploid and hyperdiploid karyotypes
probes are used, it can be shown that approximately 80% of (Fig. 15.29; see also Chap. 17, Fig. 17.11d). Occasionally,
cases with abnormal signal patterns for chromosome 13 it has also been observed together with t(11;14), which is
actually show complete loss of one copy of the chromosome, known to be a standard-risk marker [439]. Contrary to
generally in the context of a hypodiploid karyotype [434]. what is seen with deletion of 13q, deletion of TP53 has a
The significance of del(13q) as the sole abnormality, negative impact, irrespective of the presence of favorable
when detected by FISH only, is unclear. It appears that the prognostic markers.
poor prognosis originally attributed to del(13q) might be the
result of other abnormalities present in the karyotype [435– Chromosome 1 Abnormalities
437] (Fig. 15.29). In fact, studies have shown that the prog- Abnormalities involving chromosome 1 in PCM include
nostic outcome of a hyperdiploid karyotype typically deletions of 1p, gains of 1q, and/or translocations involving
associated with standard-risk myeloma is not altered by the either arm (Fig. 15.29, see also Chap. 17, Fig. 17.11b).
presence of del(13q). On the other hand, in a hypodiploid Deletions of 1p most frequently involve the segment
karyotype, del(13q) or loss of chromosome 13 indicates a between bands 1p12 and 1p31, whereas gain of 1q involves
poor prognosis. the segment q21→qter or the entire long arm [440]. Gain of
1q represents the second most frequent chromosomal
Deletion of 17p abnormality in PCM after del(13q). In fact, it has been
Deletion of 17p in PCM generally leads to deletion of observed in approximately 40% of newly diagnosed PCM
TP53 (17p13.1), and it has been observed in approximately patients and in approximately 70% of relapsed PCM cases
10% of patients [438]. This deletion, when it involves [440, 441].
350 A. Meloni-Ehrig
Fig. 15.29 Complex karyogram in plasma cell myeloma. Although of chromosomes 1 and 16, and loss of chromosome 17, are most likely
deletion 13q is thought to be one of the high-risk markers, other abnor- to blame for the poor prognosis
malities often present in such karyotypes, specifically rearrangements
Among the translocations involving chromosome 1, the to IGH@ or other Ig loci, for example, CCND1 at 11q13,
majority are derivatives of rearrangements involving vari- FGFR3/MMSET at 4p16.3, MAF at16q23.1, CCND3 at
ous chromosomes, resulting in gain of 1q. The most recur- 6p21.1, and MAFB at 20q12.
rent unbalanced translocations leading to gain of 1q are t(11;14), which is detected in approximately 20–25% of
der(1;15)(q10;q10), der(1;16)(q10;p10), and der(1;19) PCM patients, is not exactly the same as the one seen in
(q10;p10) [440]. However, other derivative chromosomes mantle cell lymphoma (MCL) [449]. In MCL, the break-
have been described. Gain of 1q can also be the result of an points on 11q13 are clustered to a specific minimal region,
isochromosome of the long arm of chromosome 1 [441]. whereas in PCM they are scattered over a large portion of
More recently, FISH probes and CGH targeting the region the q13 region [450]. Therefore, to increase the targeted
q21→q23 have detected multiple copies (amplification) of area and improve the ability to detect the CCND1-IGH@
genes located in this region, such as MUC1, BCL9, and fusion, two commercial CCND1-IGH@ fusion probes are
ARNT [442]. The segmental amplification of 1q is thought available. The one suggested for PCM includes another
to be associated with an unfavorable prognostic outcome. gene, MYEOV, which is distinct from the CCND1-IGH@
probe used for detection of t(11;14) in MCL [451]. An
IGH@ Rearrangements association has been found in PCM between the presence
Some PCM patients exhibit a rearrangement of an immuno- of t(11;14), CD20 expression, and lymphoplasmacytic
globulin (Ig) gene, most often of the heavy chain gene IGH@ morphology [452]. Therefore, a clinical laboratory should
at 14q32.3. The primary translocations are the result of consider performing FISH to investigate the presence of a
somatic hypermutation or recombination errors in the VDJ CCND1-IGH@ fusion in patients with such morphologic
portion of the switch region [440]. characteristics.
At least 20 nonrandom chromosomal partners have been t(4;14)(p16.3;q32.3) is the second most frequent translo-
found in translocations with IGH@ at 14q32.3. The most fre- cation involving IGH@. It is detected in approximately 15%
quent of these are t(11;14)(q13;q32.3), t(4;14)(p16.3;q32.3), of PCM patients and is cryptic by conventional cytogenetics
and t(14;16)(q32.3;q23.1) [443–446]. The latter two are cyto- [444, 445]. This translocation is thought to involve two genes
genetically cryptic and are detected only by FISH or PCR. located at 4p16.3, fibroblast growth factor receptor 3 (FGFR3)
Two other translocations, t(6;14)(p21.1;q32.3) and t(14;20) and the Wolf-Hirschhorn syndrome candidate 1 (WHSC1)
(q32.3;q12), have also been described [447, 448]. All of these gene, which in myeloma is known as multiple myeloma SET
translocations lead to overexpression of the gene translocated domain (MMSET)[453]. The detection of this translocation is
Fig. 15.30 Karyogram of a patient with plasma cell myeloma (PCM) with favorable or neutral prognostic outcome. However, MYC rear-
showing a MYC rearrangement, specifically t(2;8)(p12;q24.1), together rangements might be seen in these cases, and when present, they appear
with t(11;14)(q13;q32.3). PCM with t(11;14) are thought to be associated to be associated with a more aggressive course
possible with FISH using the commercially available FGFR3- Because abnormalities involving MYC are detected in
IGH@ probes, or with PCR. This translocation tends to be hypodiploid and hyperdiploid clones and in clones with
very frequent in hypodiploid karyotypes, particularly together t(11;14) (Fig. 15.30), it is believed that they are not primary
with del(13q) and/or del(17p). As such, patients with this abnormalities but rather occur during disease progression.
translocation are placed in a high-risk prognostic category MYC translocations in the presence of a highly complex
[444, 445]. karyotype have also been observed in plasmablastic lym-
t(14;16)(q32.3;q23.1) is observed in approximately 5–7% phoma, an aggressive lymphoma with plasmacytic differen-
of PCM patients [446]. This translocation is also cryptic by tiation (see next section). Other clinical, morphologic, and
conventional cytogenetics, and FISH or PCR are used to immunophenotypic data should therefore be used to differ-
determine its presence. In this case, the gene involved is entiate aggressive forms of PCM from plasmablastic
MAF at 16q23.1 [446]. Similarly to t(4;14), this transloca- lymphoma.
tion tends to occur in hypodiploid karyotypes, together with
deletions of 13q and/or 17p. As a result, it has been sug- Plasmablastic Lymphoma
gested that patients with this translocation should be placed Plasmablastic lymphoma (PBL) is an aggressive B-cell lym-
in a high-risk prognostic category. However, a recent large phoma that accounts for approximately 3% of human
study of myeloma patients indicates that the overall survival immunodeficiency virus (HIV)-related lymphomas and is
of patients with t(14;16) did not significantly differ from that characterized by plasma cell differentiation and an immuno-
of patients lacking this abnormality [454]. blastic cellular morphology [458]. The first described cases
involved the oral cavity of patients infected with HIV [459].
MYC Rearrangements However, this lymphoma also affects other sites, such as the
Approximately 15% of PCM patients, particularly those with gastrointestinal mucosa, skin, and soft tissues of HIV and
high-risk PCM, have a translocation involving MYC at 8q24.2 non-HIV patients [460].
and an immunoglobulin gene, leading to overexpression of With conventional cytogenetics, plasmablastic lymphomas
the MYC oncogene [455, 456]. Some translocations do not are quite interesting. They have chromosome abnormalities sim-
involve an immunoglobulin gene, but they still lead to overex- ilar to those observed in PCM, particularly rearrangements of
pression of MYC. In the majority of cases, however, there is chromosome 1, deletions of 13q and 17p, and simultaneous gains
gain or amplification of MYC without an apparent transloca- of odd-numbered chromosomes, specifically +3, +5, +7, +9,
tion involving this region [457]. +11, and/or +15 [461]. Invariably, they are also characterized by
352 A. Meloni-Ehrig
Fig. 15.31 Typical karyogram of a patient with plasmablastic lym- chromosomes, loss of chromosome 13, and the simultaneous presence
phoma. Of interest is the presence of chromosome gains similar to what of a MYC translocation, in this case t(2;8)(p12;q24.1)
is seen in plasma cell myeloma, in this case gain of odd-numbered
one of the MYC translocations, possibly occurring during analyzed with conventional cytogenetics. Although the majority
progression [461] (Fig. 15.31). Since plasmablastic morphology, of HL patients have a normal karyotype, a minority are found
plasma cell immunophenotype, and MYC translocation can also to have an abnormal chromosome complement. The first kary-
be observed in some aggressive (anaplastic) PCM, it is important otype was described in 1967 [465]. Subsequently, other cytoge-
to use all available clinical information to differentiate these two netic cases have been described, and still no specific
entities. Some of the main differentiating factors in PBL are the abnormalities have been detected. The common feature is that
high association with Epstein-Barr virus (EBV) and the lack of the karyotypes tend to be hyperdiploid, with 60–70 chromo-
monoclonal paraproteinemia and lytic bone lesions typically somes. Some recurrent abnormalities include losses of 1p, 6q,
seen in PCM [458]. 7q, 13q, 16q, and 17p; gains of 2p, 9p, and chromosome 12, as
well as rearrangements of 3q27 (BCL6) [466, 467].
Hodgkin Lymphoma
Hodgkin lymphoma (HL) is an indolent neoplasm of the
lymphatic system that makes up approximately 30% of all Mature T-Cell Lymphomas
lymphoma cases [462]. This lymphoma owes its name to
Dr. Thomas Hodgkin who first described it in 1832 [463]. A complete description of all mature T-cell and natural killer
HL affects individuals of all ages with two preferential peaks, cell (NK) lymphomas, some of which are quite rare, is provided
one occurring between the ages of 15 and 30 years and the by the WHO [67]. In this chapter, only the most common
other at ³60 years [462, 464]. T-cell lymphomas, particularly those that have been reported
One of the morphologic characteristics of HL is the pres- to have recurrent chromosome abnormalities, are described.
ence of giant cells called Reed-Sternberg cells. These cells These T-cell neoplasms have many characteristics in com-
comprise only approximately 1% of the affected tissue; the mon, such as location (skin is the most common site of
remaining tissue is composed of inflammatory cells [462]. involvement) and genes involved (T-cell receptors are most
HL is subdivided into two morphologically and clinically frequently rearranged) [468, 469]. Bone marrow and periph-
distinct subgroups, nodular and classic [464]. Classic HL eral blood samples usually require mitogen stimulation, the
(CHL) is itself subdivided into four histologic entities known most frequently used of which is phytohemagglutinin (PHA).
as lymphocyte rich, nodular sclerosis, mixed cellularity, and Other tissues such as lymph nodes, spleen, or liver produce
lymphocyte depleted [464]. CHL accounts for about 95% of all analyzable metaphase cells in overnight cultures without
Hodgkin lymphomas and is therefore the most common type stimulation. See Table 15.14.
Table 15.14 Biologic and genetic characteristics of mature T-cell lymphomas

Chromosome abnormalities
Neoplasm Sites involved Frequency Primary Additional
T-PLL PB, BM, LN, SP, LV 2% of lymphomas; 20% inv(14), t(7;14), t(14;14), t(X;14) i(8)(q10), t(8;8), del(6q),
of PLL del(11q), del(12p), del(17p)
NKCL PB, BM, SP, LV Rare del(6q), del(11q) del(17p)
ATLL LN, PB, BM, SK 2–5% of lymphomas Complex with + X, +3, and +7; rearrangements del(17p)
of 1q, 3q, 6q, 9p, 14q
HSTL SP, LV, BM <1% of lymphomas i(7)(q10), gain of 7q +8
MF SK, LN 50% of all cutaneous Complex with rearrangements of 1p, del(6q), Rearrangements of 3q, 10q,
lymphomas del(9p) 11q, 12p, and 14q
SS SK, PB, BM 5% of cutaneous Complex with del(1p), del(6q), del(10q), +17, del(9p), del(17p), −19
lymphomas +18
PTCL-NOS LN, BM, SP 30% of PTCL TCR gene rearrangements, very complex Variable
karyotypes with +3q, +7q, +8q, del(4q),
del(5q), del(6q), del(9p), del(10q), del(13q)
AITL LN 1–2% of T-cell TCR gene rearrangements; +X, +3, +5, +11q, Variable
lymphomas del(10q), del(12q), del(13q)
ALCL-ALK+ LN, SK, LV 3–5% adults, 15–20% t(2;5)(p23.1;q35.1), or variant translocations +X, +7, +9; variable
children among all involving 2p23 (ALK): t(1;2), inv(2), t(X:2), structural rearrangements
lymphomas t(1;2), t(2;3), t(2;17), t(2;19), t(2;22)
Abbreviations: T-PLL T-cell prolymphocytic leukemia, NKCL NK-cell leukemia (aggressive type), ATLL adult T-cell leukemia/lymphoma, HSTL
hepatosplenic T-cell lymphoma, MF mycosis fungoides, SS Sézary syndrome, PTCL-NOS peripheral T-cell lymphoma, not otherwise specified,
AITL angioimmunoblastic T-cell lymphoma, ALCL-ALK+ anaplastic large cell lymphoma, ALK-positive, PB peripheral blood, BM bone marrow,
LN lymph node, SP spleen, LV liver, SK skin
Fig. 15.32 Three common rearrangements in T-PLL. inv(14) and t(14;14) relocate the TCL1 gene from its normal location at 14q32.1 to 14q11.2
under the control of the TRA/D@ promoter. Less frequently, the MTCP1 at Xq28, instead of TCL1, relocates to the TRA/D@ locus
T-Cell Prolymphocytic Leukemia Natural Killer Cell Leukemia

T-cell prolymphocytic leukemia (T-PLL) is an aggressive Natural killer cell leukemia (NKCL) is a rare form of T-cell leu-
neoplasm that affects approximately 2% of adults aged kemia that has a strong association with the Epstein-Barr virus
30 years and over [469–471]. The most common sites of (EBV) and tends to affect younger individuals, with a median
involvement include peripheral blood, bone marrow, lymph age at diagnosis of 40 years [476]. Although a large number of
node, and other hematopoietic organs such as spleen and these patients have an abnormal chromosome complement, no
liver [469]. The most common chromosome abnormalities specific cytogenetic abnormalities have been identified. Recurrent
are inv(14)(q11.2q32.1), t(14;14)(q11.2;q32.1), and t(7;14) chromosome abnormalities include deletions of 6q and 11q.
(q34;q32.1) [472] (Fig. 15.32). All of these translocations Gain of 1q and loss of 17p are also seen [252].
involve the T-cell receptor (TCR) genes (TRA@/TRD@ at
14q11,2, and TRB@ at 7q34) and the T-cell leukemia 1 Adult T-Cell Leukemia/Lymphoma
(TCL1) gene located at 14q32.1 [472, 473]. In a minority of Adult T-cell leukemia/lymphoma (ATLL) is a lymphoid neo-
cases, translocations involve a TCR gene and MTCP1, plasm that it is known to be associated with early exposure to
located at Xq28, instead of TCL1. The most common trans- the human T-cell lymphotropic virus type 1 (HTLV-1) and
location in this group is t(X;14)(q28;q11.2) [474]. affects approximately 3% of individuals who carry the virus
Additional abnormalities are present in the majority of (median age: 60 years) [469]. Although HLTV-1 is consid-
cases; these include i(8)(q10) or other rearrangements lead- ered to be a primary insult, neoplastic transformation requires
ing to gain of 8q, deletion or rearrangements of 11q, and additional genetic changes [477]. In fact, the karyotypes of
deletions of 6q, 12p, and 17p [475] (Fig. 15.33). patients with ATLL are very complex.
354 A. Meloni-Ehrig
Fig. 15.33 Karyogram of a patient with T-cell prolymphocytic leukemia. In addition to the inv(14)(q11.2q32,1), the recurrent abnormalities
observed in the majority of cases include gain of 8q, mainly in the form of isochromosome 8q as seen here, deletion of 11q, and deletion of 17p
Fig. 15.34 Complex karyogram with several chromosome rearrangements typically seen in adult T-cell leukemia. Gain of chromosomes X, 3,
and 7 and deletions 6q and 9p are present in most cases
The most frequent genetic abnormalities include 13q, and 17p [478] (Fig. 15.34). The prognosis associated
rearrangements of the T-cell receptor genes TRG@ at with these karyotypes is considered unfavorable. In partic-
7p14.1, TRB@ at 7q34, and TRA/D@ at 14q11.2; gains of ular, abnormalities of 1p, 1q, 3q, and 14q and deletions of
the X chromosomes and chromosomes 3 and 7; rearrange- 2q, 9p, 14q, and 17p are thought to be associated with
ments of 1p, 1q, 2q, 3q, and 17q; and deletions of 6q, 9p, shorter survival [479].
Fig. 15.35 Isochromosome 7q is often the sole abnormality in the karyogram of patients with hepatosplenic lymphoma
Hepatosplenic T-Cell Lymphoma well as other numerical and/or structural abnormalities

Hepatosplenic T-cell lymphoma (HSTL) is a rare and aggres- involving chromosomes 3, 6, 10, 11, 12, and 14 [484, 485].
sive T-cell lymphoma that usually affects adolescents and
young adults [469]. A male predominance has been noted. Sézary Syndrome
As the name implies, there is obvious involvement of the Sézary syndrome (SS) is a cutaneous T-cell lymphoma sim-
spleen and liver with evident hepatosplenomegaly, neutropenia, ilar in many ways to mycosis fungoides except for the pres-
and thrombocytopenia [469, 480]. A characteristic recurrent ence of erythroderma and lymphadenopathy with circulating
abnormality of this lymphoma is the presence of an isochro- malignant cells in the peripheral blood and in the bone marrow
mosome for the long arm of chromosome 7 [i(7)(q10)] or [470, 483]. This neoplasm occurs in adults over the age of 60
less frequently alternative rearrangements leading to gain of and is associated with an unfavorable prognosis [470].
7q [481, 482] (Fig. 15.35). Additional abnormalities, most Karyotypes tend to be complex, with rearrangements involv-
commonly +8 and loss of a sex chromosome, have also been ing chromosomes 1, 6, 10, 14, 17, 18, and 19 [484–486].
reported [482].
Peripheral T-Cell Lymphoma, Not Otherwise
Mycosis Fungoides Specified
Mycosis fungoides (MF), the most common form of cutaneous Peripheral T-cell lymphoma, not otherwise specified (PTCL,
T-cell lymphoma, is characterized by an increased number of NOS), represents any nodal or extranodal mature T-cell lym-
CD4+ T-cells in the skin [470, 483]. The annual incidence is phoma that does not fit into the current system of classification.
around 0.3 cases per 100,000 in Western countries, and the These neoplasms account for approximately 30–70% of all
median age at diagnosis is between 55 and 60 years, with a 2:1 T-cell lymphomas worldwide [470, 487, 488].
male to female ratio [470]. MF is an indolent disease, and given The majority of these lymphomas have a nodal histology
the high average age at diagnosis, it therefore has a high inci- and occur primarily in adults, with a peak at around 60 years
dence among the elderly. However, more advanced cases have if age [470, 487, 488].
an unfavorable prognosis that resembles the clinical behavior These lymphomas have similar genetic abnormalities.
seen in patients with Sézary syndrome, a closely related Karyotypes are usually highly complex, with rearrangements
lymphoma (see next section) [483]. that often lead to losses of 6q, 9p, 10q, and 13q and to gains
The karyotypes of patients with MF tend to be complex of 3q, 7q, and 8q [489]. The prognosis is poor for most
and often include rearrangements of the short arm of chro- patients. However, the identification of a new recurrent trans-
mosome 1, particularly of the critical regions 1p32-p36, as location, t(5;9)(q33.3;q22.2), has given hope for the possible
356 A. Meloni-Ehrig
Fig. 15.36 t(2;5)(p23.1;q35.1) in a patient with anaplastic large T-cell lymphoma. This translocation leads to fusion of ALK at 2p23.1 and NPM1
at 5q35.1. Other translocations involving ALK and other partner chromosomes have been reported in this lymphoma (see Table 15.13)
development of a new treatment for some patients with these Approximately 60% of cases are positive for ALK (ALK+
lymphomas [490]. This translocation leads to fusion of the ALCL). This subtype tends to have an aggressive course with
inducible T-cell kinase (ITK) gene located at 5q33.3 and the extranodal involvement. Fifteen to twenty percent of patients
spleen tyrosine kinase (SYK) located at 9q22.2 and subse- are children, and 3–5% are young adults (around 30 years of
quent tyrosine kinase activation [490]. Patients with the age) [497–500]. The cytogenetic hallmark of ALK+ ALCL is
ITK-SYK fusion appear responsive to treatment with SYK the presence of specific translocations involving the anaplas-
inhibitors. tic lymphoma kinase gene (ALK) and various partner chro-
mosomes [499] (see Table 15.14). The most common ALK
Angioimmunoblastic T-cell Lymphoma translocation is t(2;5)(p23.1;q35.1) (approximately 80% of
Angioimmunoblastic T-cell lymphoma (AITL) is an aggres- cases), which fuses part of the nucleophosmin gene (NPM1)
sive nodal T-cell lymphoma that accounts for approximately located at 5q35.1 with ALK located at 2p23.1, leading to acti-
2% of all non-Hodgkin lymphomas but represents the most vation of ALK [500] (Fig. 15.36). This has promoted the
common subtype (15–20%) of peripheral T-cell lymphomas development of specific therapies with the function of inhibit-
[470]. Affected patients average 60 years of age and present ing tyrosine kinase activity [498].
with an array of symptoms including skin rash, pleural effu- ALK–ALCL on the other hand, affects older individuals
sion, and eosinophilia [470, 491]. One of the characteristics (around 60 years of age) and has a relatively favorable prog-
of this lymphoma is the presence of the EBV genome in the nosis. This subtype does not involve the ALK gene and no
lymph nodes [492]. Karyotypes are complex and often show recurrent chromosome abnormalities have been associated
gain of 11q13 and gains of chromosomes 3, 5, and an X with it [497, 498].
chromosome, as well as losses of 5q, 10q, and 12q [493,
494]. Gain of 11q13 may represent a primary event in angio-
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The Cytogenetics of Solid Tumors
16
Linda D. Cooley and Kathleen S. Wilson
Introduction toxic therapeutic regimens to increase patient survival and

quality of life. The genetic anomalies of tumors and their
All neoplasms have genetic abnormalities; most have visible correlation with response to therapy have altered therapies
nonrandom chromosome abnormalities. Clonal chromo- such that the survival rates, length of survival, and even
some aberrations in both benign and malignant neoplasms cure rates have significantly increased. The pace of discov-
define particular tumors. These can make or refine the ery has been faster for hematologic tumors due to the ease
histopathologic diagnosis, provide prognostic information, of culture and yield of genetic information through chro-
and inform therapeutic decisions. Examples include distin- mosome analysis (see Chap. 15).
guishing an atypical teratoid/rhabdoid tumor from a medullo- While slower to accumulate, the knowledge of the genetic
blastoma and documenting MYCN gene amplification in a aberrations in solid tumors has proven to be as valuable as
neuroblastoma. for those in hematologic disorders. It is vitally important to
Today, more and more therapeutic drugs are designed to continue this acquisition of genetic information to persevere
target a specific genetic anomaly. Examples include ima- toward finding effective therapies. Conventional chromo-
tinib mesylate for patients with t(9;22) or BCR-ABL1 some analysis of solid tumors is a valuable tool that contin-
fusion, ATRA (all-trans retinoic acid) for patients with ues to discover genetic anomalies that influence current
t(15;17) or PML-RARA fusion, trastuzumab for ERBB2 patient therapy. Chromosome data and that from newer tech-
amplified intraductal breast carcinoma, tyrosine kinase nologies—e.g., various types of microarrays (see Chap. 18)
inhibitors for gastrointestinal stromal tumors, and others. and molecular methods—contribute to the growing genetic
For tumors without a known targetable genetic anomaly, databases that are used to further efforts to design new thera-
chromosome aberrations provide information to determine peutic trials and discover new more effective drugs through
optimal therapies. Examples include oligodendroglial understanding the genes involved.
brain tumors with der(1;19) or 1p/19q deletion and Wilms
tumors with 1p/16q deletion. Collaborative oncology
groups have, over the past several decades, dedicated their Solid Tumor Culture and Analysis
efforts toward the discovery of the most effective, least
Cytogenetic analysis of solid tumors (STs) is challenging.
While most STs can be grown in tissue culture with good
L.D. Cooley, M.D., M.B.A. (*) results, STs require more time and effort than the typical
Cytogenetics Laboratory, Children’s Mercy Hospital, tissues like products of conception and skin that are cul-
2401 Gillham Road, Kansas City, MO 64108, USA tured and analyzed in cytogenetics laboratories. Solid
University of Missouri Kansas City School of Medicine, tumors are many and diverse, with more different names
Kansas City, MO USA than there are tumor types. Understanding the “diagnosis” is
Department of Pathology and Laboratory Medicine, fundamental to a successful outcome. The diagnosis directs
Children’s Mercy Hospital, 2401 Gillham Road, the processing of the sample, preparation, culture type
Kansas City, MO 64108, USA
(monolayer vs. suspension), growth medium, and times and
methods of harvest. ST cultures require close observation
K.S. Wilson, M.D.
for growth and growth patterns to determine when to flood
McDermott Center for Human Growth and Development and the
Department of Pathology, University of Texas Southwestern Medical the culture, change the medium, and harvest. Harvest should
Center, Dallas, TX, USA begin as soon as the cultures are mitotic in order to capture
372 L.D. Cooley and K.S. Wilson
the malignant cell population and avoid cell confluence and supplemented medium prepared for bone marrow cultures
growth of normal cells. Harvest time and method differ for (e.g., Roswell Park Memorial Institute medium, RPMI).
various cell types. • Monolayer culture on coverslips has advantages that
Chromosome analysis of STs can also be challenging. include: facilitates growth of tiny samples, avoids trypsini-
Some STs have a single diagnostic abnormality, while others zation for splitting and harvesting, avoids making slides
may be complex with many structural and numerical aberra- for banding, requires less medium, allows growth and
tions. Perseverance and experience are factors in analysis of mitotic activity to be easily monitored with an inverted
any complex hematologic or solid tumor in order to discern microscope, etc.
which anomalies have significance for diagnosis and/or ther- • Tumor disaggregation (physically with scalpel blades
apeutic management. and/or enzymatically with collagenase) usually promotes
faster growth. Some tumors grow better with physical dis-
aggregation alone. Cultures of tumor with and without
Factors Affecting Growth of Solid Tumors enzymatic disaggregation can be initiated with sufficient
sample.
Tissue culture simulates the in vivo environment of the tumor • Sample initiation into culture should occur ASAP to pre-
so as to support viability and growth. Knowledge of the tumor serve viability.
type influences how it is processed; the referring physician or • Initiation of multiple cultures provides opportunities to
pathologist can provide the tumor type or working diagnosis. feed and harvest at different times to promote growth and
The condition of the sample is also critical to culture suc- capture metaphase cells.
cess. A surgeon or pathologist obtains the specimen; thus, it • Initiation with a small amount of medium allows cells to
is imperative that he or she knows how to handle a sample for attach faster. Be patient and wait for evidence of cell
cytogenetic culture. A working relationship with the attachment before flooding. Waiting several days can
pathologist(s) can be very beneficial and is encouraged. yield success.
Critical aspects of the tumor sample collection that the • Daily observation is needed to detect optimal times for
referring physician or pathologist controls and should under- harvest and prevent confluent growth.
stand are: • Harvest with different lengths and strengths of Colcemid®
• Sterility must be maintained. Non-sterile tumors must be exposure may capture more metaphase cells.
handled in a way that minimizes further contamination. If Laboratories that grow and analyze solid tumors must rec-
the sample is contaminated, this should be communicated ognize and provide the extra time for the work it takes to cul-
to the cytogenetics laboratory. ture and analyze these samples. As noted, the genetic
• The sample needs to be tumor without surrounding nor- information gleaned from this process is critical to optimal
mal tissue. patient management. Both pathologists and oncologists use
• The sample must be viable, i.e., not necrotic, frozen, or this information to refine diagnoses and therapeutic decisions.
fixed. This chapter will cover certain solid tumors with known
• The sample must be delivered to the laboratory ASAP. chromosomal abnormalities that have documented pathologi-
The sample should be stored in supplemented culture cal and clinical significance for diagnosis, diagnostic subtype,
medium at room temperature or 37°C until delivered. prognosis, and therapeutic intervention. The chapter does not
• Adequate sample size; a 1 cm3 piece of tumor is optimal. comprehensively cover all tumors and does not present all
However, even very small pieces can be successfully chromosomal or genetic anomalies of those discussed.
cultured.
• Touch preparations of tumor often provide a good sample
for fluorescence in situ hybridization (FISH) analysis and, Central Nervous System Tumors (Table 16.1)
therefore, rapid results.
Gliomas
Tissue culture factors that impact tumor cell growth
include:
Gliomas, the most common primary central nervous system
• Type of culture: suspension or monolayer. Tumors that
(CNS) tumors, include astrocytomas, oligodendrogliomas,
need attachment and/or cell-to-cell contact for growth
and ependymomas. Histopathologic features used for patho-
require a monolayer culture, while small round cell tumors
logical classification and grading of the tumor correlate with
may grow better in suspension.
prognosis and guide therapy. The annual incidence of pri-
• Type of medium: monolayer cultures do fine with basic
mary brain tumors is ~8–12 in 100,000 and for intraspinal
supplemented medium (e.g., minimum essential medium,
tumors it is 1–2 in 100,000. CNS tumors are the most com-
MEM), while suspension cultures work well using
mon (~20%) cancers of childhood [1].
16
Table 16.1 Chromosome abnormalities with diagnostic or clinical significance in tumors of the central nervous system
Tumor Chromosomal aberration(s) Gene(s) involved Clinical significance References
Glial
Pilocytic astrocytoma Gain of 5, 7/7q common, followed by 6, 8, 11, KIAA1549-BRAF from BRAF tandem Constitutive kinase activity [3–5]
12, 17, 19, 22 duplication
Diffuse grade II/anaplastic Loss of 10q, 13q, 17p, 19q IDH1, IDH2, RB1 and TP53 mutation TP53 mutation correlates with IDH mutations [6]
astrocytoma grade III
Glioblastoma Loss of 9p, 10q; EGFR amplification, MDM2 CDKN2A/B, PTEN, EGFR, MDM2 Short survival, aggressive course [8–10]
amplification
Oligodendroglial
Oligodendroglioma (OD) der(1;19)(q10;p10), loss of 1p, 19q 1p36, 19q13.3, IDH mutation Longer survival, sensitive to therapy [11–15]
Anaplastic OD der(1;19)(q10;p10), loss of 1p, 19q, 9p 1p36, 19q13.3, CDKN2A/B, IDH Longer survival, sensitive to therapy [11–15]
Oligoastrocytic
Oligoastrocytoma (OA) der(1;19)(q10;p10), TP53 mutation 1p36, 19q13.3, TP53, IDH mutation Outcome is better with der(1;19) [1, 16]
Anaplastic OA der(1;19)(q10;p10), loss of 9p, 10q 1p36, 19q13.3, TP53, CDKN2A/B Overall survival is better with der(1;19) [20, 21]
Ependymoma
Spinal Loss of 22q More common in adults [22]
Posterior fossa Gain of 1q, balanced karyotypes Young age, unfavorable prognosis [23, 24]
Posterior fossa Loss of 6q, 22q, gain of 9q, 15q, 18q Favorable prognosis [23, 24]
Intracranial Gain of 1q, 7, loss of 6q, 9p EGFR, CDKN2A Gain of 1q, CDKN2A homozygous deletion [25]
is unfavorable
Choroid plexus
Papilloma (CPP) Normal karyotype Distinguish from atypical CPP [26–28]
Atypical papilloma Hyperdiploid with gain of 7, 12, 20 Chromosome enumeration Distinguish from CPC [26–28]
Carcinoma (CPC) Hyperhaploid with gain of 1, 12, 20, 4, 7 Chromosome enumeration Distinguish from papilloma [26–28]
Embryonal
Medulloblastoma, pediatric Gain of 6q, MYC or MYCN amplification MYC, MYCN, ERBB2 Very high risk, correlates with large cell/ [29–33]
anaplastic histology
i(17q), gain of 17q Intermediate risk [29–33]
Loss of 6 or 6q Excellent prognosis, desmoplastic histology [29–33]
Medulloblastoma, adult Loss of 10q with gain of 17q, CDK6 amplification CDK6 (7q21.2) High risk, poor OS [30]
Either loss of 10q or gain of 17q Intermediate risk [30]
Neither loss of 10q nor gain of 17q Good prognosis [30]
PNET, supratentorial Gain of 1q, loss of 14, 16, 19, rare EGFR EGFR Lacks i(17q), poor prognosis [31, 35]
amplification
Atypical teratoid/rhabdoid Loss of 22, del(22q) SMARCB1-INI1 Distinguishes AT/RT from MB, PNET, CPC [36–39]
tumor
Meningioma Loss of 22, del(22q) 22q, NF2 Primary abnormality [43, 44]
Atypical/anaplastic loss of 1p, 14/14q, 18, 10, 6q Increased risk of recurrence [43, 44]
CPC choroid plexus carcinoma, CPP choroid plexus papilloma
373
Astrocytomas Grade IV, glioblastoma (GB), is the most malignant astro-

Astrocytomas have a spectrum of histologic features that cytoma. In addition to increases in cellularity, nuclear atypia,
correlate with the clinical course [2]. Grade I, pilocytic astro- and pleomorphism, mitotic activity, vascular proliferation,
cytoma (PA), is a slow-growing, noninfiltrating tumor with a and necrosis are characteristic. Genetic changes in primary
relatively benign course. Most PAs have a normal diploid glioblastomas, which account for >90% of GBs, include loss
karyotype; ~30% show gain of one or more chromosomes. of 9p (CDKN2A deletion), loss of 10q (PTEN deletion),
Approximately half of children younger than 15 years old PTEN mutation, frequent EGFR amplification, and infre-
show a single extra chromosome, while those greater than quent MDM2 amplification. The secondary glioblastomas
15 years of age show gain of multiple chromosomes. Gains (5–10%) that result from progression of grade II–III tumors
of chromosomes 5 and 7 are the most frequent, followed by have mutations of IDH1 or IDH2 and TP53. Tumors with
chromosomes 6, 8, 11, 12, 17, 19, and 22. See Fig. 16.1. wild-type IDH1 and IDH2 have fewer TP53 mutations and
Tandem duplication of BRAF at7q34 is found in >50% of frequent alterations of PTEN, EGFR, CKDN2A, or CDKN2B
pediatric PA and in up to 66% of all PA [3, 4]. Tandem dupli- [1, 6–8]. Median overall survival in patients with IDH muta-
cation produces a novel fusion gene, KIAA1549-BRAF, tions is twice that of patients with wild-type IDH, 31 versus
which has constitutive kinase activity [4, 5]. 15 months [6]. Double minutes (dmin) with EGFR
Grade II diffuse astrocytoma and grade III anaplastic amplification have been demonstrated in up to 50% of GBs
astrocytoma show increasing cellularity and nuclear pleo- [9]. Current clinical trials are evaluating several targeted
morphism. Gliomas of World Health Organization (WHO) molecular therapies [10].
grade II or III are invasive, progress to higher-grade lesions,
and have a poor outcome. As the tumor progresses, acquired Oligodendroglial Tumors
genetic abnormalities include loss of 10q, 13q, 17p, and 19q, Oligodendroglioma (OD), a diffusely infiltrating well-
and TP53 and RB1 mutations. The majority of WHO grade II differentiated (grade II) to anaplastic (grade III) glioma, is a
and III gliomas, including astrocytomas and oligodendro- relatively rare primary brain tumor, comprising 2–5% of all
gliomas, have IDH1 (2q34) or IDH2 (15q26.1) mutations. primary brain tumors. ODs characteristically show an unbal-
Most also have TP53 mutation. Patients with mutations of anced der(1;19)(q10;p10) with loss of 1p and 19q (Fig. 16.2).
IDH genes have longer median overall survival, 65 versus These aberrations may be detected by conventional chromo-
20 months [6]. some analysis, fluorescence in situ hybridization (FISH), loss
Fig. 16.1 Pilocytic astrocytoma, grade I, from the posterior fossa of a 12-year-old boy: 47,XY,+5,der(14;21)(q10;q10)
16 The Cytogenetics of Solid Tumors 375
Fig. 16.2 Oligodendroglioma, grades II–III, from the brainstem of an 8-year-old girl: 57,XX,+der(1;19)(q10;p10)x2,+2,+3,+4,+8,+11,+16,+20,
+20,+22. Arrows indicate the der(1;19); (see text for details)
of heterozygosity (LOH) studies, or microarray compara- Ependymomas

tive genomic hybridization (aCGH) [11, 12]. 1p/19q code- Ependymomas are slow-growing neuroepithelial neoplasms
letion is associated with a longer median survival (~10 years that arise from the wall of the intracranial ventricles and the
vs. 2 years) in patients with OD and anaplastic OD [13]. central canal of the spinal cord. Little is known about the
Anaplastic oligodendrogliomas show loss of 9p (CDKN2A/B) genetics of myxopapillary ependymoma, a grade I tumor that
in additional to 1p/19q loss [14]. Recent studies have shown arises predominately in the terminal spinal cord of young
that IDH1 mutations are strongly associated with 1p/19q adults. The grade II ependymoma may arise at any site in the
codeletion (~85%) and that IDH1 mutation and 1p/19q ventricular system and spinal canal with the posterior fossa
codeletions are independent prognostic factors [6, 15]. the most common site in children. The most frequent cytoge-
netic aberrations include losses of 22q and 6q and gains of 1q
Oligoastrocytic Tumors and 9q (Fig. 16.3).
Oligoastrocytomas (OA) are composed of a mixture of dis- Alterations of 22q are more frequently identified in spinal
tinct cell types found in oligodendroglioma and astrocytoma. tumors. NF2 may be a candidate tumor suppressor gene in spi-
Genetic analysis reveals 1p/19q codeletion in ~30–50%, nal ependymoma. However, few mutations in candidate genes,
TP53 mutations in ~30%, and IDH mutations in 100% of including NF2, TP53, PTEN, MEN2, and CDKN2A, have been
OA. Progression-free survival (PFS) was 60 versus 30 months identified in pediatric intracranial tumors [1, 3, 22].
in patients with and without 1p/19q codeletion, respectively Recent studies have identified two clinically and geneti-
[1, 16]. Few studies have attempted to evaluate the genetics cally different posterior fossa subgroups [23, 24]. Group A
of the distinct cellular components. Rare tumors have been patients are younger (median age 4 years), more often male,
shown to have different genetic aberrations in the two histo- more often have higher grade tumors (grade III), have more
logic elements [17–19]. Anaplastic OA (grade III) acquire metastases at recurrence, and have a diminished prognosis
additional genetic alterations—e.g., 9p (CDKN2A/B) loss, compared with group B patients. Group A tumors have pre-
which is also implicated in the progression of astrocytoma dominately balanced karyotypes; some show gain of 1q.
and OD. Recent studies of anaplastic OD and OA confirm Group B tumors frequently show loss of 6q and 22q and gain
that presence of 1p/19q codeletion correlates with significantly of 9q, 15q, and 18q. Intracranial tumors are more common in
longer survival than presence of genetic abnormalities other children than adults, with spinal tumors more common in
than 1p/19q codeletion [20, 21]. adults. Tumors with gain of 1q correlate with higher grade,
tumor recurrence, and worse prognosis [25].
Fig.16.3 Ependymoma, grade II, from the posterior fossa of a 2-year-old girl: 46,XX,+14,−22
Choroid Plexus Tumors Embryonal CNS Tumors
Choroid plexus (CP) tumors arise from the choroid plexus in Medulloblastoma and Supratentorial
the ventricles of the brain. Choroid plexus papilloma (CPP) Primitive Neuroectodermal Tumor
is a benign (grade I) neoplasm with very low mitotic activity Medulloblastoma (MB) is a primitive small round cell tumor,
that closely resembles normal choroid plexus. Atypical CPP which may show glial or neuronal differentiation. MB, also
(grade II) is a CPP with increased mitotic activity that may referred to as an infratentorial primitive neuroectodermal
have increased cellularity and nuclear pleomorphism. Grade tumor (iPNET), is located in the cerebellum. MB is the most
III choroid plexus carcinoma (CPC) has frequent mitoses, common malignant brain tumor in children, accounting for
increased nuclear pleomorphism, cellular density, necrotic ~20% of all pediatric brain tumors. MB is rare in adults,
areas, and sheets of tumor cells. The cytogenetics of these comprising ~1% of primary adult intracranial malignant
lesions helps to distinguish them from each other and from tumors. Supratentorial primitive neuroectodermal tumors
entities with which CP tumors may be confused. The CPP is (sPNETs) are histologically very similar to MBs, but are
karyotypically normal, the atypical CPP is hyperdiploid located in the cerebrum, and are clinically more aggressive.
(Fig. 16.4), while CPC is hyperhaploid (Fig. 16.5). The most Cytogenetically, i(17q) is the most common chromosome
common recurrent chromosomal gains in atypical CPP are of abnormality in pediatric MBs, present in ~35% of cases, but
chromosomes 7, 12, and 20, followed by gains of whole it is not found in pediatric sPNET. Recent studies identified
chromosomes 8, 9, 11, 15, 17, 18, and 19. Hyperhaploid characteristic and prognostic cytogenetic subgroups [29–31].
CPCs characteristically have between 32 and 35 chromo- Highest risk pediatric MBs are characterized by gain of 6q,
somes; the most common gains (relative to a haploid back- MYC amplification, MYCN amplification, and ERBB2
ground) in decreasing frequency order are of chromosomes amplification; intermediate risk MB by 17q gain; and low risk
1, 12, 20, 4, and 7 [26–28]. MB by loss of 6q [29, 32]. High-risk genetic abnormalities
Fig. 16.4 Choroid plexus papilloma, grade II, from the lateral ventricle of an 8-month-old boy: 55,XYY,+1,+5,+7,+8,+8,+12,+19,+20. The con-
stitutional karyotype is 47,XYY
Fig. 16.5 Choroid plexus carcinoma, grade III, from the lateral ventricle of a 4-month-old boy: 30<1n>,XY,+1,+2,+4,+12,+20,+21
strongly correlate with the large cell/anaplastic histology Meningeal Tumors

found in ~7% of MBs [29, 33]. MYC/MYCN amplification is
rare (~4–10% of MBs). Patients with low-risk genetic abnor- Meningioma, a tumor that arises from membranes surround-
malities, loss of chromosome 6 or 6q, and nodular desmo- ing the brain, accounts for ~20–30% of primary intracranial
plastic histology have an excellent 5-year overall survival tumors. The World Health Organization classification of
(OS) of ~90%. Most (~90%) MBs have classic histology brain tumors recognizes three grades of meningioma: WHO
with a 60% OS. Gain of chromosome 7, common in MB, is grade I, the most common (70–80%); grade II, atypical
not associated with OS [34]. (5–25%); and grade III (1–3%), the anaplastic type [1].
Cytogenetic abnormalities associated with poor OS in Grade I tumors are slow growing and benign with a low risk
adult MBs include high-level amplification of the oncogene of recurrence, while grade II and III tumors have a greater
CDK6 at 7q21.3 (more frequent than MYC), chromosome likelihood of recurrence and/or aggressive behavior.
17 aberrations including i(17q), and loss of 10q [30]. Meningiomas occur most commonly in middle-age to older
Oncogene amplification frequently associates with aberra- patients with a peak in the sixth and seventh decades. Female-
tion of chromosome 17. Independent significant predictors to-male ratio is ~2:1, with males overrepresented in grade II
for poor prognosis are loss of 10q and gain of 17q. Combined and III tumors [40, 41]. Pediatric (<20 years old) menin-
10q loss and 17q gain show the poorest OS of ~16%; either giomas are rare, comprising ~2% of all meningiomas [42].
10q loss or 17q gain is associated with a 44% OS, and Meningioma was the first solid tumor to be associated
absence of both 10q loss and 17q gain is associated with the with a nonrandom cytogenetic abnormality (monosomy 22).
best OS (92%). Loss of or interstitial deletion of chromosome 22q is the
Supratentorial PNETs are less well characterized due to most common and often the sole anomaly in benign menin-
a smaller number of sPNETs as compared with MBs. The gioma. Atypical and anaplastic meningiomas often show
most frequent cytogenetic aberrations so far associated additional chromosome losses, i.e., 1p, 14q, 18, 10, and 6q.
with sPNET are gain of 1q and losses of 14q, 16p, and Spinal meningiomas are usually low grade with monosomy
19p/q. Gain of 17q is not found in sPNET. Rare amplifications 22 as the sole anomaly [43, 44]. Pediatric meningiomas show
of 1q, 4q12-q13, 8q22-q24, 19q12-q13, and EGFR are similar chromosome anomalies as those in adults. A com-
reported [31, 35]. plex karyotype is more common in pediatric meningiomas,
but there is insufficient data to correlate karyotype with bio-
Atypical Teratoid/Rhabdoid Tumor logic behavior in pediatric tumors [26, 42]. Mutations and/or
Atypical teratoid/rhabdoid tumor (AT/RT) is a highly deletions encompassing the NF2 gene at 22q12.2 are present
malignant tumor in young (<5 years of age) children that in NF2-associated meningiomas and in ~60% of sporadic
may arise infratentorially (posterior fossa) or supratentori- meningiomas [1].
ally. AT/RTs account for ~10% of CNS tumors in infants.
The tumor is defined by the rhabdoid cell, which resembles
a rhabdomyosarcoma cell. Pathologically, AT/RTs may be Genitourinary Tumors (Table 16.2)
difficult to recognize by histology alone due to variable
components of primitive neuroectodermal, mesenchymal, Renal Cell Carcinoma
and epithelial features. AT/RT may be distinguished from
other poorly differentiated and anaplastic CNS tumors Renal cell carcinoma (RCC) is the most common malignant
(e.g., sPNET, MB, or ependymoma) by a characteristic tumor arising from the kidney. Prognosis is related to histo-
loss of chromosome 22 or 22q deletion [36]. The region of logic subtype and tumor stage at diagnosis. Histologic sub-
loss includes the SMARCB1/INI1 gene at 22q11.23. The types include clear cell RCC (70%), papillary RCC (10–15%),
overall prognosis for patients with AT/RT is poor, particu- chromophobe RCC (4–6%), Xp11.2 translocation RCC, and
larly in patients diagnosed at <3 years of age. AT/RT may others. Different subtypes are characterized by different
be sporadic or part of the rhabdoid tumor predisposition genetic abnormalities. Hereditary syndromes with RCC as a
syndrome [36, 37]. Germline deletion or mutation of feature include von Hippel-Lindau, Birt-Hogg-Dube, tuberous
SMARCB1 has been identified in up to 35% of patients sclerosis, hereditary papillary RCC, familial clear cell RCC,
with rhabdoid tumors [38]. Investigation of familial cases hereditary leiomyomatosis and RCC, and familial oncocy-
has revealed other affected relatives, unaffected carrier toma. Hereditary RCCs account for 4% of RCCs [7].
parents, and gonadal mosaicism. Individuals with germline
mutations of SMARCB1 present at a younger age (5 vs. Clear Cell RCC
18 months), may have multiple primary tumors, or affected Clear cell RCC (ccRCC) histology shows cells with clear or
siblings as expected with a germline tumor suppressor granular cytoplasm without a papillary growth pattern. The
gene mutation [36, 38, 39]. majority of ccRCCs have deletion or rearrangement of the
16
Table 16.2 Chromosome abnormalities with diagnostic or clinical significance in genitourinary tumors
Renal
Clear cell RCC Loss of 3 or 3p VHL, PBRMI, PTH1R Non-papillary RCC [45–48]
Loss of 3p with gain of 5q Favorable prognosis [45–48]
Loss of 9p, 14q Unfavorable, shorter survival [45–48]
Papillary RCC Gain of 7, 17, loss of Y MET Adult papillary RCC [45, 49]
t(Xp11.2) RCC t(X;1)(p11.23;q23.1) TFE3-PRCC Xp11.2 RCCs infrequent in adult RCC, [50–54]
common in pediatric RCC
t(X;17)(p11.23;q25.3) TFE3-ASPSCR1 Balanced t(X;17) in RCC; Unbalanced [50–54, 184]
t(X;17) in ASPS
t(X;17)(p11.23;q23.1) TFE3-CLTC [50–54]

t(X;1)(p11.23;p34.3) TFE3-SFPQ [50–54]
t(6;11) RCC t(6;11)(p21.1;q13.1) TFEB-ALPHA Subset of RCC, children, young adults [54, 55]
Chromophobe Loss of 1, 2, 6, 10, 13, 17, 21 Distinguish from oncocytoma [56, 57]
Oncocytoma Loss of 1 or 1p, Y Distinguish from chromophobe [57]
Wilms tumor Loss of 16q, 1p, 4q, 14q, 17p, 22; gain of 1q; TP53 Unfavorable histology; augmented [58–64]
der(1;16)(q10;p10) chemotherapy if loss of 1p, 16q
Clear cell sarcoma (CCSK) t(10;17)(q22.3;p13.3), del(14)(q24.1q31.1) FAM22-YWHAE t(10;17) in 12% of CCSK; t(10;17) also [65–68]
seen in endometrial stromal sarcoma
CMN t(12;15)(p13.2;q25.3), gain of 11, 20, 17, 8 ETV6-NTRK3 Diagnostic; t(12;15) also seen in CFS/IFS [69, 70]
and secretory breast cancer
Rhabdoid tumor (RTK) Loss of 22, del(22q) SMARCB1-INBI1 Diagnostic [71–73]
Prostate Gain of 7q, 8q, loss of 8p, 10q, 13q, 17p, MYC, PTEN, TP53, Poor OS with ERG amplification, [75–80]
7p, 16q, 6q TMPRSS2-ERG TMPRSS2-ERG fusion+; poorest OS with
PTEN-, TP53-
Bladder Gain of 3, 5p, 7, 17, 20, loss of 9, 9p CDKN2A Homozygous deletion CDKN2A higher [81–87]
grade and stage; recurrence, progression
Reproductive
Endometrial stromal tumor (EST) t(7;17)(p15.2;q11.2), t(6;7)(p21.3;p15.2), t(6;10) JAZF1-SUZ12, PHF1-JAZF1, Distinguish from non-EST uterine tumors [88–91]
(p21.3;p11.22), t(10;17)(q22.3;p13.3) PHF1-EPC1, FAM22-YWHAE
Germ cell
Postpubertal GCTs 12p overrepresentation, i(12p) or 12p amplification 12p i(12p), amplification of 12p distinguishes [95, 100–104]
GCTs
Ovarian dysgerminoma; testicular, 12p overrepresentation, i(12p) or 12p amplification, Chromosome enumeration Mediastinal GCT associated with [2, 7, 95, 98,
seminoma, nonseminoma; extragonadal gain of X, 7, 8, 12, 21, loss of 1p, 11, 13, 18 Klinefelter syndrome 100, 102]
Prepubertal GCTs Loss of 1p36, 4q, 6q; gain of 1q, 3p, 16p, 20q; rare Chromosome enumeration 12p gain rare in prepubertal GCT [96, 101, 102]
gain of 12p
ASPS alveolar soft part sarcoma, CFS/IFS congenital fibrosarcoma/infantile fibrosarcoma, CMN congenital mesoblastic nephroma. CCSK clear cell sarcoma of kidney, GCT germ cell tumor, OS
overall survival, RCC renal cell carcinoma, RTK rhabdoid tumor of kidney
379
short arm of chromosome 3 that results in loss of part or all chromosomes 12, 16, and 20 are also frequent. The MET
of 3p, often including the von Hippel-Lindau (VHL) gene at proto-oncogene located at 7q31.2 is mutated in a subset of
3p25.3. Mutations of VHL are present in ~90% of sporadic sporadic pRCC and is responsible for hereditary pRCC
tumors. No association between VHL status, tumor grade, [45, 49].
and stage has been found. Other genes at 3p21.1 (PBRM1)
and 3p21.31 (PTH1R) are reported to be mutated or lost in TFE3 and TFEB Translocation RCC
40 and 76% of ccRCCs, respectively [45–47]. In addition to RCC with Xp11.2 translocations/TFE3 gene fusions is seen
3p loss, ccRCCs may show gain of chromosome 5 or gain of in children and adults, but is more predominant in the pediat-
5q, gain of chromosome 7, and loss of 9p and/or 14q [48]. ric age group. These tumors have a papillary architecture and
Recent work by Dondeti et al. further elucidated two sub- resemble pRCC. Xp11.2 RCCs have been misclassified as
types of VHL deficient ccRCC—H1H2 and H2—based on chromophobe and ccRCCs.
HIF1A expression, with each group having its own specific Several partner genes fuse with TFE3 at Xp11.2. The two
pattern of copy number alterations. The H2 subtype showed most common translocations are t(X;1)(p11.23;q23.1)/TFE3-
gain of 5q and loss of 14q more frequently than H1H2, while PRCC and t(X;17)(p11.23;q25.3)/TFE3-ASPSCR1. Variants
the H1H2 subtype more frequently showed gain of 16p and include t(X;1)(p11.23;p34.3)/TFE3-SFPQ and inv(X)
19p and loss of 6q. Gain of 5q, present in ~30% of ccRCCs, (p11.23q13.1)/TFE3-NONO and others. The Xp11.2 translo-
is reported as a favorable prognostic factor, while losses of cation tumors are reported to be aggressive in both pediatric
9p and 14q are associated with a poor outcome [45]. Two and adult patients, which may in part be due to higher stage
genes identified on 5q—STC2 at 5q35.2 and VCAN at disease at diagnosis [50–54].
5q14.3—are thought to be important in the tumorigenesis of TFE3 is a member of the MiT transcription factor fam-
ccRCCs [47]. ily. Another member, TFEB, is involved in a subset of
RCCs. These RCCs show a t(6;11)(p21.1;q13.1). TFEB at
Papillary RCC 6p21.1 fuses with alpha at 11q13.1. Tumors with the t(6;11)
Papillary RCC (pRCC) is characterized by a papillary are a distinctive subset of RCCs in children and young
growth pattern and occurs in familial and sporadic forms. adults. The tumor has nests of epithelioid cells with clear
Cytogenetically, pRCC shows gains of chromosomes 7 cytoplasm along with a second population of smaller cells
and 17 and loss of the Y chromosome (Fig. 16.6). Gains of usually clustered around hyaline nodules [54, 55].
Fig. 16.6 Papillary renal cell carcinoma from the kidney of a 67-year-old male: 48,X,−Y,+7,+12,+17
Chromophobe RCC and Oncocytoma Of 50 CCSKs studied by RT-PCR, only 12% were FAM22-
Chromophobe RCCs are composed of cells with prominent YWHAE fusion positive [67]. This same translocation and
cell membranes and eosinophilic cytoplasm. They may be gene fusion was also recently characterized and reported in
difficult to distinguish from the benign renal oncocytoma or high-grade endometrial stromal sarcomas [68].
the granular variant of ccRCC. Chromophobe RCC is char-
acteristically hypodiploid with loss of multiple chromosomes Congenital Mesoblastic Nephroma
including chromosomes 1, 2, 6, 10, 13, 17, and 21. Renal Congenital mesoblastic nephroma (CMN), the third most
oncocytomas, composed of large eosinophilic cells, show a common pediatric renal tumor, is an uncommon spindle cell
normal karyotype in 60% of tumors and partial or complete tumor diagnosed in infancy (60%) to <2 years of age. Infantile
loss of chromosome 1 in 40%. Loss of the Y chromosome or congenital fibrosarcoma (CFS) is a soft tissue spindle cell
and chromosome 14 may be seen together with chromosome tumor usually located in an extremity in children under
1 loss [56, 57]. 2 years old. Both CFS and CMN share a common transloca-
tion, t(12;15)(p13.2;q25.3), and gains, in decreasing fre-
Wilms Tumor quency order, of chromosomes 11, 20, 17, and 8 (Fig. 16.7).
Wilms tumor (WT) or nephroblastoma is the most common t(12;15) fuses ETV6 with NTRK3. These chromosomal
primary malignant renal tumor of childhood and the fourth abnormalities distinguish CFS and CMN from other child-
most common pediatric malignancy overall. The classic WT hood spindle cell tumors, such as benign infantile fibromatosis
is triphasic with blastemal, epithelial, and stromal compo- or malignant adult-type fibrosarcoma [69, 70].
nents [2]. Most tumors are sporadic and unilateral.
Approximately 5–10% of patients with Wilms tumor have Rhabdoid Tumor of the Kidney
a germline predisposing gene mutation. WT is associated Rhabdoid tumor of the kidney (RTK), a neoplasm different
with congenital syndromes including Wilms tumor, aniridia, from Wilms tumor, was given the name “rhabdoid” because
genitourinary anomalies, and mental retardation (WAGR) microscopically it resembled a rhabdomyosarcoma (see later).
syndrome, Denys-Drash syndrome, Perlman’s syndrome, RTK, a highly malignant neoplasm that occurs perinatally, dur-
and Beckwith-Wiedemann syndrome. Patients with a ger- ing the first year of life, and occasionally in older individuals,
mline mutation come to medical attention ~10 months earlier is characterized by early metastases and a high mortality rate.
than patients with sporadic tumors and may have bilateral Malignant rhabdoid tumors (MRT) occur in soft tissues, skin,
tumors. With the treatment protocols of two large coopera- CNS, and other extrarenal sites. Concomitant brain tumors are
tive groups that prospectively study children with Wilms present in about one third of fetuses and neonates with RTK
tumor (the National Wilms Tumor Study Group and the [71]. Loss of chromosome 22, 22q deletions, SMARCB1/INI1
International Society of Paediatric Oncology), survival of mutations, and lack of INI1 immunostaining in histopathologic
patients with WT is now >85%. Prognosis correlates with sections facilitate the diagnosis of MRTs [72, 73].
histopathologic features; survival rates are lower with unfa-
vorable histology (UFH) versus favorable histology (FH).
Cytogenetic analysis and aCGH and LOH studies of Wilms Prostate Cancer
tumor tissue have found that loss of 16q consistently corre-
lates with UFH and higher mortality [58, 59]. Loss of 16q Prostate adenocarcinoma is the most common cancer in
often results from an unbalanced der(1;16)(q10;p10). Deletions males, representing 29% of cancers and causing 9% of can-
of 1p, 4q, 14q, 17p, and 22q and gain of 1q are associated with cer deaths in men [74]. Prognosis for patients with prostate
adverse outcome [60–63]. Mutations of TP53 at 17p13.1 are cancer correlates with stage and grade of disease at diagno-
associated with anaplasia, a feature of UFH tumors [63, 64]. sis. Frequently reported chromosome alterations include loss
of 8p, 10q (PTEN), 13q (RB1), 17p (TP53), 7p, 16q, 6q, and
Clear Cell Sarcoma of the Kidney gain of 8q24.1 (MYC) and 7q31. Hemizygous or homozy-
Clear cell sarcoma of the kidney (CCSK), the second most gous deletion of PTEN at 10q23.3 correlates with disease
common pediatric renal tumor, is an unfavorable histology stage, disease progression, and survival [75–77]. PTEN loss
tumor with a propensity for recurrence and metastasis to is among the most frequent recurring abnormalities in pros-
bone, brain, and soft tissue. Little is known about the genet- tate cancer and is seen in preinvasive prostatic intraepithelial
ics of CCSK. Several individual cases have been reported with neoplasia as well as in invasive prostate cancer.
t(10;17)(q22.3;p13.3) and/or del(14)(q24.1q31.1) [65, 66]. In 2005, TMPRSS2-ETS gene fusions were discovered
O’Meara et al. recently identified the genes involved in the in prostate cancer. TMPRSS2, transmembrane protease
t(10;17) as FAM22 at 10q22.3 and YWHAE at 17p13. The serine 2 gene, is androgen-regulated. The fusion of
translocation produces an in-frame fusion gene that is TMPRSS2 at 21q22.3 with an ETS gene results in overex-
comprised of exons 1–5 of YWHAE and exons 2–7 of FAM22. pression of the 5¢ truncated ETS oncogene. The most common
Fig. 16.7 Cellular congenital mesoblastic nephroma, grade III, from seen in congenital/infantile fibrosarcoma and in secretory breast carci-
the kidney of a 6-week-old boy: 48,XY,+11,+11,t(12;15)(p13.2;q25.3). noma (see text for details)
This same t(12;15), which results in ETV6-NTRK3 fusion, can also be
fusion (TMPRSS2-ERG, present in >50% of prostate recurrence. Primary noninvasive (Ta) or superficially invasive
specific antigen-screened localized cancers) results from (T1) transitional cell carcinoma of the bladder can be monitored
an interstitial ~2.8 Mb deletion within 21q22; ERG is using FISH with centromeric and locus-specific DNA probes.
located at 21q22.2 [78, 79]. TMPRSS2-ERG fusion is asso- UroVysion®, a US Food and Drug Administration (FDA)-
ciated with PTEN deletion and with earlier disease recur- approved FISH probe set, detects aneusomy of chromosomes 3,
rence of localized prostate cancer after surgical resection. 7, and 17, and 9p21 (CDKN2A) loss in patients with hematuria
A study by Markert found that the poorest overall survival or a history of bladder cancer [81, 82]. Recent studies support
(57 months mean OS) correlated with TP53 and PTEN FISH analysis for use in monitoring and predicting recurrence
inactivation. TMPRSS2-ERG fusion positive tumors had a risk in patients with non-muscle-invasive bladder cancer and
mean OS of 93 months compared with a mean OS of predicting residual tumor load after transurethral resection
>103 months in the most favorable group. Data from this [83–86]. FISH analysis in combination with cytology and the
study and others suggests that a subset of TMPRSS2-ERG telomeric repeat amplification protocol (TRAP) assay to detect
fusion-positive tumors with amplification of ERG have a telomerase activity increases the sensitivity of detection in
poorer outcome [80]. low-grade and early-stage cancers [87]. See also Chap. 17,
Fig. 17.16.
Bladder Cancer
Tumors of the Reproductive
Bladder cancer can generally be divided into superficial, inva- System (Table 16.2)
sive, and in situ categories, which correspond to the TNM
(“tumor, nodes, metastasized”) staging scheme. Tumor stage is Endometrial Stromal Tumor
the most important independent predictor of patient prognosis.
Most tumors are superficial at diagnosis. However, during the Endometrial stromal tumors (EST) are rare uterine mesen-
course of the disease, multiple recurrences are common, with a chymal tumors that occur in women of reproductive and
minority of superficial tumors progressing to muscle invasion or postmenopausal age. In 2003, the World Health Organization
metastatic disease. Identification of genetic changes in exfoli- (WHO) divided ESTs into three categories: endometrial
ated cells from the bladder has shown utility in detecting disease stromal nodules (ESN), endometrial stromal sarcomas (ESS),
and undifferentiated endometrial sarcomas (UES). ESNs are GCTs account for 15–20% of all ovarian tumors. Most
benign, circumscribed tumors. ESSs are low-grade, malig- are benign cystic teratomas. The remaining, primarily found
nant tumors that invade the myometrium. Both can have in children and young adults, may be malignant with histo-
variant histologic features, but most have a classic morphol- logic types similar to those in the testis. The counterpart to
ogy. UESs are highly aggressive, malignant tumors with the testicular seminoma is the ovarian dysgerminoma, which
cytologic atypia and high mitotic activity. ESSs may be pri- is always malignant. Dysgerminomas account for ~2% of all
mary uterine, metastatic uterine, or primary extrauterine. ovarian tumors, but ~50% of GCTs. They may occur in
The tumors are characterized by reciprocal translocations patients with gonadal dysgenesis.
and gene fusions with t(7;17)(p15.2;q11.2)/JAZF1-SUZ12 in
ESN, ESS, and rarely UES, and t(6;7)(p12.3;p15.2)/JAZF1-
PHF1 or t(6;10)(p12.3;p11.22)/EPC1-PHF1 and variants Extragonadal Germ Cell Tumors
in ESS [88–91]. Lee et al. recently identified a t(10;17)
(q22.3;p13.3)/FAM22-YWHAE in high-grade ESS [68]. This Rare primary brain GCTs occur in the midline, pineal (male
same t(10;17) is found in clear cell sarcoma of the kidney predominance), or suprasellar regions. They account for
(see earlier) [67]. 0.2–1% of brain tumors in those of European descent, but up
to 10% in those of Japanese origin.
Primary mediastinal germ cell tumors account for 10–15%
Germ Cell Tumors (Table 16.2) of all mediastinal tumors [97]. Pediatric mediastinal GCTs
represent ~5% of all GCTs [98]. These tumors have the same
Germ cell tumors (GCTs) are a heterogeneous group of rare histologies as gonadal GCTs, but have a worse prognosis.
benign and malignant tumors. GCTs may arise in the The mediastinum is the most common site of extragonadal
gonads and at extragonadal sites found primarily in the tumors in young males. Mediastinal NSGCTs are associated
body midline (intracranial, mediastinal, retroperitoneal, with Klinefelter syndrome in ~20% of cases [7]. Patients
sacrococcygeal, and others). Primordial germ cells are with Klinefelter syndrome have a 50-fold higher risk for a
thought to give rise to these tumors with aberrant germ cell mediastinal GCT, but do not develop testicular GCT [2].
migration responsible for the extragonadal tumors. While Sacrococcygeal GCTs, the most common extragonadal
the tumors may arise from the same cell type, the clinical GCT in children, present prenatally to ~4 years of age. Most
course and outcome of the various GCTs differ depending (~90%) external lesions are benign, while intrapelvic or
on tumor site and histology [92–94]. Tumors are found pre- intra-abdominal tumors are more likely to be malignant (60–
natally into old age with diverse groups: neonates, infants 90%) [99].
and children £5 years of age, postpubertal to the fifth
decade, and older age.
Chromosome Abnormalities in Germ Cell Tumors
Gonadal Germ Cell Tumors Cytogenetically, additional copies of all or part of 12p are
the characteristic chromosome abnormality associated
Testicular GCTs (TGCT), divided into seminomas and non- with GCTs in adults and postpubertal children [95, 100].
seminomas, are the most common tumor of men in the sec- Additional copies of 12p are present as i(12p) in 80–90%
ond to fourth decades and are responsible for ~10% of cancer of cases, while the remaining tumors show 12p
deaths of men in this age bracket. The US incidence is amplification. Additional cytogenetic anomalies in this
~6/100,000 with a 5:1 white: black ratio. Cryptorchidism is group include gains of an X chromosome and chromo-
associated with ~10% of TGCTs. Seminomatous GCTs, somes 7, 8, 12, and 21 and loss of 1p, 11, 13, and 18
~40–50% of GCTs, are composed of cells that resemble pri- (Fig. 16.8).
mordial germ cells. Nonseminomatous GCTs (NSGCT) may GCTs in infants and prepubertal children characteristi-
be composed of undifferentiated cells that resemble embry- cally show loss of 1p36, 4q, and 6q, and gain of 1q, 3p, 16p,
onic stem cells. Malignant cells can differentiate to generate and 20q. Gain of 12p is rarely reported in prepubertal chil-
yolk sac (endodermal sinus) tumor, embryonal carcinoma, dren and infants [101, 102]. Array CGH has shown proximal
choriocarcinoma, teratomas, and mixed malignant GCTs. 12p11.2-p12.1 gain associated with adult TGCTs and distal
Approximately 60% of TGCTs are composed of more than 12p12-pter gain in yolk sac tumors of very young children
one of these cell types [95]. TGCT of infants are rare neo- [102–104]. GCTs are rare in children between 5 and 9 years
plasms occurring in boys 0–4 years of age, the majority of of age; thus there is a paucity of tumor genetic information in
which are pure yolk sac tumors [96]. this age range.
Fig. 16.8 Malignant mixed germ cell tumor from the mediastinum of an results in gain of 12p in the form of isochromosomes for the short arm
8-year-old boy: 96<4n>,XXYY,+X,+X,+1,idic(1)(p22),−4,−5,−7,+i(12) (lower left) (see text for details)
(p10)x4,+21,+21,−22. In addition to other abnormalities, this karyotype
Gastrointestinal Tumors (Table 16.3) and some have KIT-PDGFRA genomic amplifications,
which are responsible for therapeutic resistance [105].
Gastrointestinal Stromal Tumors Disease-free survival correlates with mutation (KIT = poor),
site (stomach = best), cytogenetic complexity (³3 abnor-
Gastrointestinal stromal tumors (GIST), the most com- malities = poor), and losses of 1p and/or 22q and gains of
mon mesenchymal tumor of the gastrointestinal (GI) tract, 1q and 12q (shorter survival) [110, 111]. GISTs in patients
arise from the connective tissue of the GI wall. with neuro-fibromatosis type I (NF1) lack KIT and
Approximately 90% of GISTs have activating mutations PDGFRA mutations. Rare families have been reported
of the KIT or PDGFRA receptor tyrosine kinase genes with germline KIT or PDGFRA mutations [105, 110].
[105]. In addition to KIT or PDGFRA mutations, other
genetic events involved in tumorigenesis include primar-
ily chromosomal losses (14q, 22q, 1p, 15q, 13q), nuclear/ Liver Tumors
mitochondrial microsatellite instability, LOH at 9p21
(CDKN2A), methylation of CDKN2B (p15), homozygous Hepatoblastoma
loss of TLX2 (HOX11L1), and rare gene amplification Hepatoblastoma (HB) is the most common primary malig-
(MYC, MDM2, EGFR, CCND1, KIT) [106–109]. The KIT nant tumor of the liver in children. This rare tumor accounts
and PDGFRA mutations are diagnostic, and tyrosine for ~1% of all pediatric malignancies, with ~100–150 new
kinase inhibitors (TKIs) are used as targeted therapy. The tumors per year in the United States.
molecular aberrations of KIT-PDGFRA are correlated Cytogenetic analysis of hepatoblastomas has found that
with cell histomorphology, metastasis, prognosis, and the most common anomalies are numerical, with gain of
efficacy of targeted therapy. Genotyping these tumors whole chromosomes, specifically of chromosomes 20, 2, and
helps to guide therapy, as the effects of TKIs vary with the 8, in decreasing order of frequency (Fig. 16.9). The most
presence or absence and site of KIT-PDGFRA mutation. common structural abnormalities result in gain of chromo-
Metastatic GISTs often have secondary KIT kinase mutations, some 1 long-arm material. An unbalanced der(4)t(1;4) that
Table 16.3 Chromosome abnormalities with diagnostic or clinical significance in gastrointestinal and other tumors
GIST Loss of 14q, 22q, 1p, 15q, 13q; gain KIT, PDGFRA KIT, PDGFRA mutation diagnostic, [105–111]
of 1q, 12q response to TKIs
Liver
Hepatoblastoma Gain of 1q, 2, 8, 20, der(4)t(1;4), Distinguish from HCC, HMH [112, 113]
loss of 4q
Hepatic mesenchymal t(11;19)(q13;q13.4), t(19q13.4) Unknown genes Distinguish from hemangioma or [120]
hamartoma (HMH) malignant tumor
Salivary gland
Pleomorphic adenoma t(3;8)(p22.1;q12.1), t(12q14.3), +8 CTNNB1-PLAG1, Diagnostic; benign tumor [127–129]
HMGA2
Ca-ex-PA HMGA2, MDM2 amplification HMGA2, MDM2 Amplification contributes to [128]
malignant transformation of PA
Mucoepidermoid cancer t(11;19)(q21;p13.11) in 40–80%; MAML2-CRTC1 Malignant; t(11;19) assoc with [130, 131]
gain of 7, 8, X, loss of 6q better outcome
Warthin’s tumor t(11;19)(q21;p13.11) in low percentage MAML2-CRTC1 Benign; t(11;19) w/metaplasia [132]
Dermal
DFSP and variants (GCF, t(17;22)(q22;q13.1), der(22)t(17;22) COL1A1-PDGFB Diagnostic for DFSP; response [135–140]
Bednar, other) or r(22)t(17;22) to TKIs
Hidradenoma t(11;19)(q21;p13.11), gain of 7, 8, MAML2-CRTC1 Clear cell variant [133, 134]
X, loss of 6q
Cutaneous melanoma Gain of 6p, 1q, 7, 8q, 17q, 11q, 20q; CDKN2A, BRAF, CDKN2A [143, 144]
loss of 9, 9p, 10q, 6q PTEN
Uveal melanoma Loss of 3, gain of 8q GNA11, GNAQ Monosomy 3 correlates with [145, 146]
metastatic disease
Breast
Invasive intraductal dmin, hsr (ERBB2 amplification) ERBB2 Improved outcome with targeted [149–151]
therapy
Secretory breast t(12;15)(p13.2;q25.3) ETV6-NTRK3 Favorable; distinguish from other [152]
breast lesions
Lung
NSCLC EGFR high copy number or EGFR Response to TKIs [155]
amplification, loss of 3p, gain of 7
inv(2)(p21p23.2) EML4/ALK Response to TKIs [156, 157]
Ca-ex-PA Carcinoma ex Pleomorphic Adenoma, DFSP dermatofibrosarcoma protuberans, dmin double minutes, GCF giant cell fibroblastoma,
GIST gastrointestinal tumor, HCC hepatocellular carcinoma, hsr homogeneously stained regions, NSCLC non-small cell lung carcinoma, TKI
tyrosine kinase inhibitor
results in gain of 1q and loss of 4q is the most common recur- Beckwith-Wiedemann syndrome (BWS), familial adenoma-
ring structural abnormality [112]. Rare genomic and expres- tous polyposis, and Li-Fraumeni syndrome. HB can also be
sion profiling studies have confirmed these abnormalities seen in glycogen storage disease type I. Premature infants,
and further refined the regions of gain and loss. Single nucle- particularly those with low or very low birth weight, are at
otide polymorphism (SNP) array analysis revealed paternal increased risk of developing hepatoblastoma [115].
11p uniparental disomy (UPD, see Chap. 20) that included Familial adenomatous polyposis (FAP), a syndrome of
the IGF2-H19 region at 11p15.5 [113]. Molecular studies early-onset colonic polyps and adenocarcinoma, results from
have discovered mutations in key genes that are important in germline mutations in the APC tumor suppressor gene at
the genetic pathways of the developing liver [114]. These 5q22.2. Children with a family history of FAP have a
studies may help elucidate the pathogenetic mechanisms significantly increased risk for hepatoblastoma [116]. One
responsible for the development of hepatoblastoma. study estimated that 1 in 20 hepatoblastomas is probably asso-
SMARCB1 (INI1) testing helps differentiate hepatoblastoma ciated with FAP. APC mutations, common in patients with
from a more aggressive variant that mimics rhabdoid tumor. hepatoblastoma and FAP, are rare in patients with sporadic
Most cases of HB are sporadic. However, HB is associ- hepatoblastomas [117, 118]. Children who survive HB should
ated with several cancer predisposition syndromes including be considered for evaluation of FAP, and those patients found
Fig. 16.9 Hepatoblastoma, mixed embryonal and fetal, from the liver of a 9-year-old extremely premature boy: 50,XY,del(1)(q32q42),add(2)
(q23),+add(2)(q31),add(3)(p21),add(6)(q23),+8,+12,add(14)(q13),+20
to carry an APC mutation need close surveillance because of 19q13.4 with t(11;19)(q13;q13.4) so far the most common
their increased risk for colonic polyps and adenocarcinoma. reciprocal translocation [120].
Patients with hemihypertrophy or Beckwith-Wiedemann HMH has been associated with placental mesenchymal
syndrome should be screened using a-fetoprotein (AFP) as dysplasia (PMD), an uncommon disorder of the placenta.
a marker to detect hepatoblastoma. AFP monitoring should Placental changes include cystic villi with dilated/thick-
be performed every 3 months until the child is at least walled vessels, which can mimic a partial hydatidiform
4 years. Loss of heterozygosity of 11p markers occurs com- mole. In contrast to a partial mole, PMD can coexist with
monly in hepatoblastoma associated with BWS and a normal viable fetus [121]. Both HMH and PMD have
hemihypertrophy. been considered developmental aberrations rather than
Children with HB often have very elevated AFP levels true neoplasms [120].
and may have anemia and thrombocythemia. Complete sur- Further analysis suggests PMD may be a disease of
gical removal effects a cure. However, ~70% of tumors are dysregulated imprinting with mosaic placental and fetal
metastatic or unresectable at diagnosis. Even with aggressive paternal UPD [122–124]. HMH and PMD have been asso-
chemotherapy, 25–30% remain resistant. AFP levels return ciated with Beckwith-Wiedemann syndrome (BWS), an
to normal with tumor removal and rise if the tumor returns, imprinting disorder [125]. One cause of BWS is paternal
thus providing a monitor for disease [115, 119]. UPD at 11p15.5. A paternally imprinted gene, PEG3, is
within the locus at 19q13.43 that is commonly disrupted
Hepatic Mesenchymal Hamartoma in HMH [123].
Mesenchymal hamartoma of the liver (HMH) is a rare benign While HMH is considered to be benign, there is a low risk
lesion that occurs mainly in infants. Histologically, the lesion of malignant transformation. Several malignant tumors of
has cystic and solid areas with islands of hepatocytes and poorly undifferentiated embryonal sarcoma (UES) of the liver have
defined or dilated biliary ducts in a myxoid stroma. Complete been reported to arise from HMH. Cases of UES have been
surgical removal is curative. In the few reported cases, a com- reported to have involvement of the same 19q13.4 locus as
mon denominator has been involvement of chromosome band that of HMH [120, 126].
Salivary Gland Tumors but not exclusively in the parotid gland. A t(11;19)(q21;p13.11)
that results in CRTC1-MAML2 fusion is found in 40–80% of
Pleomorphic Adenoma tumors (Fig. 16.11). Evidence from several studies found that
Pleomorphic adenoma (PA), a benign mixed salivary gland fusion positive tumors are less likely to recur or metastasize
tumor, has been associated with abnormal karyotypes in the and are associated with an overall better survival [130, 131].
majority of cases, with nonrandom involvement of 8q12.1 t(11;19) negative cases show gain of chromosomes 7, 8, and
(locus of the pleomorphic adenoma (PLAG1) gene), 3p22.1 X and 6q deletion [131].
(CTNNB1), 12q14.3 (HMGA2), and gain of chromosome 8
[127, 128]. t(3;8)(p22.1;q12.1) is the most common translo- Warthin’s Tumor
cation (Fig. 16.10). Reported partner genes for PLAG1, Warthin’s tumor, the second most common salivary gland
CTNNB1, and HMGA2 vary. tumor, is a benign neoplasm that arises almost exclusively
Few PAs (~6%) undergo malignant transformation to car- in the parotid gland. Warthin’s tumor, also referred to as
cinoma ex pleomorphic adenoma (Ca-ex-PA). Recent work papillary cystadenoma lymphomatosum, is composed of
has shown that HMGA2 translocations are often associated polyclonal lymphoid cells and neoplastic epithelium.
with gene amplification. There is an increased risk of malig- Recurrence and malignant transformation occur very rarely
nant transformation with HGMA2 amplification. HGMA2 is [2]. t(11;19)(q21;p13.11), which results in CRTC1-MAML2
usually co-amplified with others genes, most often MDM2. fusion, is found in a low percentage of Warthin’s tumors
Other genetic abnormalities thought to contribute to [132]. Tumors that exhibit the translocation or are fusion-
malignant transformation of PA to Ca-ex-PA include dele- positive characteristically have metaplasia of the oncocytic
tions of 5q23.2-q31.2 and TP53, gains of 8q12.1 (PLAG1) epithelium. There is ongoing discussion regarding the asso-
and 8q22.1-q24.1 (MYC), and ERBB2 amplification [129]. ciation of the t(11;19) CRTC1-MAML2 fusion with both
Warthin’s tumor and MEC; there is morphologic overlap
Mucoepidermoid Carcinoma between metaplastic Warthin’s tumor and MEC [130].
Mucoepidermoid carcinoma (MEC) accounts for ~15% of Further, clear cell hidradenoma, a benign sweat gland
salivary gland tumors and is the most common primary malig- tumor, also demonstrates the t(11;19) and CRTC1-MAML2
nant tumor of the salivary gland. MEC arises predominantly, fusion [133, 134].
Fig. 16.10 Pleomorphic adenoma, submandibular, from a 10-year-old girl: 46,XX,t(3;8)(p22.1;q12.1). The translocation results in CTNNB1-
PLAG1 fusion
Fig. 16.11 Mucoepidermoid carcinoma from the parotid gland of a 9-year-old boy: 46,XY,t(11;19)(q21;p13.11). This same t(11;19), which
results in MAML2-CRTC1 fusion, can be seen in hidradenoma, a benign sweat gland tumor
at chromosome 22q13.1. Variability of the COL1A1 break

Dermal Tumors (Table 16.3) point has no correlation with any clinical or histological
parameter [135, 137–139]. However, cytogenetically, ring
Dermatofibrosarcoma Protuberans chromosomes are common in adult DFSP, while the translo-
cation derivatives are seen in all childhood cases [140]. The
Dermatofibrosarcoma protuberans (DFSP) is an intermedi- COL1A1-PDGFB chimeric gene protein causes unregulated
ate-grade soft tissue malignancy that usually arises in the expression of platelet-derived growth factor leading to abnor-
dermis and subcutaneous tissue of adults and rarely in chil- mal activation of the platelet-derived growth factor receptor
dren [135]. DFSP is a slow-growing infiltrative dermal neo- beta (PDGFRB) tyrosine kinase through an autocrine loop.
plasm with a propensity to recur locally after surgical Tyrosine kinase inhibitors are the current therapy for recur-
resection, but is rarely metastatic (1–4%). Tumor-related rent, metastatic, or inoperable tumors [141, 142].
deaths are very rare. There are several histologic variants of Demonstration of the COL1A1-PDGFB fusion is necessary
DFSP, such as giant cell fibroblastoma, Bednar tumor, and for the diagnosis of DFSP or DFSP variants [137].
other fibrohistiocytic tumors, which should be considered in
the differential diagnosis. Variants of DFSP
DFSP is characterized by a balanced or unbalanced form Several histologic variants of DFSP are described including
of a translocation t(17;22)(q21.3;q13.1) or by supernumer- giant cell fibroblastoma (GCF), pigmented Bednar tumor
ary ring chromosomes derived from this chromosome 22 (BT), DFSP with fibrosarcoma (FS)-like changes (DFSP-FS),
[der(22)r(17;22)] that contain low-level amplified sequences and others.
from 17q21.31-qter and from 22q11.1-q13.1 [136]. The Giant cell fibroblastoma (GCF), also called juvenile DFSP,
unbalanced form is usually a der(22)t(17;22) (Fig. 16.12). more commonly affects infants and children. Bednar tumor, a
Both the ring and linear forms of the derivative chromosome pigmented form of DFSP with additional melanin-containing
22 result in fusion of the a(alpha)-1 chain of type 1 collagen dendritic cells, occurs in early to middle adulthood. DFSP-FS
gene (COL1A1) at chromosome 17q21.3 with the second is a more cellular form with higher mitotic rate. All variants
exon of the platelet-derived growth factor–b (PDGFB) gene are characterized by COL1A1-PDGFB fusion [137, 140].
Fig. 16.12 Dermatofibrosarcoma protuberans from the breast of a 2-year-old girl: 50,XX,+4,+11,+18,+der(22)t(17;22)(q21.3;q13.1). The arrow
indicates the chromosome 22 derived from the translocation, which results in COL1A1-PDGFB fusion (see text for details)
Hidradenoma nevi. Individuals with dysplastic nevus syndrome have a

50% risk for developing melanoma by 60 years of age.
Hidradenoma, a benign sweat gland tumor, often presents as a Frequent aberrations found in melanomas include gains of
solitary, slow-growing, solid, or cystic intradermal nodule. 6p, 1q, 7p, 7q (BRAF), 8q, 17q, 11q, and 20q and losses of
Malignant transformation is uncommon. t(11;19)(q21;p13.11) 9p (CDKN2A), 9q, 10q (PTEN), 10p, and 6q [143, 144].
is characteristic of a subset of these tumors (Fig. 16.11). The Uveal melanoma, the most common form of primary eye
CRTC1-MAML2 fusion was demonstrated in 50%, specifically cancer, is characterized by loss of chromosome 3 in ~50% of
in tumors with clear cells, representing the clear cell variant tumors; metastasis is correlated with such loss. Chromosome
of hidradenoma [133]. As noted earlier, salivary gland 3 loss is often accompanied by i(8q); tumors without loss of
Warthin’s tumor and mucoepidermoid carcinoma also dem- chromosome 3 have 6p abnormalities.
onstrate the t(11;19)/CRTC1-MAML2 fusion. The common Two regions of chromosome 3, 3p25 and 3q24-26, appear
glandular denominator for these different tumors suggests they to harbor tumor suppressor genes. More than 80% of uveal
may originate from a common progenitor cell in salivary, bron- melanomas have been found to have a constitutively active
chial, and sweat glands [130, 133]. somatic mutation of one of two genes, GNA11 at 19p13.3
and/or GNAQ at 9q21.2. These genes appear to contribute to
the development of these tumors [145, 146].
Malignant Melanoma
Melanomas are malignant lesions, primarily cutaneous, but Epithelial Cancer (Table 16.3)
may occur on mucosal surfaces and in the eye. Cutaneous
malignant melanoma, a pigmented skin lesion, may be lethal Breast Cancer
if not recognized and completely excised prior to metastasiz-
ing. Malignant melanomas spread superficially before pro- Breast carcinoma is the most common cancer in women and
gressing to invade the deeper dermal tissues. Malignant the second leading cause of cancer deaths in women. The
melanoma may be recognized by visible changes of a pig- lifetime risk for breast cancer for women in the general popu-
mented lesion (mole) characterized by changes in size and lation is 1 in 8. A positive family history of breast cancer
color and irregular borders, and may evolve from dysplastic increases this risk. A germline mutation of one of the known
breast cancer predisposing genes greatly increases risk [2]. Non-small Cell Lung Carcinoma
Currently known breast cancer genes explain only ~30% of Adenocarcinoma is the most common type of lung cancer in
the heritability. Mutations of the breast cancer predisposing women and nonsmokers. KRAS mutations occur primarily in
genes BRCA1 and BRCA2 account for ~16–20% of the famil- adenocarcinoma, while TP53, RB1, and CDKN2A mutations
ial risk of breast cancer in the general population [147, 148]. occur in squamous cell and adenocarcinoma. EGFR muta-
tions and amplification occur more frequently in patients
Invasive or Metastatic Breast Cancer with adenocarcinoma histology, no history of smoking, East
Prognosis for patients with breast cancer correlates with Asian ethnicity, and female gender [2].
stage, histologic type and grade, hormonal (estrogen and Up to 20% of non-small cell lung carcinomas (NSCLC) have
progesterone) receptor, and ERBB2 (HER2) status. EGFR mutations and/or amplification, and ~80–85% of patients
Amplification and/or protein overexpression of ERBB2, with EGFR mutations respond to therapy with tyrosine kinase
found in ~20% of new diagnosis breast cancer, is associated inhibitors. FISH analysis detects EGFR amplification (defined
with more aggressive disease and decreased survival time. as a gene:chromosome ratio ³2, or ³15 copies per cell in ³10%
Accurate assessment of ERBB2 oncogene status is critical to cells) and polysomy (defined as ³4 copies in ³40% of cells)
care of the patient with invasive or metastatic breast cancer [153, 154]. Fukuoka et al. reported that EGFR mutation was the
as it is used in selection of therapy. The risk of recurrence strongest predictive biomarker for benefit of gefitinib over car-
and mortality are reduced by ~50% and ~33%, respectively, boplatin/paclitaxel on progression-free survival (PFS) and over-
in patients with early-stage ERBB2-positive tumors treated all response rate (ORR). PFS was significantly longer with
with trastuzumab (Herceptin®). Data indicate that patients gefitinib for patients whose tumors had both high EGFR gene
with tumors that show ERBB2 overexpression (3+ by IHC) copy number and EGFR mutation [155].
or gene amplification (by FISH) be considered a candidate A subset of NSCLCs exhibit ALK gene rearrangement,
for anti-ERBB2 therapy [7] (see also Chap. 17, Fig. 17.15). e.g., EML4-ALK, which results from an inv(2)(p21p23.2). The
Because correlation between the IHC and FISH is <100%, EML4-ALK fusion is found predominantly in younger (aver-
guideline recommendations for ERBB2 testing were estab- age 52 years) nonsmokers with adenocarcinoma histology.
lished by an expert panel of members from the American The fusion protein causes constitutive activation of the ALK
Society of Clinical Oncology (ASCO) and the College of tyrosine kinase. Most reports show no overlap with EGFR or
American Pathologists (CAP) [149–151]. RAS gene mutations. ALK positive patients have shown
significantly better overall survival at 1 and 2 years when
Secretory Breast Carcinoma treated with crizotinib, a drug targeted against the constitu-
Secretory breast carcinoma (SBC), a rare subtype of breast tively active tyrosine kinase. The FDA has approved treatment
cancer, is characterized by abundant eosinophilic secretions with crizotinib with FISH testing as a companion diagnostic
in intracellular vacuoles and intercellular spaces. SBC occurs test for ALK detection [156, 157]. See also Chap. 17.
in both sexes and in children and adults, but is most often
seen in young adult females. Most tumors are hormone
receptor and ERBB2 negative. SBC is associated with a Bone and Soft Tissue Tumors (Table 16.4)
favorable prognosis, even in cases with local recurrence or
£3 positive lymph nodes. SBC is characterized by t(12;15) Congenital Fibrosarcoma/Infantile Fibrosarcoma
(p13;q26)/ETV6-NTRK3 fusion, which results in a chimeric
tyrosine kinase fusion product. This same t(12;15) ETV6- Congenital fibrosarcoma (CFS), also referred to as infantile
NTRK3 fusion is also seen in congenital (infantile) fibrosarcoma (IFS), is a rare soft tissue tumor composed of
fibrosarcoma and congenital cellular mesoblastic nephroma malignant fibroblasts in a collagen background. It is highly
[152] (see Fig. 16.7). cellular and composed of spindle cells arranged in fascicles.
Tumor cells are diffusely positive for vimentin and may show
focal positivity for actin but are negative for S-100, desmin,
Lung Cancer and myoglobin [158]. The cellular variant of congenital meso-
blastic nephroma (CMN) is histologically similar to CFS, and
Lung cancer, the most common cancer worldwide, is largely both tumors share a similar cytogenetic aberration, t(12;15)
due to tobacco products. Incidence and mortality rates of (p13.2;q25.3), that rearranges the ETV6 and NTRK3 genes
lung cancer have been declining since ~1990 secondary to (Fig. 16.7). Because of the histologic and cytogenetic similari-
decreased smoking rates over the past 30 years. The most ties, as well as the fact that both tumors are low-grade and
common types are non-small cell carcinomas (which include highly responsive to chemotherapy, cellular CMN is consid-
adenocarcinoma, squamous cell carcinoma, and large-cell ered a visceral form of CFS. Fusion of the promoter region of
carcinoma) and small-cell carcinomas [7]. the transcription factor ETV6 with the tyrosine kinase receptor
16
Table 16.4 Chromosome abnormalities with diagnostic or clinical significance in bone and soft tissue tumors
CFS/IFS t(12;15)(p13.2;q25.3), gain of 11, 20, 17, 8 ETV6-NTRK3 Distinguish from malignant adult-type [69, 158–161]
fibrosarcoma
Synovial sarcoma t(X;18)(p11.23;q11.2) SSX1-SS18, SSX2-SS18 Biphasic, unfavorable monophasic [162–165]
Gain of 12q, 21q22 TSPAN31 amp Unfavorable prognosis [166]
Lipoma
Conventional 12q14.3 65% of lipomas; t(3;12)(q28;q14.3) HMGA2 Distinguish from LPS [167, 168]
in 25%; many variant translocations
Loss of 13q, 6p21 or 6p rearrangement 15–20% lipomas w/o 12q abnormalities [167, 168]
Spindle/pleomorphic Loss of 13q, 16q, karyotype more complex Distinguish from LPS [167]
Atypical lipoma Rings, giant markers Deep seated, larger tumors, older age [167, 168]
Lipoblastoma 8q12.1 rearrangements, gain of 8 PLAG1 Distinguish from LPS [169]
Liposarcoma (LPS)
Myxoid or myxoid round cell t(12;16)(q13.3;p11.2), t(12;22)(q13.3;q12.2) DDIT3-FUS, DDIT3-EWSR1 Diagnostic of M/RC LPS [171, 172]
Well-differentiated (WDLPS) Rings, giant markers, dmin MDM2, CDK4, HMGA2 Distinguish from lipoma [171, 173–175]
Dedifferentiated (DDLPS) Rings, giant markers, dmin MDM2, CDK4, HMGA2 Aggressive, poor prognosis [171, 173–175]
Pleomorphic LPS Loss of 13q, 17p, 17q RB1, TP53, NF1, MAD2L1 amp Aggressive, myxofibrosarcoma-like histology [171, 173]
Leiomyoma t(12;14)(q14.3;q24), loss of 7q22-q31.1, +12 HMGA2 Distinguish from leiomyosarcoma, 7q, 12 [176–178]
associated w/tumor size
Leiomyosarcoma Loss of 17p TP53 Higher tumor grade [179]
Alveolar soft part sarcoma der(17)t(X;17)(p11.23;q25.3) ASPSCR1-TFE3 Diagnostic of ASPS [182–184]
(ASPS)
Osteosarcoma Loss 6q, 10, 13, 18; gain/amp 17p11.2, 6p12, RB1, TP53, MYC amp TP53 mutation correlates with genomic [185, 186]
1p32p36, 8q24 instability
Aneurysmal bone cyst t(16;17)(q21;p13.2), t(1;17)(p34.3;p13.2), CHD11-COL1A1, THRAP3-USP6, CNBP- Benign, locally aggressive, recurrences [188–192]
t(3;17)(q21.3;p13.2), t(9;17)(q22.31;p13.2), USP6, OMD-USP6, USP6-COL1A1 common
inv(17)(p13.2q21.33)
Extraskeletal myxoid t(9;22)(q22;q12.2), t(9;17)(q22;q12), t(9;15) NR4A3-EWSR1, NR4A3-TAF15, NR4A3- Diagnostic [195, 196]
chondrosarcoma (q22;q21.3) TCF12
Angiomatoid fibrous t(2;22)(q33.3;q12.2), t(12;16)(q13.12;p11.2), CREB1-EWSR1, ATF1-FUS, ATF1-EWSR1 t(2;22) in majority; t(12;16) is a variant; [197–199]
histiocytoma t(12;22)(q13.12;q12.2) t(12;22) atypical presentation in organs
Inflammatory myofibroblastic t(1;2)(q21.3;p23.2), t(2;19)(p23.2;p13.12), TPM3-ALK, ALK-TPM4, ALK-CLTC, May recurrence, rarely metastatic [200, 201]
(IMT) t(2;17)(p23.2;q23.1), t(2;2)(p23.2;q12.3) ALK-RANBP2
Epithelioid variant t(2;2)(p23.2;q12.3) ALK-RANBP2 Rapid recurrence, poor prognosis [202]
Malignant rhabdoid tumor Loss of 22q, rare t(1;22)(p36;q11.2) SMARCB1-INI1 Rare tumor prenatal/perinatal; poor prognosis [38, 71, 203, 204]
(MRT)
aCGH array comparative genomic hybridization, CFS/IFS congenital fibrosarcoma/infantile fibrosarcoma, dmin double minute, M/RC LPS myxoid/round cell liposarcoma, w/ with, w/o without
391
NTRK3 results in dysregulated NTRK3 tyrosine kinase activity may form glands. The spindle cells are arranged in fascicles
and is postulated to be the initial transforming event [69, 159]. and surround the epithelial cells. Monophasic synovial sar-
Both CFS and CMN have a favorable prognosis and are docu- coma is composed of only spindle cells, or rarely only epi-
mented to have the potential for spontaneous regression [160]. thelial cells. Tumors composed of only spindle cells can be
Congenital fibrosarcomas usually present in the perinatal histologically misdiagnosed as fibrosarcomas or malignant
period in the subcutaneous tissues of the extremities. Wide peripheral nerve sheath tumors. Immunohistochemically,
local excision is the treatment of choice unless size, anatomic synovial sarcoma shows positivity for keratin and epithelial
location, or metastases dictate otherwise [69, 161]. membrane antigen, distinguishing this entity from other sar-
Cytogenetic aberrations in addition to t(12;15)(p13.2;q25.3) comas. These tumors require aggressive treatment. Common
include gains of chromosomes 8, 11, 17, and 20. Tumors with sites of metastases include lung, bone, and lymph nodes [2].
these additional chromosome aberrations often have a more A characteristic cytogenetic aberration, t(X;18)
cellular histology, and acquisition of these polysomies has been (p11.23;q11.2), is present in greater than 95% of tumors
associated with progression from a classic to cellular histology (Fig. 16.13). This translocation fuses SS18 at 18q11.2 with
in mixed histology CMN. Therefore, these chromosomal poly- SSX1, SSX2, or SSX4, all in Xp11.2, producing chimeric tran-
somies are considered secondary oncogenic events [69]. scription factors. Both RT-PCR and FISH utilizing a breaka-
part probe for SS18 may be helpful adjuncts in diagnosing
this entity [162, 163] (see also Chap. 17, Fig. 17.14). The two
Synovial Sarcoma most common fusion transcripts appear to be associated with
specific histologic subtypes, the SS18-SSX2 rearrangement
Synovial sarcomas (SS) account for approximately 10% of presents in the monophasic tumors, and the more aggressive
all soft tissue sarcomas and are the fourth most common sar- biphasic tumors are associated with the SS18-SSX1 transcript
coma. The lower extremity is involved in 60–70% of cases. [164, 165]. Whether the fusion transcript type has prognostic
There are two histologic subtypes of synovial sarcoma: significance as an independent variable remains unclear.
monophasic and biphasic. Biphasic tumors have two types of Studies by aCGH have associated tumors with gains of 12q
differentiation: epithelial and mesenchymal. The epithelial and 21q22 and gain and/or amplification of TSPAN31 at
cells are cuboidal to columnar and grow in solid cords or 12q14.1 with unfavorable clinical outcomes [166].
Fig. 16.13 Synovial sarcoma from the thigh of a 46-year-old man: 45,Y,t(X;18)(p11.23;q11.2),tas(13;19)(p13;p13.3). The translocation (arrows)
results in SSX1-SS18 fusion (see text for details)
Lipoma Liposarcoma
Lipomas are benign neoplasms of fat. They are the most Liposarcoma (LPS) is the most common soft tissue sarcoma,
common soft tissue tumors of adults and are categorized accounting for approximately 20% of mesenchymal malig-
based on morphologic features. Subtypes include conven- nancies [170]. The tumor is most common in adults, present-
tional lipoma, fibrolipoma, angiolipoma, spindle cell ing between 40 and 60 years of age. Tumors arise in the deep
lipoma, myelolipoma, and pleomorphic lipoma. The most soft tissues of the proximal extremities and retroperitoneum
common subtype is the conventional lipoma. Histologically, and may become large masses [2]. There are three histologic
it is a well-encapsulated mass of mature adipocytes with subtypes: myxoid and round cell, well-differentiated, and
variability in size. It most frequently presents in mid- pleomorphic [171].
adulthood and arises in the subcutis of the proximal Myxoid LPS accounts for one third of all LPS. It is a
extremities and trunk. Lipomas are soft, mobile tumors malignant tumor composed of round- to oval-shaped primi-
that are usually cured by complete excision. Some of the tive nonlipogenic mesenchymal cells with small signet-ring
morphologic subtypes are associated with particular cyto- lipoblasts in myxoid stroma with a characteristic branching
genetic aberrations, distinguishing them from liposarcoma vascular pattern [170]. Some myxoid liposarcomas undergo
[2]. Conventional lipomas are associated with rearrange- histologic change to a hypercellular, round cell morphology,
ment of 12q14.3, most commonly as a result of a t(3;12) which is associated with a poorer prognosis than those with
(q28;q14.3) resulting in rearrangement of HMGA2. pure myxoid histology. Tumors with myxoid histology only
Rearrangements of 13q and 6p21 are also common aberra- or those with myxoid and round cell histology are character-
tions seen in these tumors. Supernumerary rings or giant ized by a t(12;16)(q13.3;p11.2) that fuses DDIT3 at 12q13.3
marker chromosomes, which characterize atypical lipoma- and FUS at 16p11.2. Rare cases of myxoid LPS demonstrate
tous tumors, are associated with an older age and larger a variant translocation, t(12;22)(q13.3;q12.2), which fuses
tumor size at presentation as well as with a propensity to DDIT3 with EWSR1 [171]. FISH analysis using a breakapart
recur locally. Atypical lipomatous tumor is also called probe set for DDIT3 has been shown to have high sensitivity
well-differentiated liposarcoma. Spindle cell and pleomor- and specificity in the diagnosis of this neoplasm [172].
phic lipomas are characterized by losses in 13q and 16q. Well-differentiated liposarcoma (WDLPS), also referred
The presence of these genetic aberrations aids in distin- to as atypical lipomatous tumor, comprises 40–45% of all
guishing this benign neoplasm from the malignant liposar- LPS. WDLPS is a low-grade tumor that may recur after
coma [167, 168]. removal, but rarely metastasizes. It occurs in the retroperito-
neum and deep soft tissues of the extremities—commonly
the thigh. It has a peak incidence in the sixth decade. WDLPS
Lipoblastoma is characterized cytogenetically by supernumerary ring chro-
mosomes, giant marker chromosomes, and double minutes
Lipoblastoma, a rare benign soft tissue mesenchymal that are associated with amplification of MDM2 and CDK4
tumor, arises from fetal white fat and occurs almost exclu- (Fig. 16.14). The modal chromosome number of abnormal
sively in children under 3 years of age. Lipoblastomas are clones in WDLPS is usually near-diploid but may be near-
superficial, lobulated, small, localized masses. When tetraploid [171]. Dedifferentiated liposarcoma (DDLPS)
diffuse, rather than localized, the tumor is referred to as represents progression from low- to high-grade nonlipogenic
lipoblastomatosis. The tumors are most often found in the morphology within a WDLPS. DDLPS is more aggressive,
extremities, but may involve other sites and organs. They with a metastatic rate of 10–20% and overall mortality of
have no malignant potential, but may recur with incom- 50–75%. Cytogenetically, DDLPS shows the same supernu-
plete surgical excision. Histologically, lipoblastoma may merary ring and giant marker chromosomes as WDLPS.
be difficult to distinguish from an atypical lipo- Several studies have now compared the genetic aberra-
matous tumor/well-differentiated liposarcoma or myxoid tions of WDLPS and DDLPS, seeking differences to eluci-
liposarcoma. date the genes that influence progression. The majority of
For cases with ambiguous clinical or histologic fea- WDLPS and DDLPS show amplification of MDM2, CDK4,
tures, chromosomal analysis provides the only unequivo- HMGA2, and TSPAN31 within the 12q13-q15 region.
cal diagnostic confirmation. Cytogenetically, ~70% of Chromosome regions with identified differences so far
lipoblastomas have 8q12 rearrangements with involve- include 1p32.2 amplification (JUN), 6q23 amplification
ment of PLAG1 that results in transcriptional upregulation (MAP3K5 [ASK1]), loss of 11q23-24, loss of 19q13, and
of the PLAG1 oncogene. Another ~20% have trisomy or amplification of GLI1 at 12q13.3 detected in DDLPS but not
polysomy of chromosome 8 with or without 8q12 rear- in WDLPS by aCGH studies [173–175]. Tap et al. found
rangement [169]. amplification of GLI1, JUN, and MAP3K5 to be mutually
Fig. 16.14 Dedifferentiated liposarcoma from the retroperitoneum FISH analysis was positive for amplification of DDIT3, CDK4, and
of a 76-year-old male: 47,XY,t(3;20)(q27;q12),+r(12),add(16) MDM2 (not shown). This same karyotype and amplification may also
(p13.3). The arrow indicates the ring chromosome 12. Interphase be seen in well-differentiated liposarcoma (see text for details)
exclusive [174]. Loss of 11q23-24 was associated with histo- from small, barely visible, to large tumors that fill the pelvis.
logic features similar to undifferentiated pleomorphic sar- Histologically, they are characterized by a whorled pattern of
coma and myxofibrosarcoma, while loss of 19q13 was smooth muscle bundles. Two rare benign variants include
associated with an unfavorable outcome [175]. metastasizing leiomyoma, a uterine tumor that metastasizes
Pleomorphic LPS, comprising 5% of all LPS, is an aggres- hematologically, and disseminated peritoneal leiomyomato-
sive, high-grade sarcoma with histologic features similar to sis. Both are considered benign despite their biologic behav-
malignant fibrous histiocytoma (MFH) with the addition of a ior. Neither has been associated with specific genetic
variable number of pleomorphic lipoblasts. Pleomorphic LPS aberrations [2]. Approximately 40% of leiomyomas harbor
usually arises in patients older than 50 years of age with an cytogenetic aberrations that include t(12;14)(q14.3;q24),
equal gender distribution. Tumors commonly present in the deletion in the 7q22 to q31.1 band regions, gain of chromo-
extremities. Cytogenetically, this tumor type is characterized some 12, rearrangements of 6p21 and 10q, and deletion in 3q
by complex structural rearrangements, often with high chro- [176, 177]. t(12;14)(q14.3;q24) characterizes the leiomyoma
mosome counts. Deletions of 13q encompassing RB1 are and is useful in distinguishing it from leiomyosarcoma.
found in ~60% of pleomorphic LPS. Additional described Cytogenetic aberrations involving 7q and gain of chromo-
abnormalities include amplification of MAD2L1 at 4q27 and some 12 have been associated with tumor size, those tumors
deletions of 17p (TP53) and 17q (NF1). Aberrations of the with deletions in 7q being of smaller size than those with an
12q14-q15 region with associated MDM2 amplification are additional copy of chromosome 12 [178].
not characteristic of pleomorphic LPS [171, 173].
Leiomyosarcoma
Leiomyoma
Leiomyosarcomas account for approximately 10% of mes-
Uterine leiomyomas are one of the most common tumors in enchymal neoplasms, have a female predilection, and can
women. They are benign smooth muscle tumors that usually occur in a number of body sites. Tumors are characterized
occur in multiple uterine sites. The tumors are sharply cir- by smooth muscle differentiation. Immunohistochemical
cumscribed, round, firm neoplasms that may vary in size and ultrastructural evaluation play an important role in the
diagnosis. The tumors can be categorized into three groups: found gain and losses in all chromosomes, with the exception
those that arise in deep soft tissues such as the retroperito- that no losses were seen on chromosome 21. Recurrent losses
neum and female genital tract, subcutaneous and cutaneous most commonly involved chromosomes 18, 6q, 10q, and 13;
tumors, and those tumors with a vascular presentation. Both recurrent gains or amplifications most commonly involved
conventional cytogenetic and aCGH analyses have identified 17p11.2, 6p12, 1p32p36, and 8q24 [185, 186]. Osteosarcoma
recurrent aberrations in these tumors including losses at in young children may warrant consideration of a predisposing
1p36, 2p, 11q23-q24, 13q, and 17p. Aberrations at 17p that germline gene mutation. Children in families with a germline
cause TP53 mutations or loss have been associated with RB1 mutation have a 2,000-fold risk of developing OS. Three
high-grade leiomyosarcomas and a poor prognosis [179]. percent of OSs are found in families with Li-Fraumeni syn-
Upregulation of a receptor tyrosine kinase, ROR2, in leio- drome and a germline TP53 mutation [187].
myosarcomas has been associated with a poor clinical out-
come. Because of the tyrosine kinase receptor activity, it is
a potential therapeutic target [180]. Aneurysmal Bone Cyst
Aneurysmal bone cyst (ABC) is a benign, locally aggressive

Alveolar Soft Part Sarcoma bone lesion found in all age groups, with most occurring in
the first two decades of life. The lesion is characterized by
Alveolar soft part sarcoma (ASPS) is a rare tumor of young multiloculated blood-filled cystic spaces that rapidly expand
adults. It occurs in the soft tissues of the extremities and has the bone and look aggressive radiologically. Surgical removal
a unique histology with nest-like or organoid patterns of cells is curative, though recurrences are frequent. ABC was con-
in fibrovascular septae. The tumors also commonly have sidered nonneoplastic until a clonal t(16;17)(q22;p13) was
cytoplasmic crystals identified with periodic acid-Schiff discovered in separate tumors [188]. The t(16;17) was shown
staining with diastase digestion or by electron microscopy to result in CDH11-USP6 fusion. Other chromosomal
[181]. The diagnosis of ASPS may be difficult, however, in translocations, e.g., t(1;17)(p34;p13.2), t(3;17)(q21;p13,2),
tumors that arise in an atypical location or lack the typical t(9;17)(q22;p13.2), and t(17;17)(q22;p13.2), involve
crystal morphology. Distinguishing ASPS from tumors that THRAP3 (TRAP150), CNBP (ZNF9), OMD, and COL1A1,
can mimic ASPS histology, such as granular cell tumor, respectively, with USP6 at 17p13.2. [189–191]. Variants of
paraganglioma, clear cell sarcoma, and metastatic clear cell the t(17;17) include inv(17)(p13.2q11) and inv(17)
renal cell carcinoma, is important because the treatment is (p13.2q21.33) [192] 1 (see Fig. 16.15). The common theme
different for each tumor type. in each of the 17p13.2 translocations is juxtaposition of the
The der(17)t(X;17)(p11.23;q25.3) that fuses TFE3 and USP6 coding sequences to the noncoding promoter regions
ASPSCR1 is considered diagnostic of the entity and has of highly expressed genes. These fusion oncogenes result in
significant diagnostic utility in cases that lack typical presen- USP6 transcriptional upregulation [190].
tation and histology [182, 183]. RT-PCR for the tumor-
specific fusion transcript ASPSCR1-TFE3 has proven to be
an important diagnostic adjunct for this entity. Extraskeletal Myxoid Chondrosarcoma
Immunohistochemical evaluation for nuclear immunoreac-
tivity of TFE3 can also be a useful diagnostic adjunct, Extraskeletal myxoid chondrosarcoma (EMC) is a rare malig-
although it is considered to be less sensitive [184]. nancy that comprises 2.5% of soft tissue sarcomas. It has a
male predilection with 50% of cases occurring between the
fifth to sixth decades of life [193]. The tumor is more indolent
Osteosarcoma than skeletal myxoid chondrosarcoma, is late to metastasize,
and has favorable survival rates [194]. Current treatment
Osteosarcoma (OS), the most common primary malignant includes wide local excision as the principal modality.
bone tumor, is a high-grade malignant mesenchymal tumor The majority of EMC have the translocation t(9;22)
that produces bone matrix. OS occurs at all ages, but 75% (q22;q12.2) or variants t(9;17)(q22;q11.2) and t(9;15)
occur prior to age 20 years, with males more commonly (q22;q21.3). Each of these translocations involves NR4A3
affected than females. OS arises in the metaphyseal regions of resulting in the chimeric fusion genes of NR4A3-EWSR1,
the extremity long bones with ~50% involving the knee [2]. NR4A3-TAF15, and NR4A3-TCF12, respectively. These
The karyotype of most osteosarcomas is very complex. translocations are considered to be diagnostic of extraskeletal
Recurrent chromosome abnormalities are difficult to detect; myxoid chondrosarcomas [195, 196].
however, 13q (RB1) and 17p (TP53) deletions are most frequent.
1
Comparative genomic hybridization analysis of primary OS and personal observation of LDC.
Fig. 16.15 Aneurysmal bone cyst from the olecranon of a 5-year-old boy: 46,XY,t(2;3)(q11.2;q25),inv(17)(p13.2q21.33). The inversion (arrow)
results in USP6-COL1A1 fusion (see text for details)
Angiomatoid Fibrous Histiocytoma plasma cells, and peripheral fibrosis [2]. IMTs may recur
but rarely metastasize. Approximately 50% of IMTs har-
Angiomatoid fibrous histiocytoma (AFH) is a rare, slowly bor a cytogenetic aberration that rearranges the ALK gene
growing multinodular tumor of the deep dermis or subcutis at 2p23.2. The clonal chromosome abnormalities indicate
in children and young adults. The extremities are the most this is a neoplastic rather than a reactive inflammatory pro-
common location, followed by the trunk, head, and neck cess. The ALK gene has a diversity of partner genes, e.g.,
regions. Three characteristic cytogenetic aberrations have t(1;2)(q21.3;p23.2)/TPM3-ALK, t(2;19)(p23.2;p13.12)/
been reported in AFH: t(2;22)(q33.3;q12.2) that rearranges ALK-TPM4, t(2;17)(p23.2;q23.1)/ALK-CLTC, and t(2;2)
CREB1 with EWSR1 (Fig. 16.16), t(12;22)(q13.12;q12.2) (p23.2;q12.3)/ALK-RANBP2 [200]. ALK rearrangement
that rearranges ATF1 with EWSR1, and t(12;16)(q13.12;p11.2) results in upregulation of ALK expression and constitutive
that rearranges ATF1with FUS [197, 198]. Tumors bearing gene activation [200].
the ATF1-EWSR1 fusion and/or the concomitant t(12;22) Absence of ALK expression in IMTs has been associated
(q13.12;q12.2) are associated with rare presentation outside with higher age and a higher mortality rate in younger
of soft tissues in organs such as lung, mediastinum, and ret- patients from disease or distant metastases [201].
roperitoneum. Such AFH tumors with mediastinal/retroperi- Recently, a subgroup of IMTs with aggressive behavior
toneal presentation demonstrate a higher mean age, larger and poor outcome has been described [202]. These IMTs
size, higher frequency of systemic symptoms, higher recur- had epithelioid morphology, were intra-abdominal, arose
rence rate, and a higher incidence of myxoid change [199]. in the mesentery or omentum, and were often multifocal at
diagnosis. Neutrophils and lymphocytes were prominent,
but plasma cells were absent. All were ALK positive by
Inflammatory Myofibroblastic Tumor immunohistochemistry and/or FISH analysis. All patients
had rapid local recurrences and most died of disease.
Inflammatory myofibroblastic tumor (IMT) is a rare mes- RT-PCR identified RANBP2-ALK chimeric fusion with
enchymal neoplasm that is more common in the pediatric exon 18 of RANBP2 fused to exon 20 of ALK in three
and young adult population with an equal gender ratio. It tumors with available DNA. One patient treated with an
is characterized by a proliferation of spindle-shaped ALK inhibitor had no evidence of disease. The authors
fibroblasts and myofibroblasts admixed with lymphocytes, propose this group of tumors be designated as “epithelioid
Fig. 16.16 Angiomatoid fibrous histiocytoma from the inguinal region of a 12-year-old girl: 46,XX,t(2;22)(q33;q12.2). The translocation results
in CREB1-EWSR1 fusion (see text for details)
inflammatory myofibroblastic sarcoma” to convey the Small Round Cell Tumors (Table 16.5)
malignant behavior of this tumor type and its close rela-
tionship with IMT [202]. Rhabdomyosarcoma
Rhabdomyosarcoma (RMS) usually presents before 20 years

Malignant Rhabdoid Tumor of age and is the most common sarcoma of children and ado-
lescents. It most commonly occurs in the head, neck, or geni-
Malignant rhabdoid tumors (MRT) are uncommon highly tourinary tract, with a paucity of normal skeletal muscle in
aggressive tumors that occur in the neonate, infant, or young the tumor mass. Morphologically, rhabdomyosarcomas are
child and occasionally at older ages. MRTs occur in many characterized by the rhabdomyoblast. Ultrastructural evalua-
body sites, including the kidney, central nervous system, soft tion shows the presence of sarcomeres, and the tumors stain
tissues, skin, liver, lungs, and others [71, 203]. A 40-year with antibodies to the myogenic markers desmin, MYOD1,
review of reported cases of fetal and neonatal MRTs found and myogenin [2]. There are three histologic subtypes of
that of 72 cases, 12 presented prenatally and 60 in the neona- RMS: alveolar (ARMS), embryonal (ERMS), and pleomor-
tal period. There were 12 CNS, 27 renal, and 33 non-CNS, phic, which are associated with differences in outcome and
non-renal tumors. Metastatic disease was present at diagnosis prognosis. The ARMS and ERMS subtypes are character-
in 33% of CNS, 52% of renal, and 70% of non-CNS, non- ized by certain molecular cytogenetic aberrations. Because
renal tumors. There was a concomitant renal tumor in 25% of the subtypes may have overlapping histologic features, par-
patients with CNS tumors, a concomitant CNS tumor in 30% ticularly between ERMS and solid pattern ARMS, molecular
of patients with renal tumors, and a concomitant CNS tumor cytogenetic analysis has diagnostic utility [205, 206].
in 6% of patients with non-renal, non-CNS tumors. MRTs,
non-renal non-CNS, occur more often in the perinatal period Alveolar Rhabdomyosarcoma
than in older children. Prognosis was poor, with ~10% overall Alveolar rhabdomyosarcoma accounts for 20% of all RMS,
survival [71]. Cytogenetic and molecular analysis of MRTs presents in adolescence, and most commonly arises in the
shows 22q11.23 deletion, loss of chromosome 22, or extremities. Histologically, the tumor has a crude resem-
SMARCB1/INI1 gene deletion or mutation [38, 204]. blance to pulmonary alveolae, with a network of fibrous
Table 16.5 Chromosome abnormalities with diagnostic or clinical significance in small round cell tumors
Rhabdomyosarcoma
Alveolar RMS t(2;13)(q36.1;q14.11), t(X;2) PAX3-FOXO1, FOXO4-PAX3, Older youth, unfavorable, poor [207–210]
(q13.1;q36.1), t(2;2)(p23.3;q36.1) NCOA1-PAX3 outcome with metastatic disease
t(1;13)(p36.13;q14.11) PAX7-FOXO1 Younger, extremity location, 75% [207–211]
survival with metastatic disease
PAX fusion negative Favorable, comparable to ERMS [210]
Embryonal RMS Gain of 2, 7, 8, 11, 12, 13, 20 Chromosome enumeration Distinguish from alveolar RMS [212]
15q26.3 IGF1R amplification Gene amplification with anaplasia [212]
11p15.5 Loss of heterozygosity Implicates imprinting [213]
Neuroblastoma del(1p) with or without MYCN 1p Unfavorable [215–217]
amplification
2p24.3 MYCN amplification Unfavorable [214, 217]
11q 11q23 band region Unfavorable; inversely associated [215, 217]
with MYCN amplification
Gain of 17q with or without MYCN 17q Unfavorable [214, 219]
amplification
Triploidy without above abnormalities Chromosome enumeration Favorable [217]
EWS/pPNET t(11;22)(q24.3;q12.2), t(21;22) FLI1-EWSR1, ERG-EWSR1, Diagnostic, distinguish from other [220, 221]
(q22.3;q12.2), t(7;22)(p21.2;q12.2), ETV1-EWSR1, ETV4-EWSR1, SRCTs
t(17;22)(q21.31;q12.2), t(2;22) FEV-EWSR1
(q35;q12.2)
del(9p), 17p-,der(1;16)(q10;p10) CDKN2A, TP53 Unfavorable prognosis [220]
DSRCT t(11;22)(p13;q12.2) WT1-EWSR1 Distinguish from other SRCTs; [225, 226]
peritoneal location
Clear cell sarcoma t(12;22)(q13.12;q12.2) ATF1-EWSR1 Absent in cutaneous MM [228, 229]
Retinoblastoma del(13q14.2) RB1 Retinoblastoma hallmark [233]
Gain of 1q21-22; gain of 1q32.1q32.2 SHC1; MDM4, GACI Implicated in cellular proliferation [234]
Gain of 6p22 DEK, E2F3 Potential therapeutic targets [235, 236]
16q24 loss Multiple genes Associated with vitreous seeding [237]
Adrenal cortical Complex karyotype, loss of 11q22.3 ATM Common to ACC, not adenoma; [239, 240]
carcinoma associated with hereditary cancer
syndromes
ACC adrenal cortical carcinoma, DSRCT desmoplastic small round cell tumor, ERMS embryonal rhabdomyosarcoma, EWS/pPNET Ewing sar-
coma/peripheral primitive neuroectodermal tumor, MM malignant melanoma, RMS rhabdomyosarcoma, SRCT small round cell tumor
septae traversing the tumor mass [2]. The tumor is character- amplification of the fusion gene than PAX3-FOXO1 cases
ized by three genetic subgroups: PAX3-FOXO1 fusion and (Personal communication). FISH analysis of a FOXO1
variants (60% of cases), PAX7-FOXO1 fusion (20% of cases), breakapart probe set will show amplification of the 3¢ FOXO1
and PAX-FOXO1 negative (20%) [207]. t(2;13)(q36.1;q14.11) probe in cases with fusion gene amplification. Concurrent
fuses PAX3 to FOXO1 (Fig. 16.17). Variant translocations chromosome analysis is needed to confirm which fusion
that also rearrange PAX3 include t(X;2)(q13.1;q36.1) with gene is amplified (Fig. 16.18). The prognostic significance of
concomitant FOXO4-PAX3 fusion and t(2;2)(p23.3;q36.1) fusion gene amplification still requires elucidation.
with PAX3-NCOA1 fusion [208]. PAX7 is fused to FOXO1
by the variant t(1;13)(p36.13;q14.11) (Fig. 16.18). In patients Embryonal Rhabdomyosarcoma
with metastatic ARMS, a translocation involving PAX3 has Embryonal rhabdomyosarcoma accounts for 60% of all RMS.
been associated with a significantly shorter overall survival The tumor occurs in young children and commonly presents
than those with PAX7 translocations [207, 209]. Patient out- in the nasal cavity, orbit, middle ear, prostate, and paratesticu-
comes for PAX fusion negative ARMS are comparable to lar region. The tumors are soft, gray infiltrative masses that
those of ERMS patients [210]. histologically resemble skeletal muscle with the presence of
About 20% of PAX-FOXO1 fusion positive tumors show round and spindled cells in a myxoid stroma [2]. ERMS is
amplification of the fusion gene [211]. Data indicate a characterized by segmental or whole chromosome gains, in
significantly larger percentage of PAX7-FOXO1 cases show decreasing order of frequency, of chromosomes 2, 7, 8, 11,
Fig. 16.17 Alveolar rhabdomyosarcoma from the hand of a 14-year-old boy: 47,XY,t(2;13)(q36.1;q14.1),+der(13)t(2;13). The translocation
(arrows) results in PAX3-FOXO1 fusion (see text for details)
Fig. 16.18 Alveolar rhabdomyosarcoma from the thigh of a 4-year-old girl: 91,<4n>,XXXX,t(1;13)(p36.1;q14.1),der(1)t(1;13),−13. The transloca-
tion (arrows) results in PAX7-FOXO1 fusion (see text for details). FISH analysis was positive for amplification of the fusion product (not shown)
Fig. 16.19 Embryonal rhabdomyosarcoma from the nasal cavity of an 8-year-old boy: 59,XY,dup(1)(q23q43),+der(1;19)(q10;p10),+6,+7,+7,+7,
+8,+8,+12,+13,+13,+19,+20,+21
12, 13, and 20 [212] (Fig. 16.19). Amplification of the insu- 18 months of age have an excellent prognosis as do those
lin-like growth factor 1 receptor (IGF1R) at 15q26.3 has been patients with ipsilateral disease (no extension across the mid-
associated with progression of ERMS with development of line). Patients are grouped into low-, intermediate-, or high-
anaplastic histology [212]. Loss of heterozygosity in 11p15.5 risk groups based on age, stage, and genetic characteristics.
is also a frequent finding, implicating genomic imprinting as Overall survival for younger children and those with ipsilat-
a potential mechanism of tumorigenesis [213]. eral disease is 80–90%, while OS for those with higher stage
and metastatic disease is 60% and <15%, respectively [7].
A number of specific genetic aberrations have prognostic
Neuroblastoma significance in NB. Amplification of MYCN at 2p24.3 is asso-
ciated with a poor prognosis and puts the patient in the high-
Neuroblastoma (NB) is the most common extracranial pedi- risk category regardless of age, stage, or histology [214].
atric solid tumor. Approximately 700 cases are diagnosed Specific segmental chromosomal aberrations (SCAs) that are
annually in the United States. It originates from neural crest evaluated by conventional cytogenetic analysis can also be
cells within the sympathetic nervous system, and approxi- characterized by aCGH analysis. Deletion of the distal short
mately 40% of NBs arise in the adrenal medulla. arm of chromosome 1 (1p36.3) and deletion of the long arm
Histologically, the classic neuroblastoma exhibits solid of chromosome 11 in a minimal deletion interval including
sheets of small cells with dark nuclei, scant cytoplasm, and band 11q23 are associated with a poor prognosis as indepen-
poorly defined cell borders. Cytoplasmic catecholamine- dent prognostic variables, i.e., in the absence of MYCN
containing granules are often present by ultrastructural anal- amplification [215, 216]. In fact, deletion in 11q is inversely
ysis, consistent with the fact that 90% of NBs produce associated with MYCN amplification and has emerged as an
catecholamines [2]. A morphologic classification has been important prognostic marker [217]. Gain of the long arm of
developed, which associates specific morphology with prog- chromosome 17 is often present together with loss of 1p and
nostic features. The presence of Schwannian stroma and is also associated with a poor prognosis [214, 218]
gangliocytic differentiation and low number of mitotic or (Fig. 16.20a). Deletion of 1p is strongly associated with
karyorrhectic cells is favorable. Metastases can develop early MYCN amplification [219] (Fig. 16.20b). The presence of an
with pronounced hematologic spread. A standardized stag- abnormal clone with a triploid or near-triploid chromosome
ing system has been developed which is widely used interna- number is associated with a more favorable prognosis, while
tionally. Age and stage at diagnosis are considered the two near-diploid clones are generally associated with more aggres-
most important prognostic features. Children younger than sive disease and a poorer prognosis [217]. The presence of
Fig. 16.20 (a) Neuroblastoma from the adrenal gland of a 2-year-old girl: 45,XX,der(1;17)(q10;q10),der(2)(qter → q24.2::p16 → q11.2::?),−10,
+17,>100dmin. (b) The double minutes are shown in the metaphase image. FISH analysis was positive for MYCN amplification (not shown)
genome-wide SCAs, with or without MYCN amplification, soft tissue. EWS and pPNET share a similar neural pheno-
has been shown to be a significant predictor of relapse [219]. type and genetic features and thus are viewed as variants of
the same tumor, differing in their degree of neural differen-
tiation. pPNET has a greater degree of neural differentiation,
Ewing Sarcoma/Peripheral Primitive while tumors with less differentiated histology are desig-
Neuroectodermal Tumor nated as Ewing sarcoma. The EWSFTs comprise the third
most common sarcoma of bone, accounting for approxi-
The Ewing sarcoma family of tumors (EWSFT) includes mately 10% of primary bone tumors. There is a slight male
Ewing sarcoma (EWS) and peripheral primitive neuroecto- preponderance, with the majority of all patients presenting
dermal tumor (pPNET), small round cell tumors of bone and between the ages of 5 and 30 years [7].
Fig. 16.21 Ewing sarcoma from the thoracic spine of a 14-month-old girl: 46,XX,t(11;22)(q24.3;q12.2). The translocation (arrows) results in
FLI1-EWSR1 fusion (see text for details)
EWS arises from the medullary canal of bone and Desmoplastic Small Round Cell Tumor
usually presents in the diaphysis of long tubular bones,
particularly the femur or the flat bones of the pelvis. Classic Desmoplastic small round cell tumor (DSRCT) is a rare
Ewing sarcoma is composed of sheets of monotonous aggressive tumor present in the pediatric and young adult
small round cells with faintly eosinophilic cytoplasm, population. Patients are most commonly 5–30 years of age at
nuclei with granular chromatin and inconspicuous nucle- presentation and 90% are male [222]. DSRCT was first
oli, and a low mitotic rate. The tumors are vimentin posi- described in 1991, and fewer than 200 cases have subse-
tive, contain cytoplasmic glycogen, and strongly express quently been reported [222, 223]. Overall survival at 5 years
the cell-surface glycoprotein CD99 in a “chain-mail” pat- is approximately 15% and no consensus has evolved regard-
tern. pPNETs often exhibit positivity for neuron-specific ing optimal treatment because of the rarity of the tumor and
enolase (NSE) and exhibit rosette formation, characteris- the aggressive clinical course [224].
tics of neural differentiation [2, 220]. Clinical presentation usually includes significant peritoneal
The tumor is characterized by cytogenetic aberrations or retroperitoneal involvement with hundreds of tumors stud-
involving the EWSR1 gene at 22q12.2 that fuses EWSR1 to ding the peritoneal cavity. Treatment requires a multidisciplinary
various genes on different partner chromosomes. The most approach and aggressive surgical extirpation in conjunction
common is the FLI1-EWSR1 rearrangement present in with adjuvant chemotherapy. Morphologically, the tumor con-
85% of tumors due to a t(11;22)(q24.3;q12.2) (Fig. 16.21). sists of small round blue cell nests that are separated by desmo-
EWSR1 fusion to ERG is present in 10% of tumors and is plastic stroma. Immunohistochemical findings demonstrate the
due to a t(21;22)(q22.3;q12.2). Additional rearrangements trilinear coexpression of the epithelial marker keratin, the mes-
include fusion of EWSR1 to ETV1 at 7p21.2, ETV4 at enchymal markers desmin and vimentin, and occasionally the
17q21.31, or FEV at 2q35 (<1% each). The resulting fusion neuronal marker neuron-specific enolase [225].
genes differ by partner gene and also by intragenic vari- The diagnostic cytogenetic finding of t(11;22)(p13;q12.2)
ability in break point location. While the prognostic impact results in fusion of exon 7 of the Ewing sarcoma gene (EWSR1)
of these various fusion transcripts has been evaluated, no and exon 8 of the Wilms tumor gene (WT1). The EWSR1-WT1
clear impact of the partner gene or intragenic fusion archi- chimeric gene encodes a transcriptional activator protein that
tecture on disease progression and relapse has been eluci- fails to suppress tumor growth. RT-PCR shows high sensitivity
dated [221]. for detection of the fusion transcript [225, 226].
Clear Cell Sarcoma do not carry the same increased risk for other malignancies,
as they lack the inherited RB1 mutation [7].
Clear cell sarcoma (CCS) of tendons and aponeuroses or Additional molecular cytogenetic aberrations reported in
malignant melanoma of soft parts is a rare soft tissue tumor retinoblastoma patients include a gain in the 1q21-q22 band
of mesenchymal derivation. It most commonly occurs in the region, encompassing the SHC1 gene, and a gain in the 1q32.1-
tendons and aponeuroses of young adults, has a propensity to q32.2 band region, encompassing MDM4 and LRRN2. These
metastasize to bone, and has a poor overall prognosis. genes are implicated in cellular proliferation pathways [234].
Histologically, the tumor cells may demonstrate morpho- A 0.6 Mb copy number gain in 6p22 encompassing the DEK
logic characteristics similar to malignant melanoma: the and E2F3 genes has also been identified as a recurrent aberra-
presence of melanin as well as positivity for the melanocyte tion. E2F3 is a cell cycle promoting gene and DEK encodes for
immunohistochemical markers HMB-45 and S-100. It is a nuclear protein. These genes are thought to be potential thera-
therefore important to distinguish between malignant mela- peutic targets [235, 236]. A 6.6 Mb region of loss in 16q24
noma and CCS since the treatment and prognosis of the two involving multiple genes including cadherin 13 (CDH13) has
is different [227]. been associated with diffuse vitreous seeding [237].
t(12;22)(q13.12;q12.2), which characterizes CCS, fuses Hereditary and nonhereditary retinoblastomas are mor-
the ATF1 and EWSR1 genes to create a chimeric EWSR1- phologically indistinguishable. The tumors may contain both
ATF1 protein that constitutively activates transcription. undifferentiated and differentiated elements. Undifferentiated
Molecular evaluation for EWSR1 rearrangement by either a areas include small, round cells with hyperchromatic nuclei.
breakapart FISH probe set specific for EWSR1 or RT-PCR Well-differentiated tumors may have rosettes reflecting pho-
for the fusion transcript is recommended to definitively dis- toreceptor differentiation [2]. Leukocoria (cat’s eye reflex or
tinguish clear cell sarcoma from malignant melanoma in white pupil) and strabismus are the two most common pre-
melanotic tumors of soft tissue [228, 229]. senting signs of retinoblastoma, present in 55 and 20% of
patients, respectively [238]. Retinoblastoma is now consid-
ered curable if diagnosed early [7].
Retinoblastoma
Retinoblastoma is the most common pediatric intraocular Adrenal Cortical Carcinoma

neoplasm. It is reported to have an incidence of 6 in 100,000
live births and comprises approximately 10% of cancers dur- Adrenal cortical carcinomas (ACC) are rare and occur in all
ing the first year of life. Approximately 300 new cases are age groups. They are commonly functional and are associ-
diagnosed in the United States each year [230, 231]. One ated with characteristics of hyperadrenalism including
third of retinoblastoma tumors are bilateral [232]. virilism. The tumors are usually large and invasive. Metastases
Retinoblastoma arises as a result of mutational inactiva- by hematogenous spread to lungs and other viscera are com-
tion of both retinoblastoma (RB1) alleles. The RB1 gene mon. The tumors can show histologic variability ranging
maps to 13q14.2, and gene inactivation by cytogenetic rear- from well-differentiated tumors with cells that resemble cor-
rangement is a major mechanism for retinoblastoma devel- tical adenomas to undifferentiated tumors with significant
opment. RB1 encodes a tumor suppressor gene that regulates anaplasia and large giant cells [2].
the cell cycle at the G1/S checkpoint, blocking entry into S Carcinomas that metastasize to the adrenal gland are more
phase and therefore cell growth. Inactivation of RB1 func- common than primary adrenal cortical carcinomas. They
tion as a tumor suppressor gene results in uncontrolled cell may be difficult to distinguish histologically from primary
growth and the malignant phenotype. The Knudson “two- ACC, and a distinguishing genetic aberration would have
hit” model posits that retinoblastoma develops when both diagnostic utility. Deletion of the ATM gene at 11q22.3 as
RB1 alleles are inactivated, most commonly by a chromo- well as lower gene expression is more commonly present in
somal mechanism. Patients with hereditary retinoblastoma ACC than in the benign adrenal cortical adenoma, implicat-
inherit their first “hit” as a germline mutation in every cell. ing ATM in the oncogenesis of ACC [239].
The second hit is somatic, with loss of the second RB1 allele Cytogenetically, ACCs generally have complex karyo-
by mutation or deletion. Patients with a germline RB1 muta- types without a diagnostic nonrandom recurrent abnormality.
tion have a 10,000-fold increased risk for retinoblastoma ACC is a feature of patients with hereditary tumor syndromes
over the general population and a significantly increased risk with germline mutations: Li-Fraumeni syndrome and TP53,
for other neoplasms including sarcomas, lymphomas, and as well as Beckwith-Wiedemann syndrome and 11p15.5 rear-
brain tumors [7, 233]. Patients with sporadic retinoblastoma rangement. Both of these genetic aberrations, at TP53 and
have two somatic RB1 mutations in the tumor. These patients 11p15.5, may also occur in sporadic forms of ACC [240].
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Part V
Adjunct Technologies
Fluorescence In Situ Hybridization
(FISH) 17
Daynna J. Wolff
Introduction Methodology
Dr. Seuss’s eloquent One Fish, Two Fish, Red Fish, Blue Fish Basic Procedure
may have been describing one of the most significant advance-
ments in clinical cytogenetics, fluorescence in situ hybridiza- The FISH method that is widely employed in clinical labora-
tion or FISH [1]. While the basic in situ technology was tories involves the hybridization of a labeled DNA probe to
developed more than 30 years ago, the application involving an in situ chromosomal target. Probe and target DNAs are
fluorescent detection of probe DNA hybridized to chromo- denatured using high temperature incubation in a formamide/
somal target sequences was introduced to the clinical cytoge- salt solution. The probe is applied in great excess, ensuring
netics laboratories in the late 1980s [2, 3]. The overall that the probe anneals to the specific target DNA. Probe
hybridization process was essentially the same as that used for detection is accomplished by ultraviolet (UV)-light excite-
radioactive probes, but the major advantage was the incorpora- ment of a fluorochrome, such as fluorescein-5-thiocyanate
tion of fluorescent detection of the probe sequences that allowed (FITC) or rhodamine, that is directly attached to the probe
for high sensitivity in a simple and quick assay. In the ensuing DNA or by incubation of a hapten (biotin or digoxigenin)-
years, “molecular cytogenetics,” as it has come to be called, labeled probe with a fluorescent conjugate (Fig. 17.1). FISH
has become an integral part of the clinical cytogenetics labora- signal patterns may be scored manually by qualified tech-
tory and has been accepted as standard-of-care for the study of nologists, or computerized automated “spot” counting may
a host of chromosomal aberrations. FISH allows for the study be incorporated into the analysis (see also Chap. 7).
of genetic aberrations that are too small to visualize by routine
cytogenetic studies and too large to detect using standard DNA
sequencing. In addition, as the complexity of copy number Probe Types
variation in the human genome has been appreciated, FISH has
become an important tool for visualizing the copy number Given the abundance of sequence data available from the
changes, determining the etiology of the change, and correlat- Human Genome Project, probes amenable for FISH proce-
ing the clinical significance. Standardized nomenclature rules dures may be produced for the study of almost any human
for FISH were published in An International System for chromosomal site. However, the majority of probes used for
Cytogenetic Nomenclature [4] (see Chap. 3). In addition, the clinical purposes are commercially manufactured and sold as
American College of Medical Genetics and Genomics analyte-specific reagents (ASRs) that must be validated by
(ACMG) and the Clinical Laboratory Standards Institute each laboratory. Most FISH probes fall into one of three cat-
(CLSI), formerly the National Committee for Clinical Labo- egories: repetitive sequence, whole chromosome, or unique
ratory Standards (NCCLS), have developed standards and sequence. The most widely used repetitive sequence probes
guidelines for the use of FISH in clinical laboratory testing [5, 6]. are for the alpha satellite sequences located at the centromeres
of human chromosomes. Alpha satellite DNA is composed
D.J. Wolff, Ph.D. (*) of tandemly repeated monomers, thus the sequences targeted
Department of Pathology and Laboratory Medicine,
by the probes are present in several hundreds or thousands of
Medical University of South Carolina,
165 Ashley Avenue, Suite 309, Charleston, SC 29425, USA copies, producing strong signals. Each chromosome’s alpha
satellite sequence (with the exception of chromosomes 13
Cytogenetics and Molecular Genetics, Medical University of South
Carolina, 165 Ashley Avenue, Suite 309, Charleston, SC 29425, USA and 21 and chromosomes 14 and 22) is sufficiently divergent
e-mail: [email protected] to allow for the development of centromere-specific probes.
416 D.J. Wolff
Fig. 17.1 Schematic representation of the

basic steps of the FISH procedure. Both the
probe and chromosomal target are heat-
denatured. Probe sequences hybridize to the
complementary target sequences, and
nonspecific binding is eliminated via stringent
washing. The probe hybridization is detected
with fluorescence microscopy
These probes are particularly useful for detection of aneuploidy

in both metaphase and interphase cells. In addition, alpha
satellite probes are useful for the detection of acquired mono-
somy or trisomy in malignancies, such as trisomy 12 in chronic
lymphocytic leukemia, or monosomy 7 or trisomy 8 in myeloid
disorders (see Chap. 15). Other types of repetitive sequences
for which probes have been developed include the beta satellite
sequences (located in the short arms of the acrocentric chro-
mosomes), “classical” satellite sequences (found at various
locations including the heterochromatic region of the Y chro-
mosome), and telomeric repeat sequences (TTAGGG) that
mark the physical ends of each human chromosome. These
latter probes are not as routinely used in the clinical setting
but are valuable for the study of structural aberrations.
Whole chromosome probes (WCP), also known as
chromosome libraries or chromosome “painting” probes, are
composed of unique and moderately repetitive sequences Fig. 17.2 Characterization of a structurally abnormal chromosome 7
in a patient with an unbalanced translocation. A chromosome 17
from an entire chromosome or chromosomal region. The genera- library (“painting” probe) was applied to peripheral blood metaphase
tion of this type of probe requires that DNA from a particular cells. Both normal chromosomes 17 hybridized entirely, and the
chromosome be isolated from the rest of the genome. This may unidentifiable material attached to the short arm chromosome 7 (arrow)
be accomplished using flow sorting, somatic cell hybrids is also painted. The patient therefore has three copies of sequences
from chromosome 17
containing a single human chromosome or area of a chromo-
some, or microdissected chromosomes and subsequent
amplification of the dissected DNA sequences via the poly- The third and most widely used type of probe is for unique
merase chain reaction (PCR) [7]. WCPs are commercially sequence DNA. These probes are generated from regions of
available for each human chromosome and are most fre- the genome that are either cloned into various vectors (e.g.,
quently used for the study of structural aberrations. For cosmids, yeast artificial chromosomes [YACs], bacterial
example, WCPs may be used to identify the chromosomal artificial chromosomes [BACs]) or are made by PCR using
origin of additional unknown material of derivative chromo- sequence-specific primers. Some probes include extraneous
somes and also to confirm the cytogenetic interpretation of repetitive sequences, and Cot-1 DNA must be added to the
translocations (Fig. 17.2). hybridization mixture to block nonspecific binding so that
17 Fluorescence In Situ Hybridization (FISH) 417
only the unique sequences are visualized. Other probes, chromosome analysis, paraffin-embedded tissue block
termed single-copy probes, are designed and developed sections, disaggregated cells from paraffin blocks, and touch
based on genomic sequences that are devoid of repetitive preparations of cells from lymph nodes or solid tumors.
sequences [8, 9]. Unique sequence probes, which range in For cases in which metaphase chromosomes are limited, of
size from approximately 1 kilobase (Kb) to >1 megabase poor quality, or unavailable, FISH provides a means for
(Mb), may be used to examine a particular area for copy assessment when the routine chromosome analysis would
number or location. For example, probes developed to span a otherwise be considered a failure. Analysis of interphase
translocation breakpoint, such as a probe for the 5¢ and 3¢ cells also allows for an increased number of cells to be
regions of the MLL gene, allow for detection of cryptic trans- assessed. Given that interphase studies cannot be verified by
locations involving this important cancer locus. These probes visualization on in situ chromosomes, interpretation may be
are also useful for delineating chromosomal breakpoints and compromised by hybridization inefficiency, and quality
for allowing the visualization of copy number changes assurance is of the utmost importance.
detected by genomic microarray hybridization.
Clinical Applications
Labeling and Detection
Constitutional FISH Studies
The majority of probes that are used in the clinical cytogenetics
laboratory are directly labeled and commercially available. One major advantage of FISH is its ability to detect and
However, probes can be indirectly labeled via incorporation characterize chromosomal abnormalities that are not routinely
of a hapten (such as biotin or digoxigenin) into the DNA via delineated with standard banding studies. This technology
nick translation or PCR. The haptens are attached to the allows for the detection of subtle deletions or duplications,
probe nucleotides and are detected by a secondary reaction identification of marker chromosomes, and characterization of
using a fluorescently labeled antibody. The most common other chromosomal rearrangements. In addition, FISH is used
indirect systems involve biotin-streptavidin or digoxi- to visualize aberrations that are detected by copy number
genin-antidigoxigenin. Fluorochromes, such as rhodamine, microarray analyses and to assess parental samples for the copy
Texas Red, or fluorescein, may be conjugated to the strepta- number change and/or definition of a balanced rearrangement.
vidin or antidigoxigenin and detected upon excitation with a
fluorescence microscope. Alternatively, directly labeled Microdeletions and Microduplications
probes, with the fluorochrome attached to the probe nucleotides, Microaberrations or contiguous gene syndromes are caused
require no secondary detection and may be directly visualized by the deletion or duplication of genetic material, usually
after fluorescent excitation. involving multiple contiguous genes on a chromosome
(Table 17.1). Breaks often occur at consistent locations and
are mediated by low copy repeats (LCRs) that permit nonal-
Specimen Types lelic homologous recombination. These contiguous gene
syndromes, which often involve deletions or duplications
FISH can be applied to a variety of specimen types depend- that are 2 Mb or less in size, cannot be identified with routine
ing upon the study of interest. Metaphase preparations from chromosome studies. Therefore, FISH analysis provides a
cultured cells (amniocytes, chorionic villous cells, lympho- definitive diagnostic test for these disorders.
cytes, cells from bone marrow aspirates or solid tumors, Angelman and Prader-Willi syndromes, each of which
fibroblasts) that are routinely utilized for cytogenetic analy- occurs in approximately 1/10,000 individuals, involve the
sis are optimal preparations for FISH studies as well. FISH loss of expression of the maternal or paternal genes, respec-
on metaphase cells is considered the “gold standard” because tively, in 15q11.2–15q13. Approximately, 70% of cases are
the chromosomes and the exact position of the signals can be due to a deletion (Fig. 17.3a). Other causes include uniparen-
visualized directly. However, one major advantage of FISH tal disomy (UPD), imprinting mutations (see Chap. 20), and,
is that it can also be performed on interphase cells. Interphase for Angelman syndrome, mutations of the UBE3A gene. The
nuclei assessment from uncultured preparations allows for deletions involve approximately 2–5 Mb of DNA and may be
rapid screening for prenatal diagnosis (amniocytes for ploidy detected by FISH with a probe for the SNRPN gene or other
analysis), for newborn studies (peripheral blood smears for genes in the region. Approximately 90% of the deletions
ploidy analysis), or for cancer studies (bone marrow aspirate occur at the same distal breakpoint and involve one of two
smear or direct harvest for translocation or copy number proximal breakpoints [10, 11]. The reciprocal product of the
analysis). In addition to uncultured cells, interphase analysis unequal crossing-over event, resulting in duplications of
may also be performed on slides prepared for routine 15q11-q13, has been associated with autism (Fig. 17.3b).
418 D.J. Wolff
Table 17.1 Microdeletion syndromes

Syndrome Deletion Probea Phenotype
Angelman 15q11.2–15q13 SNRPN Severe mental retardation; hypotonia; ataxia; lack of speech; hypopigmentation; seizures;
D15S10 inappropriate laughter; and dysmorphic features
DiGeorge 22q11.2 D22S75 Dysmorphic features; congenital heart disease; absence of thymus; growth failure;
HIRA cognitive deficits
Miller-Dieker 17p13.3 PAFAH1B1 Severe mental retardation; lissencephaly; dysmorphic facial features
Prader-Willi 15q11.2–15q13 SNRPN Mental retardation; hypotonia; feeding difficulty; genital hyperplasia; obesity; hyperphagia;
and dysmorphic features
Smith Magenis 17p11.2 SHMT1 Mental retardation; speech delay; bizarre behavior; peripheral neuropathy; and dysmorphic
TOP3A facial features
FLI1
LLGL1
Velo-Cardio Facial 22q11.2 HIRA Delayed development; pharyngeal deficiency; abnormal facies; palatal defects; and
congenital heart defects
Williams 7q11.2 ELN Mental retardation; hypercalcemia; elfin facies; gregarious personality; and congenital
heart disease
a
The FISH probes used to diagnose the syndrome are listed in this column and are all commercially available
Fig. 17.3 Example of FISH to a single-copy

target using a cosmid (SNRPN) to the
Prader-Willi “critical region” localized to
15q11-13. (a) A metaphase in which one normal
chromosome 15 has three hybridization signals
from a centromeric control probe (green), a
distal control probe (red), and a probe to the
critical region (red). The other chromosome 15
(arrow) revealed hybridization signals only for
the two control probes. Thus, this chromosome
was deleted for the critical region, and this
patient was diagnosed with Prader-Willi
syndrome. Chromosomes were counterstained
blue with DAPI. (b) In this partial metaphase, a
SNRPN probe and a control probe (both yellow)
were utilized (current standards and guidelines
require the use of different fluorochromes; see
Chap. 6). Chromosomes were counterstained
orange with propidium iodide. The arrow
indicates the chromosome 15 with a duplicated
SNRPN signal. This patient was referred for a
diagnosis of autism. See text
Several disorders involving unequal crossing-over medi- nosed using FISH with probes for the critical region [14].
ated by LCRs in the short arm of chromosome 17 are rou- Similarly, interphase FISH with a probe for a 1.4 Mb area of
tinely studied by FISH analysis [12]. Miller-Dieker syndrome 17p12 may be used to detect the duplication associated with
involves the loss of ~2 Mb of DNA in 17p13.3 including the Charcot-Marie-Tooth disease 1A. This same region is deleted
PAFAH1B1 (formerly LIS1 or lissencephaly 1) gene and in patients with hereditary neuropathy with liability to
other gene(s) responsible for the dysmorphic features [13]. pressure palsies (HNPP).
FISH with a probe for the PAFAH1B1 gene allows for the Microdeletions of 22q11.2, resulting in velocardiofacial
detection of the Miller-Dieker syndrome deletion and may (VCF) or DiGeorge syndrome, are seen in about 1/2,000–
also be useful for a proportion of cases with isolated lissen- 1/3,000 individuals. Because of the relatively high frequency
cephaly. Another LCR-mediated mechanism results in a of this syndrome and its association with congenital heart
deletion of chromosome 17 at band p11.2 causing Smith- disease, fetuses and newborns with a heart defect routinely
Magenis syndrome or a duplication of this region resulting in undergo FISH testing for a 22q deletion. This syndrome,
dup(17)(p11.2p11.2) syndrome, both of which can be diag- in contrast to other microdeletion syndromes, is inherited in
about 10% of the cases. Therefore, FISH studies are recom-

mended for parents of an affected individual. Most patients
with VCF or DiGeorge syndrome have an LCR-mediated
deletion of ~3 Mb; some patients have a smaller ~1.5 Mb
deletion that is caused by an internal LCR [15]. Mutations in
the TBX1 gene have been correlated with the abnormal phe-
notype, and this gene is a candidate for the psychiatric dis-
ease associated with VCF and DiGeorge syndrome [16, 17].
The reciprocal syndrome caused by duplication of 22q11.2 is
associated with dysmorphic features, growth failure, cogni-
tive deficits, hearing loss, and velopharyngeal insufficiency
[18]. Both the microdeletion and the microduplication of
22q11.2 are easily detected by FISH with a probe for the
HIRA gene or a probe for the DNA segment D22S75.
Williams syndrome involves the loss of genes in the long
arm of chromosome 7 at band q11.23. The deletion has two Fig. 17.4 Partial metaphase spread from a patient with a duplication
major breakpoints that are mediated by LCRs. The deletion involving chromosome 11. A BAC localized to chromosome 11p15.5
usually cannot be detected by G-banding, but can routinely produced one signal on the normal chromosome 11 and a double signal
be detected by FISH with a probe for the elastin (ELN) gene. on the duplicated chromosome 11 (arrow). The duplication in the short
arm of chromosome 11 was detected in a newborn that was large for
Phenotypic features seen in this syndrome elegantly demon- gestational age. The infant also had an omphalocele and was diagnosed
strate the definition of a contiguous gene syndrome, as with Beckwith-Wiedemann syndrome
Williams syndrome involves both the central nervous system
and connective tissue abnormalities. Abnormalities include While FISH has historically been the method of choice to
mental retardation, infantile hypercalcemia, elfin facies, dys- study the subtelomeric areas, chromosomal microarray anal-
morphic facial features, a gregarious personality, premature ysis that targets the entire genome, including the subtelo-
aging of the skin, and a congenital heart disease (supravalvu- meric areas, has largely replaced FISH testing for patients
lar aortic stenosis) [19, 20]. with mental retardation/development delay and autism (see
Chap. 18). Subtelomeric FISH probes are still valuable, how-
Cryptic Subtelomeric Rearrangements ever, as an aid.
It is generally accepted that even with “high-resolution”
chromosome analysis, alterations of chromosomal material Duplications and Marker Chromosomes
of less than 2–4 Mb cannot be detected. In particular, due to Characterization of de novo duplications and marker
the small size of aberration and exchange of similarly banded chromosomes has valuable implications with respect to phe-
(G-negative) material, visualization of abnormalities in the notype/karyotype correlation. Approximately 70% of chro-
telomeric regions is difficult. Given that these regions are mosomal duplications are intrachromosomal (Fig. 17.4),
gene-rich, they have particular relevance for clinical studies. while 30% involve a nonhomologous chromosome [27].
Aberrations of the subtelomeric regions have been docu- Although chromosomal microarray studies are the optimal
mented in a significant percentage of patients with idiopathic method to determine the genomic content of duplicated seg-
mental retardation with an overall frequency of approximately ments, FISH with chromosomal paints probes, locus-specific
5% (range of 0–13.3%) [21, 22]. The majority of subtelomeric probes, and/or M-FISH (see section “Specialized and
studies have been performed using FISH, and, in general, Evolving FISH Technologies” later in this chapter) may also
these studies have confirmed the efficacy of using subtelo- be used to identify the chromosomal origin of extra material.
meric probes for the assessment of individuals with mental Chromosomes that are unidentifiable by routine banding
retardation, with some cautionary notes. Not all studies used are termed “markers” (see Chaps. 3 and 8). Marker chromo-
the same set of probes, and depending on the location of some somes represent a heterogeneous group and are typically extra
probes, there was a high likelihood of detection of polymor- structurally abnormal chromosomes (ESACs). The most
phisms with no clinical significance, skewing the detection common types of markers, for which clinical phenotypes have
rates reported. Polymorphisms resulting in deletions, duplica- been defined, may be fully characterized using FISH
tions, and other rearrangements of subtelomeric regions have (Table 17.2). Other types of markers may be partially defined
been confirmed with family studies. Of note, small terminal by FISH, and the impact of these chromosomes on the clinical
deletions detected cytogenetically are also commonly detected phenotype often cannot be reliably predicted. Many marker
by subtelomeric FISH probes. These areas of involvement chromosomes are present in mosaic form and cannot be
include 1p, 1q, 2q, 8p, 10q, and 22q [23–26]. characterized by use of chromosomal microarray analysis.
420 D.J. Wolff
Table 17.2 Marker chromosome assessment

Type of marker FISH probe result Associated syndrome/phenotype (estimated risk for abnormality)a
ESAC Pan-centromeric, no alpha satellite High risk for abnormality; phenotype dependent upon
euchromatin present
Bisatellited/monocentric Alpha satellite +: 13/21, 14/22, 15 General risk for bisatellited = 11%
idic(15) 95% – MR
SNRPN – positive ~0% risk
SNRPN – negative 5% – MR (Usually due to UPD)
idic(22) ATP6V1E1 – present Cat eye syndrome
Monosatellited Alpha satellite +: 13/21, 14/22 No general risk, dependent on whether euchromatic material
present
Nonsatellited Alpha satellite present General risk for nonsatellited = 11%
metacentric Alpha satellite present for 8, 9, 12, or 18 centromere If metacentric, risk for MR = ~100%
Sex chromosome DXZ1 (X centromere) +
XIST – positive Turner syndrome only >95%
XIST – negative Majority – MR
DYZ3 (Y centromere)
SRY – positive Male phenotype
SRY – negative Female phenotype
a
From [101, 102]
Identification of chromosomal origin can be accomplished

by using M-FISH (see later in this chapter), or utilizing indi-
vidual chromosomal libraries or alpha satellite DNA probes.
Characteristics, such as shape and size of the marker chromo-
some, determine what probes are best for FISH studies. If
the marker is metacentric, it is likely to be an isochromo-
some (see Chap. 3) and should be studied with alpha satel-
lite probes from chromosomes 8, 9, 12, and 18, as these are
the most likely isochromosomes to be present. These are all
associated with an abnormal phenotype. If the marker is sat-
ellited (or bisatellited), DNA probes from the centromeres
of chromosomes 13/21, 14/22, and 15 should be used. Once
the origin is determined, that information, along with the
structure, dictates the additional studies to be done. For
example, regardless of its origin, a monocentric, bisatellited
chromosome is often not associated with an abnormal phe-
notype, whereas a monocentric, monosatellited chromo-
some may be. If a satellited marker is derived from a
chromosome 15, SNRPN status should be determined
(Fig. 17.5). If SNRPN is present, the karyotype would be
associated with an abnormal phenotype [28]. Fig. 17.5 A dicentric chromosome hybridized with dual-color chro-
Sex chromosome markers are usually found in individu- mosome 15 probes, including both an alpha satellite DNA probe
(green) and a single-copy SNRPN probe (red). Signals from both
als who have 46 chromosomes, with only one normal X and
probes are present on the normal chromosomes 15. The marker chro-
a marker chromosome in place of a second sex chromosome. mosome (arrow) has two alpha satellite DNA signals, confirming that
These abnormal chromosomes should be initially studied it is dicentric. In addition, the marker contains two copies of the
with X and Y alpha satellite probes. If the marker originates SNRPN probe. A control probe for the distal long arm was also
included; signals are only present on the normal chromosomes 15 and
from an X chromosome, it should be studied with a probe for
not on the marker chromosome. This abnormality was ascertained in a
the XIST gene (the gene responsible for initiation of X inac- 6-year-old female with hypotonia, behavior and learning problems,
tivation; see Chap. 10). If XIST is absent, the phenotype will and autism
likely be associated with mental retardation/developmental Table 17.3 Prenatal ploidy analysis
delay [29]. If the marker originates from a Y chromosome, Study No. False (+) False (−) Uninformative
FISH with a probe for SRY should be performed to better Ward et al. 4,500 .1% .2% 6.1%
understand the marker’s effect on phenotype. Patients with (1993) [31]
marker chromosomes that are Y-derived are at risk for gonad- Mercier et al. 630 0 (1) .2%
oblastoma; thus, it is of significant importance to document (1995) [32]
Bryndorf et al. 2,000 0 0 7%
the origin of sex chromosome markers.
(1997) [33]
The last category of markers involves ring or marker chro- Jalal et al. 508 0 0
mosomes that cannot be placed into any of the other groups. (1998) [34]
M-FISH or FISH along with each alpha satellite probe is useful Eiben et al. >3,000 0 0
for determining the chromosomal origin of such markers. (1999) [35]
However, this information does not usually allow for specific Weremowicz et al. 911 (1) .1% (5) .5% 3.0%
(2001) [36]
clinical risk estimations for genetic counseling (see Chap. 21).
Tepperberg et al. 5,197 (1) .003% (7) .024%
(2001) [37]
Follow-Up Studies for Copy Number Aberrations Sawa et al. 2,639 0 0 6.0%
Detected by Microarray Analysis (2001) [38]
Microarray analysis, using comparative genomic hybridiza- Witters et al. 5,049 0 0 0.26%
tion or single nucleotide polymorphism platforms, has proven (2002) [39]
to be the most sensitive and highest resolution assessment of
copy number changes in the genome, detecting aberrations
in approximately 20% of individuals referred for develop- will ascertain numerical abnormalities for these chromo-
mental delay [30] (see Chap. 18). This genomic analysis can somes (Fig. 17.6) and will also detect triploidy.
detect gains and losses of chromosomal material but cannot While FISH assessment on uncultured cells can provide
identify the mechanism underlying the change. Thus, while answers within 24 h of obtaining a sample, these studies are
microarrays offer high-resolution analysis, further studies limited in that only aneuploidies for a select number of chro-
are often necessary to assist with the interpretation of the mosomes (13, 18, 21, X, and Y) can be detected. In a 5-year
result. FISH with a probe or probes contained within the collaborative study, a total of 146,128 amniocenteses were
region designated on the microarray can be used to evaluate performed revealing a total of 4,163 abnormalities; however,
members of the proband’s family for the presence of bal- only 69.4% of these would have been detected using inter-
anced rearrangements or to detect familial copy number phase analysis of uncultured amniotic fluid cells [40].
changes that are likely of no clinical significance. A detection rate (65–70%) has been proposed in a position
statement by the American College of Medical Genetics
Prenatal Studies (ACMG)/American Society of Human Genetics (ASHG).
FISH has been widely used for the detection and analysis of The statement indicates that the sensitivity would increase to
prenatal chromosomal abnormalities (see Chap. 12). One major 80% with increasing age because of the association of
advantage of FISH technology is the ability to study uncultured increased age and nondisjunction.
material to produce a rapid result. In addition, FISH is useful to Overall, prenatal FISH technology has been found to be
characterize or detect subtle abnormalities not delineated by effective, sensitive, and specific. Tepperberg et al. reported
routine banding (e.g., deletions, markers, or duplications). on a 2-year multicenter retrospective analysis and review of
literature of the AneuVysion assay (Abbott Molecular) [37].
Ploidy Analysis Of the 29,039 studies able to be documented, there was only
The vast majority of abnormalities detected prenatally are one false-positive (0.003%) and 7 false-negative (0.024%)
aneuploidies, involving chromosomes 13, 18, 21, or the sex results. The results suggested that this was an effective test
chromosomes. FISH provides rapid ploidy assessment of for aneuploidy of the testable chromosomes in cases of
these chromosomes by utilizing probes on uncultured inter- advanced maternal age or pregnancies indicated to be at
phase cells from amniotic fluid or chorionic villi [31–39] increased risk due to maternal screening results or ultra-
(Table 17.3). In most cases, five probes are used and applied sound findings. As this test is an adjunct test to standard cyto-
to two different slides (or two different sections of a single genetic analysis, the position statement by the ACMG/
slide). a-satellite DNA for the X chromosome and chromo- ASHG states that decisions to act on laboratory test infor-
some 18 is used together with a classical satellite probe for mation should be supported by two of three possible pieces
the Y chromosome, using three different probe colors. The of information, i.e., (1) FISH results, (2) routine chromosome
other mix consists of single-copy probes for both chromo- analysis, and (3) clinical information (e.g., ultrasound
somes 13 and 21, using two different colors. These studies examination).
422 D.J. Wolff
Fig. 17.6 Prenatal ploidy assessment utilizing Abbott Molecular probes for chromosomes 13 (2 green signals) and 21 (3 orange signals). Right:
AneuVysionTM analysis of uncultured amniotic fluid cells using unique copy probes for chromosomes 18 (2 aqua signals), X (green signal), and Y (orange
probes for the long arms of chromosomes 13, 18, 21, X, and Y. The results in signal). Nuclei are counterstained blue with DAPI
these interphase cells are consistent with a XY fetus with trisomy 21. Left:
Although much less common, these probes are also used

with chorionic villus samples (CVS), in vitro fertilization
(IVF) specimens, and fetal cells found in maternal blood.
They can also be utilized to detect aneuploidy in paraffin-
embedded specimens from pregnancy losses.
Preimplantation/Embryo Studies
Preimplantation genetic diagnosis (PGD) is the early diagno-
sis of genetic disorders, prior to the onset of pregnancy.
Embryos or oöcytes are biopsied during culture in vitro and
genetic analysis is performed on the blastomeres or polar
bodies. Embryos shown to be free of the genetic disease
under investigation are transferred to the uterus. Multicolor
FISH may be used to diagnose numerical and certain struc-
tural abnormalities of chromosomes in the embryo, and this
methodology has been adopted by most PGD centers world-
wide as the method of choice for sex determination and for
diagnosis of aneuploidy [41–43]. Some test centers use only
five probes (for chromosomes 13, 18, 21, X, and Y) for ploidy
assessment, but most centers increase accuracy by using
12–24 probes. For translocation carriers, FISH with subtelo-
meric probes is useful for detecting unbalanced zygotes. Fig. 17.7 Metaphases from an XX sex-reversed male were hybridized
with probes for the X centromere (green) and a probe for the SRY gene
Although FISH is the most widely used method for PGD (red). Results demonstrated a cryptic translocation in which SRY was
for some genetic diagnoses, there are several limitations with present on the short arm of one X chromosome. Chromosomes were
this technology [41]. FISH is generally limited to diagnosis at counterstained blue with DAPI
the chromosome level rather than the single-gene level.
Therefore, other methods are needed for single-gene defects assessing embryo sex and chromosome number so that selective
such as cystic fibrosis. Also, misdiagnosis (both false-positive abortion and/or the birth of an affected child can be avoided.
and false-negative) is relatively common and has been reported
in as many as 21% of single cell assessments [44]. In addition, Sex Chromosome Abnormalities
analysis is often limited due to the restricted number of Certain sex chromosome abnormalities, such as the XX male
fluorochromes and the need to eliminate technical artifact (see Chap. 10), cannot be satisfactorily diagnosed with cyto-
(overlapping signals) in a single cell. Even with these limi- genetics alone. Because most such patients are SRY positive,
tations, for couples with a high risk of having a child with FISH analysis with probes for the X chromosome and SRY
a genetic disease, PGD using FISH is very valuable for is typically necessary to confirm the diagnosis (Fig. 17.7).
For patients with a 45,X karyotype, FISH studies are recom- abnormalities; this was updated in 2008 [46] (see also Chap. 15
mended to determine if there is hidden mosaicism for and Fig. 15.8). For each category, classical cytogenetics
Y-chromosomal material that could predispose the patient to identifies the majority of aberrations; however, FISH may be
gonadoblastoma [45]. used to detect cryptic abnormalities and variant rearrangements
and to monitor disease states during and following treatment.
The t(8;21) juxtaposes the RUNX1 (AML1) gene on chro-
FISH Applications for Studies of Acquired mosome 21 and the RUNX1T1 (ETO) gene on chromosome
Chromosomal Aberrations 8. A dual color, dual fusion (DCDF) probe set has been
developed to detect the fusion products on the derivative 8
One major area that has been advanced greatly by FISH is the and the derivative 21 chromosomes (Fig. 17.8). Similarly, a
study of chromosomal abnormalities associated with cancer DCDF probe may be used for AML with t(15;17) in which
(see Chaps. 15 and 16). Probes have been developed for the there is a juxtaposition of the retinoic acid receptor alpha
majority of recurrent translocations found in hematologic (RARA) gene at 17q21.1 and the PML (promyelocytic leuke-
malignancies, and there are many probes for the genetic study mia) gene at 15q24.1. FISH with the dual fusion probes pro-
of solid tumors. Cancer-specific FISH probes and their charac- vides a definitive diagnostic test and a sensitive assay for
teristics are presented in Table 17.4. Several of these diseases minimal residual disease assessment. Rapid FISH diagnosis
and appropriate probes are discussed in detail as follows. (8–48 h) of the PML-RARA fusion is of utmost importance,
so that patients may begin receiving appropriate therapy with
Acute Myeloid Leukemia all-trans retinoic acid (ATRA). In addition, FISH studies
Approximately 40–60% of AML patients exhibit genetic aber- allow for the differentiation of promyelocytic leukemia with
rations that are readily detected by FISH, and in 2001, the t(15;17), as opposed to a variant such as t(11;17). This is
World Health Organization (WHO) established an AML clinically significant, since patients with variant transloca-
classification system that was based on recurrent genetic tions may not respond to ATRA treatment. The t(11;17) and
Table 17.4 FISH for hematologic malignancies

Chromosomal aberrationa Chromosome – gene(s) involved Disease associationb Probe type(s)c
t(9;22)(q34;q11.2) 9 – ABL1 CML, ALL, AML DCDF, DCSF, DCES,
22 – BCR FCDF
t(15;17)(q22;q21.1) 15 – PML AML DCDF, DCSF, BAP
17 – RARA
t(*;11)(*.*; q23) 11 – MLL ALL, AML BAP
t(8;21)(q22;q22) 8 – RUNX1T1 AML DCDF
21 – RUNX1
inv(16)(p13q22) or t(16;16)(p13;q22) 16q22 – CBFB AML BAP
t(12;21)(p13;q22) 12 – ETV6 ALL DCES
21 – RUNX1
Trisomy 8 8 – 8cen AML, CML SC
t(8;14)(q24;q32) 8 – MYC ALL, NHL DCDF
14 – IGH@
t(11;14)(q13;q34) 11 – CCND1 NHL, PCM DCDF
14 – IGH@
t(14;18)(q32;q21) 14 – IGH@ NHL DCDF
18 – BCL2
t(*;14)(*.*;q32) 14 – IGH@ NHL, PCM BAP
del(13)(q14) or −13 MIR16-1, MIR15A (CLL); unknown for PCM CLL, PCM SC, PP
Trisomy 12 12 – 12cen CLL SC, PP
unknown gene(s)
del(11)(q23) ATM CLL SC, PP
del(17)(p13.1) TP53 CLL, PCM, NHL SC, PP
a
An asterisk (*) is used to delineate multiple loci or breakpoints
b
Abbreviations include ALL acute lymphoid leukemia, AML acute myeloid leukemia, CLL chronic lymphocytic leukemia, CML chronic myelog-
enous leukemia, NHL non-Hodgkin lymphoma, PCM plasma cell myeloma
c
Abbreviations include BAP break-apart probe, DCDF dual color, dual fusion, DCES dual color, extra signal, DCSF dual color, single fusion,
FCDF four color, dual fusion, PP probe panel, SC single color (Fig. 17.8)
424 D.J. Wolff
using routine cytogenetics, particularly for cases with

suboptimal chromosome preparations. Thus, FISH or other
molecular techniques are recommended for definitive
diagnostic and residual disease assessments.
Abnormalities of the MLL gene are seen in a small per-
centage of AML and are common in acute lymphoid leuke-
mias (ALL). The majority of rearrangements of 11q23
involve the translocation of the 5¢ region of MLL to the 3¢
region of a partner gene. More than 60 different partner
genes have been identified, and FISH provides an efficient
screen for detection of all aberrations involving MLL. Dual-
color break-apart probes that span the 5¢ and 3¢ regions of
the gene produce a yellow fusion signal for the normal situ-
ation with no disruption of the MLL gene or a single red
and a single green signal when any translocation involving
MLL has occurred (Fig. 17.8). In addition, the break-apart
probe allows for the assessment of copy number of MLL to
determine if deletions or duplications/amplifications of the
gene have occurred.
Chronic Myelogenous Leukemia

The t(9;22)(q34;q11.2) (see Chap. 15, Fig. 15.4) fuses the 5¢
region of the BCR (breakpoint cluster region) gene at 22q11.2
to the 3¢ region of the Abelson (ABL1) proto-oncogene at
9q34, producing a novel protein with tyrosine kinase activity.
Multiple commercial FISH probe combinations are available
to detect the BCR-ABL1 fusion in situ including a dual color,
single fusion (DCSF) probe set that detects BCR-ABL1 on
the “Philadelphia chromosome” [der(22)], a dual color, sin-
gle fusion with an extra signal (DCES) probe set that detects
the der(22) BCR-ABL1 fusion and a residual signal on the
der(9), and a DCDF probe set that detects the fusion products
on both derivative chromosomes (Fig. 17.8). Each probe set is
useful for identifying the BCR-ABL1 fusion event in diagnostic
samples. However, the ES and the DCDF probe sets offer
Fig. 17.8 Examples of normal (column A) and abnormal (column B)
results for hematologic malignancies with various FISH probe types.
increased sensitivity for posttreatment residual disease detec-
The probe type and a chromosomal abnormality exemplifying typical tion since the abnormal signal patterns produced by the latter
results are given probes rarely occur by random chance. The dual fusion probe
format is particularly useful for detection of the 10–20% of
other RARA variants may be identi fi ed with a RARA patients with a t(9;22)(q34;q11.2) with atypical FISH patterns,
break-apart (BAP) probe. including those with a deletion on the derivative chromosome
Acute myeloid leukemia with inv(16)(p13q22) or t(16;16) 9 [47]. Among these patients, there is loss of a portion of
(p13;q22) results from the fusion of the core-binding factor BCR or ABL1 or both of these hybridization sites normally
b(beta) (CBFB) gene at 16q22 to the muscle myosin heavy associated with the break and fusion point on the abnormal
chain (MYH11) at p13. The fusion product interferes with chromosome 9. The loss of DNA associated with the break
the core-binding transcription pathway that is needed for and fusion point on chromosome 9 in cells with a t(9;22)
normal hematopoiesis. Break-apart FISH probes have been (q34;q11.2) has been associated with an adverse prognosis
developed that bind to the 3¢ and 5¢ regions of the CBFB and reduced response to treatment, although more recent
gene, producing a yellow fusion signal in the normal situa- studies do not support this relationship [48–51]. Nevertheless,
tion and a single red and a single green signal when the gene FISH for CML often includes a probe for argininosuccinate
is disrupted by inversion or translocation. Given that the synthetase (ASS1) at chromosome 9q34 to detect such dele-
inversion produces a subtle change in the banding pattern of tions and/or help clarify the signal patterns if possible. See
chromosome 16, the aberration is often difficult to distinguish Fig. 17.9.
Fig. 17.9 Detection of deletions of the derivative chromosome 9, which chromosome (Ph) results in a BCR/ABL1 (green/orange) fusion, and
have been associated with decreased long-term survival in CML, using the derivative chromosome 9 [der(9)] produces all three signals. b: A
dual color dual fusion BCR/ABL1 probes along with a probe for the positive cell with a deletion of the aqua signal from the der(9). c: A posi-
argininosuccinate synthase gene (ASS1) on 9q34 (Abbott Molecular). tive cell with deletions of both the orange and aqua signals from the
The probe for ABL1 is labeled orange, BCR is green, and the ASS1 der(9). d: A positive cell with loss of the green signal from the der(9). It
probe, labeled with an aqua fluorophore, hybridizes adjacent to ABL1 at should be noted that with all three deletions the derivative chromosome
9q34. a: A positive cell with no deletion of the der(9). The normal chro- 9 cannot be distinguished from either the Philadelphia chromosome (b),
mosome 9 shows orange and aqua signals, and the single green signal the normal chromosome 22 (c), or the normal chromosome 9 (d) (Images
represents BCR on the normal chromosome 22. The “Philadelphia” provided by Melissa Anderson)
Acute Lymphoblastic Leukemia (B Lymphoblastic newly diagnosed ALL patients to undergo both conventional
Leukemia/Lymphoma) cytogenetic testing as well as molecular cytogenetic char-
Routine cytogenetic studies for acute lymphoblastic leukemia acterization utilizing a FISH panel to identify ETV6-RUNX1
(ALL) often produce suboptimal preparations and chromo- and/or BCR-ABL1 fusions, MLL gene rearrangements, and
somal aberrations may be missed; therefore, FISH is a useful chromosomes 4 and 10 double trisomy. Other probes that
and necessary adjunct to routine testing. Most cases of ALL some clinical laboratories offer as part of an ALL FISH
are pediatric, and the identification of recurrent chromosomal screening panel include probes for MYC and a common
aberrations is important for risk stratifying these patients. ALL-associated deletion in 9p21-22 [52, 53].
One of the most important probe sets is for the (12;21) For adult patients with ALL, BCR-ABL1 fusion and MLL
translocation that juxtaposes the ETV6 (TEL) gene at 12p13 rearrangement are most commonly assessed by FISH. ALL
and the RUNX1 (AML1) gene at 21q22. This translocation is FISH panels will detect the majority of genetic aberrations asso-
present in approximately 30% of childhood precursor ciated with ALL, particularly since the screen is also useful for
B-ALL, and it cannot be detected by standard cytogenetics unmasking hidden numeric abnormalities (i.e., hyperdiploidy).
unless a more complex rearrangement is present. Therefore, t(1;19) juxtaposes TCF3 at 19p13 with PBX1 at 1q23.
FISH with probes for ETV6 and RUNX1 provides a definitive This translocation can be present in balanced form or as an
diagnostic assay, as well as a means for treatment monitor- unbalanced rearrangement in which only the derivative chro-
ing, for this subgroup of patients. mosome 19 is present. A TCF3 break-apart probe and probes
Clinical trials, including those established by the for the TCF3-PBX1 fusion are both available.
Children’s Oncology Group (COG), typically require all See also Chap. 15, Figs. 15.10, 15.11, 15.12, 15.13, and 15.14.
426 D.J. Wolff
Table 17.5 Cytogenetic aberrations in CLL

Percent (%) cases detected
Cytogenetic aberration Gene(s) involved by FISH [56, 103] Prognosis (median survival) [56]
del(13)(q14) MIR16-1, MIR15A 55–64 Good (133 months)
Trisomy 12 Unknown 16–25 Intermediate (114 months)
del(6)(q21–q23) Unknown 0–6 Intermediate
del(11)(q22.3–q23.1) ATM 15–18 Poor (79 months)
del(17)(p13) TP53 7–8 Poor (32 months)
B Cell Neoplasms approximately 20% of B-CLL cases studied using FISH.

B cell disorders have traditionally presented challenges to This aberration has been associated with an intermediate
the cytogenetics laboratory. The mitotic index of the cells in prognosis in some patients presenting with what appears to
question is usually quite low, and while they have improved be advanced stage disease [55]. del(6q) is seen in 5–10% of
in recent years, the mitogens available to augment this can be B-CLL patients, and in a similar percentage, the TP53 gene
expensive, toxic, and frequently marginally effective. The at 17p13 is deleted. The latter confers the worst prognosis for
use of appropriately constructed panels of FISH probes that CLL patients and is associated with decreased survival and
target the common changes seen in these diseases without increased drug resistance [59]. Since it is hypothesized that
adding the unnecessary cost of routinely attempting to diag- CLL clones accumulate genetic aberrations as the disease
nose rare events can detect chromosome abnormalities in the advances, FISH is appropriate for initial and follow-up stud-
majority of patients. ies [56]. See also Chap. 15, Fig. 15.22.
Chronic Lymphocytic Leukemia/Lymphoma Plasma Cell Myeloma (Multiple Myeloma)/

CLL is a chronic lymphoproliferative disorder, primarily of Plasmacytoma
B cell origin. As a result of the low mitotic rate of affected Chromosomal abnormalities have been reported in approxi-
cells in CLL, metaphase cytogenetics traditionally only mately 30–50% of plasma cell disorders using routine band-
detects aberrations in approximately 40–50% of cases, ing studies, while interphase FISH detects deletions and
although use of the synthetic oligonucleotide DSP-30 has translocations in at least 90% of cases studied [60]. Since
improved this [54]. Interphase FISH is a more sensitive plasma cell myeloma can be stratified based upon the
assay, and FISH has largely replaced conventional cytoge- cytogenetic findings, FISH is an important test to determine
netics for the detection of prognostically significant genetic prognosis and therapy [61]. Plasma cells are typically either
aberrations in CLL. FISH studies reveal that the most com- hyperdiploid with extra copies of the odd-numbered chro-
mon abnormalities include deletions of 13q14, gain of chromosomes or pseudodiploid with rearrangements involving
mosome 12, deletions of 11q22.3-q23.1, deletions of 17p13, the immunoglobulin heavy chain gene [62]. As with B-CLL,
and deletions of 6q21-q23 (Table 17.5). These genomic aber- a panel of probes may be useful for defining the subgroup of
rations confer important independent predictors of disease myeloma and for staging of disease in patients with plasma
progression and survival, thus, FISH analysis with a panel of cell diseases as the frequency and extent of genetic aberra-
probes for relevant aberrations is recommended for CLL tions appears to correlate with clinical disease state [62]. See
patients [55]. A commercially available panel includes Fig. 17.11. Translocations involving the immunoglobulin
probes used to detect deletions of 13q14, 11q22-23 (ATM), heavy chain locus (IGH@) include t(4;14)(p16.3;q32) (~20%
and 17p13 (TP53) and gain of chromosome 12. A probe for of cases), t(6;14)(p21;q32) (~3%), t(11;14)(q13;q32)
MYB on chromosome 6 can also be used to detect deletions (~15%), t(14;16)(q13;q21) (~8%), and t(14;20) (rare) [63].
of this chromosome. See Fig. 17.10a, b. Abnormalities of Patients with t(4;14), t(14;16), and t(14;20) fall within a poor
13q14 are present in approximately 50–60% of CLL patients, prognosis subgroup, while those with t(11;14) have a standard
and this deletion is associated with a favorable prognosis risk [62, 63]. A FISH analysis with a commercially available
[55, 56]. This region contains microRNAs that are thought to break-apart probe specific for the 3¢ and 5¢ ends of the IGH@
upregulate important cell proliferation genes resulting in gene provides an efficient screen for these rearrangements,
CLL [57]. Deletions of the ATM gene at 11q22-23 have been while DCDF probes have been developed that detect
identified in 13–18% of CLL cases assessed by FISH. Loss rearrangements involving 4, 11, and 16. Deletions of 13q14
of ATM is associated with an advanced disease state and are found in approximately 40% of cases had been associ-
relatively rapid rate of disease progression [55, 58]. Gain of ated with a poor prognosis; however, more recent data has
chromosome 12, originally thought to be the most common shown that this deletion is only associated with an adverse
genetic aberration by routine cytogenetic analysis, is seen in prognosis when it occurs in conjunction with t(4:14) or a
Fig. 17.10 FISH panels for B cell disorders. Results from a peripheral the locus on 13q34) and a normal signal pattern for the chromosome 12
blood sample from a patient with CLL, hybridized with the Abbott centromere probe. Right panel shows deletion of TP53 (single red sig-
Molecular CLL probe panel with addition of an MYB probe. Left panel nal), deletion of ATM (single green signal), and a normal signal pattern
shows a deletion of 13q14 (single red signal and two aqua signals for for the MYB probe at 6q23
Fig. 17.11 Example of results from a panel of probes on a patient diag- (red) gene on 1q and normal CDK2NC (green) gene on 1p, (c) loss of one
nosed with plasma cell myeloma with (a) a fusion (yellow) of the FGFR3 copy of the D13S319 (red) probe (13q14), and (d) a normal signal pattern
(red) and IGH@ (green) genes [t(4;14)], (b) an extra copy of the CKS1B for TP53 (red) gene—control BCR gene (green)
428 D.J. Wolff
deletion of 17p [61, 64, 65]. Two important regions that con- FISH is useful for establishing the diagnosis for NHLs on
fer a poor prognosis are deletion 17p (TP53) and duplication primary lymph node tissue, both in cultured cells and with
of 1q (possibly CKS1B) [61, 64]. FISH with a probe for TP53 touch preparations, paraffin-embedded tissues, and in bone
and for loci on 1q21are needed to identify patients for whom marrow to assess for involvement of this tissue.
more aggressive treatment may be needed [61, 64]. Given
that hyperdiploidy with extra copies of the odd-numbered Chronic Disorders with Eosinophilia
chromosomes is associated with a favorable prognosis, FISH Chronic eosinophilic leukemia and hypereosinophilic syn-
with probes for chromosomes 1q, 11 (CCND1), 13q14, and drome are primary eosinophil disorders characterized by
17 (TP53) is useful for the detection of probable hyperdip- marked and persistent blood eosinophilia and organ damage.
loidy [64, 67]. Probes for chromosomes 5, 9, and 15 are also Mast cell disease is a clinically heterogeneous disorder with an
sometimes added to confirm hyperdiploidy in these cases. It accumulation of mast cells that also often has marked eosino-
is important to note that the number of plasma cells in a sam- philia. A subset of patients with these difficult to diagnose dis-
ple can be low, so enrichment techniques may need to be orders have a deletion in the CHIC2 region and fusion of the
employed prior to performing FISH. Two methods have been FIP1L1 and PDGFRA genes on chromosome 4q12 [68, 69].
successfully used to enrich for plasma cells: the cIg FISH Identification of the FIP1L1-PDGFRA rearrangement is a use-
method that utilizes a fluorescently labeled antibody to ful diagnostic tool, but more importantly, the rearrangement
cytoplasmic immunoglobulins and magnetic-assisted cell has been shown to be a therapeutic target of the tyrosine kinase
selection that allows cells to be separated based upon binding inhibitor imatinib mesylate (Gleevec®). Thus, FISH detection
of cells to magnetic CD 138 positive particles [66, 67]. of the CHIC2 region deletion leads to appropriate therapy for
this subset of patients [68]. As discussed in Chap. 15, rear-
Non-Hodgkin Lymphoma rangements involving PDGFRB on chromosome 5 and FGFR1
The genetic hallmarks of many non-Hodgkin lymphomas on chromosome 8 are now included in this category. See
(NHLs) are translocations involving the immunoglobulin (IG) Fig. 17.12 and Chap. 15, Fig. 15.17.
and T-cell receptor (TCR) genes resulting in inappropriate
expression of genes at the reciprocal breakpoints, and FISH Sex-Mismatched Bone Marrow or Stem Cell
presents an effective test for rearrangement assessment. Transplant
A break-apart probe can be used to screen for recurrent For many hematologic malignancies, bone marrow or
chromosomal aberrations associated with the tumorigenesis stem cell transplantation may be a reasonable treatment
of subtypes of B-cell lymphomas involving the immuno-
globulin heavy chain gene (IGH@) at 14q32. Several trans-
locations represent the primary event producing the initial
disease state. t(14;18)(q32;q21) (see Chap. 15, Fig. 15.16a),
which juxtaposes the IGH@ locus with the BCL2 gene, is
virtually pathognomonic for follicular lymphoma and may
also be seen in a percentage of diffuse large cell lymphomas.
For mantle cell lymphoma, IGH@ is positioned next to the
CCND1 (BCL1) gene by a t(11;14)(q13;q32) (see Chap. 15,
Fig. 15.19). FISH with DCDF probes provides the most sen-
sitive diagnostic assay for these rearrangements, detecting
the specific gene fusions in an estimated 95–100% of cases
[60]. Burkitt lymphoma, an aggressive disease of B-cell ori-
gin, harbors a t(8;14)(q24;q32) or variant translocation
[t(8;22)(q24;q11), t(2;8)(p11;q24)] in all cases (see Chap.
15, Fig. 15.17). Juxtaposition of an immunoglobulin gene
and MYC at 8q24 results in overexpression of the MYC tran-
scription factor. In high-grade lymphomas, the utility of
FISH with DCDF probes for MYC-IGH@ or with a BAP
probe for MYC is in the rapid diagnosis of rearrangements
involving MYC, since treatment strategies differ between Fig. 17.12 Tricolor FISH probes for detection of the PDGFRA-FIP1L1
Burkitt lymphoma and other high-grade lymphomas. A dual fusion associated with hypereosinophilia. The green probe is centromeric
to FIP1L1, the orange probe is telomeric to FIP1L1 and centromeric to
color break-apart probe for the ALK gene at 2p23 may be
PDGFRA and contains LNX, and the aqua probe begins in PDGFRA,
used to detect the t(2;5) or variant translocations involving extends toward the telomere, and contains KIT. Deletion of the orange
ALK that are characteristic of anaplastic large cell lymphoma. probe is a surrogate for the PDGFRA-FIP1L1 fusion
Fig. 17.13 FISH is the most

sensitive assessment for opposite
sex bone marrow transplantation
engraftment status studies. For
this female patient who was
transplanted with marrow from a
male, both cell types (two red
signals consistent with two X
centromeres; one red signal and
one green signal, consistent with
one X centromere and one Y
heterochromatic region) were
seen, consistent with partial
engraftment
and/or the only hope to cure the patient of disease. FISH Soft Tissue Tumors
is particularly useful for patients who receive bone mar- Many soft tissue tumors have characteristic chromosomal rear-
row cells with an opposite sex chromosome complement. rangements or gene amplifications that can be detected using
Most often, dual color probes for the X centromere FISH, which is important given that these are often difficult to
(DXZ1) and the Y heterochromatic region (DYZ1) diagnose by morphology alone [70]. In particular, FISH with
(Fig. 17.13) are employed in the analysis of interphase break-apart probes has been used to detect rearrangement of
cells to assess for bone marrow engraftment, or engraft- genes involved in the t(11;22)(q24;q12) that fuses the FLI1 and
ment status. This methodology provides a very sensitive EWSR1 genes associated with Ewing sarcoma [64] (see also
and specific assay. Chap. 16, Fig. 16.21), the t(X;18)(p11.2;q11.2) that juxtaposes
the SS18 (SYT) and SSX1 or SSX2 genes in synovial sarcoma
[65] (Fig. 17.14; see also Chap. 16, Fig. 16.13), and the t(2;13)
Solid Tumors (q35;q14) or t(1;13)(p36;q14) that fuses PAX3 or PAX7 with
Conventional cytogenetic studies of solid tumors are lim- FOXO1 in rhabdomyosarcomas [71] (see also Chap. 16, Figs.
ited by the ability to culture appropriate cells and to obtain 16.17 and 16.18). In addition, FISH has been used to identify
metaphases for chromosome analysis. Analyses from amplifications of the MYCN oncogene on chromosome 2p that
tumors often reveal complex karyotypes with multiple are associated with a poor prognosis in children with neuro-
numerical and structural aberrations that may not be well blastoma [72, 73] (see also Chap. 16, Fig. 16.20a, b). Molecular
defined by banding. FISH is a useful tool for detecting cytogenetic techniques can detect chromosomal translocations
abnormalities that allow for proper diagnosis of tumors and/or other rearrangements that are used to stratify/diagnose
and/or for providing prognostic information. One major various types of adipocytic tumors. Atypical lipomatous tumor/
advantage of FISH is the ability to study interphase nuclei well-differentiated liposarcoma and dedifferentiated liposar-
of touch preparations and paraffin-embedded tissue, allow- coma contain amplification of 12q13-15 involving MDM2,
ing for assessment of fresh and archival samples. M-FISH while myxoid/round cell liposarcoma is characterized by a
and/or comparative genomic hybridization (CGH) (see translocation t(12;16)(q13;p11) that fuses the DDIT3 (CHOP)
section “Specialized and Evolving FISH Technologies” and FUS genes. As more genes that play a role in the pathophys-
later in this chapter) has proven particularly useful for iology of solid tumors are identified, it is likely that clinical
characterizing the complex karyotypes associated with FISH applications for these neoplasms will be developed and
some solid tumors. marketed.
430 D.J. Wolff
Fig. 17.14 FISH for rearrangement of the SS18 (SYT) locus in a syn- green-red probe signals is split apart in each cell, due to rearrangement
ovial sarcoma. Probes on the telomeric and centromeric sides of SS18 of the SS18 gene
are detected with FITC (green) and Texas Red, respectively. One pair of
Fig. 17.15 ERBB2 (HER2) analysis for carcinoma of the breast. Green represents amplification of the ERBB2 gene. See text for details. (a)
signals represent the chromosome 17 centromere probe, while the Normal cells, with two red and two green signals. (b) ERBB2
ERBB2 probe signals are red. An ERBB2:17 centromere ratio of ³2.0 amplification
ERBB2 and Breast Cancer HER2 detection [ 76 ] . FISH with a probe for ERBB2
Ampli fi cation of ERBB2 ( HER2 ) and/or overexpression (17q11.2) and, usually, an alpha satellite probe for the
of the protein product, which has been demonstrated in centromere of chromosome 17 (in a second color) are
approximately 25% of breast cancers, has been associ- hybridized to 4- m m sections of paraf fi n-embedded tumor
ated with poor prognosis, increased risk for recurrence, samples. The invasive component of the cancer, as
and shortened survival in breast cancer patients [ 74, identi fi ed by a pathologist, is scored for the number of
75 ] . HER2 assessment is useful for prognosis, chemo- signals, and an ERBB2 :17 centromere ratio is calcu-
therapy responsiveness, and selection for targeted mono- lated. A ratio of ³ 2.0 indicates ERBB2 gene ampli fi cation
clonal antibody therapy—trastuzumab (Herceptin ®) [ 75 ] . (Fig. 17.15a , b). These results are used in conjunction
FISH is the most sensitive and specific US Food and with clinical fi ndings to guide treatment options for the
Drug Administration (FDA)-approved methodology for patients [ 77, 78 ] .
Fig. 17.16 Examples of normal (a) and abnormal (b) results for the two aqua signals for the chromosome 17 centromere. These cells were
Abbott Molecular UroVysion assay used to monitor for bladder cancer from a male with microhematuria. The abnormal cell exhibits aneu-
recurrence in urine or bladder wash samples. The normal signal pattern ploidy for chromosomes 3 (red), 7 (green) and 17 (aqua), consistent
reveals two red signals for the chromosome 3 centromere, two green with urothelial carcinoma. These results confirmed a recurrence in a
signals for the chromosome 7 centromere, two gold signals for 9p21, and 70-year-old male with a history of bladder cancer
Urothelial Cancer Screening method to detect this aneusomy and to distinguish 1p/19q
Bladder cancer is a relatively common cancer that has a loss in a polysomic background and in many cases can
>70% chance of tumor recurrence [79]. A multitarget FISH be superior to molecular detection [84 ] . It is important to
assay consisting of alpha satellite probes for chromosomes determine the 1p/19q status in brain tumors to provide
3, 7, and 17 and a locus-specific probe for 9p21 (Fig. 17.16) the correct diagnostic and prognostic information and
can be used in conjunction with cystoscopy to assess for to allow for the appropriate therapy. See also Chap. 16 ,
bladder cancer (UroVysion®, Abbott Molecular, Des Fig. 16.2.
Plaines, IL) [80]. The probes are hybridized to cells from
voided urine or bladder-washing samples and are used to
detect aneuploidy for chromosomes 3, 7, and 17 and Non-small Cell Lung Cancer
homozygous loss of the 9p21 locus. The overall specificity As the molecular basis of non-small cell lung cancer
is estimated to be greater than 94% in patients without (NSCLC) has become better understood, several genetic
bladder cancer, and the sensitivity is approximately 71%, mutations have been elucidated that stratify patients for
which is considerably better than the standard cytology therapy. Approximately 2–7% of patients with NSCLC,
testing that has an estimated 40% overall sensitivity. The and as high as 20–30% of never smokers with NSCLC,
FISH methodology has been shown to be particularly use- have a rearrangement of the ALK gene resulting in the
ful for detection of transitional cell carcinoma in cytologi- production of an aberrant tyrosine kinase fusion protein
cally equivocal and negative urine samples, often providing [86, 87]. Eligibility for crizotinib, a targeted inhibitor of
the earliest measure of bladder cancer recurrence (anticipa- the abnormal tyrosine kinase, is dependent on demonstra-
tory positives) [81]. tion of rearrangement of the ALK gene [88, 89]. Currently,
the only FDA-approved method for detection of ALK rear-
1p/19q Loss in Brain Tumors rangement in NSCLC is FISH with an ALK break-apart
Loss of sequences on 1p and 19q, typically mediated by probe that detects the most common EML4-ALK fusion,
a whole arm translocation between chromosomes 1 and as well as several other less common rearrangements [90,
19 with subsequent loss of the (1;19)(p10;q10) deriva- 91]. The clinical trial results showed a remarkable response
tive chromosome, is a hallmark feature of most oligo- rate in ALK-rearranged patients who were treated with
dendrogliomas, where it is associated with response to crizotinib, and FISH testing is now considered part of the
chemotherapy and radiation and improved survival routine screening performed on patients with NSCLC
[82–85]. FISH with probes for 1p36 and 19p13 is a sensitive [89–92].
432 D.J. Wolff
(CGH), multiplex FISH (M-FISH), fiber FISH, m-banding,

Special Quality Considerations for FISH primed labeling (PRINS), and reverse hybridization [93].
While these techniques can be used for clinical analysis,
Although a few commercially manufactured probe kits typically for identification of abnormalities that cannot be
have been approved by the FDA for in vitro diagnostic elucidated with chromosome analysis, most are more regu-
FISH testing, the majority of materials used for clinical larly used on a research basis.
FISH studies are considered analyte-specific reagents In addition, while this chapter deals exclusively with
(ASRs) that are exempt from the FDA and must be inde- fluorescence in situ hybridization, there are several other
pendently validated in each laboratory. In accordance with technologies that use a different method to detect the
the standards and guidelines for Clinical Genetics hybridization of probes, including chromogenic in situ
Laboratories from the American College of Medical hybridization (CISH) and silver precipitation (SISH) [94,
Genetics and Genomics (ACMG) and the CLSI document, 95]. These technologies have the advantages of allowing
prior to utilizing a probe for clinical purposes, probe vali- for permanent storage of material and the use of bright-
dation must be performed [5, 6]. The validation should con- field microscopy but lack the sensitivity and/or specificity
sist of localizing the probe to the correct chromosomal band of most fluorescent probes. These ISH techniques have
on normal metaphase spreads and determining sensitivity not been widely applied to clinical laboratory testing and
and specificity. For probes that will be used for interphase are not discussed in detail here.
analysis, normal ranges must also be calculated from a
database of cytogenetically characterized cases to establish
the percent of cells with an apparent “abnormal” pattern
that might occur randomly. Thus, depending on the normal Comparative Genomic Hybridization (CGH)
cut-off point, probes may or may not be useful for detecting on Metaphase Cells
aberrations for interphase cell analysis. Biannual or con-
tinuous evaluation of performance characteristics of each Comparative genomic hybridization is a technique that uses
probe is required. DNA from the cells of interest, rather than using a standard
It is recommended that FISH tests be analyzed by two or karyotype, for chromosomal analysis. This can be very use-
more non-color-blind technologists who have been trained ful, especially in some cancers when only DNA is available
in the scoring of the resulting signal patterns. For metaphase rather than any growing cells. DNA from the sample of inter-
studies, at least 10 intact cells should be scored with one est (e.g., tumor DNA) that is labeled in one color and DNA
image saved for documentation. A large number of nuclei from a normal control labeled in a different color are hybrid-
(~200) are generally scored for an interphase study, with at ized, in equal proportions, to metaphase chromosomes from
least one image documenting results. Many commercially a normal control. The ratio of fluorophores generated by
available probe mixes contain internal control probes that photometric analysis of the two samples of DNA as deter-
identify the chromosome of interest. In addition, the normal mined by a computer algorithm indicates gains and/or losses
homolog signal may often be used as a control as well. For of material from the DNA being examined. Where there is a
tests without internal controls, for example, a Y chromo- normal amount of genetic material, the equal hybridization
some probe on a newborn with ambiguous genitalia, a con- of patient and control DNA will give a 1:1 ratio; the ratio will
trol sample (for the example given, a sample known to have be skewed to the patient color if there is a gain of material in
a Y chromosome present) needs to be studied along with the the sample of interest or be skewed to the control color if
test case. Reports should include the names of probes used there is loss of genetic material in the patient sample. This
and proper ISCN nomenclature (see Chap. 3). technology has been used successfully for clinical analysis,
When ASRs are employed for FISH studies, the disclaimer particularly with cases that have a low (or no) mitotic index;
“This test was developed and its performance characteristics however, it is limited in that its resolution is about 10–20 Mb.
determined by <laboratory name>. It has not been cleared or It is not useful for detecting balanced rearrangements [96].
approved by the U.S. Food and Drug Administration” must be See Fig. 17.17.
included on the final report.
M-FISH
Specialized and Evolving Technologies
Multiplex FISH (M-FISH) (Fig. 17.18a, b; see also Chap. 7,
There are a number of technologies that involve variations of Fig. 7.17) is a technique that allows the investigator to view a
the standard FISH applications already discussed in this karyotype so that each chromosome is “painted” with a dif-
chapter. These include comparative genomic hybridization ferent color. Combinatorially or ratio-labeled probes are used
Fig. 17.17 The utility of metaphase CGH is illustrated by the a gain of 15q (highlighted in orange ) (This fi gure was kindly pro-
CGH pro fi les of a case with an insertion of unknown material into vided by Dr. Brynn Levy, Columbia University)
the short arm of chromosome 4. The chromosomal pro fi les reveal
to create a distinct computer-generated false color for each especially useful for complex rearrangements, such as
chromosome [97]. those seen in neoplastic disorders and solid tumors. As
As described earlier, the use of different fluorophore described earlier, this technology is also very useful for
colors (and the appropriate bandpass filters, see Chaps. 5 determining the origin of duplications and marker
and 7) allows for the examination of more than one probe chromosomes.
simultaneously. For example, using two colors (red and
green) permits the examination of three probes at the same
time (red, green, and red + green = yellow). The addition of mBAND Analysis
a third color (e.g., blue) increases the number of colors to
7 (red, green, blue, red + green = yellow, red + blue = magenta, Multicolor banding uses chromosome-specific mixtures of
green + blue = cyan, and red + green + blue = white). The partial chromosome paints that are labeled with various
formula for the number (n) of possible combinations is fluorochromes [98]. A computer program analyzes meta-
n = 2c−1, where c is the number of colors used. In order, phase chromosome data and produces a pseudocolored,
therefore, to “paint” each of the 24 human chromosomes a banded karyotype with an estimated resolution of 550 bands,
different color, five different fluorophores are needed. regardless of chromosome length. This methodology is
Specific computer software analyzes the data acquired advantageous for the determination of breakpoints and the
from the probes and pseudocolors the chromosomes for analysis of intrachromosomal rearrangements (Fig. 17.19a, b)
analysis (the multiple colors can only be detected and ana- and can be particularly useful in preparations with shorter
lyzed by utilizing such software). This type of analysis is chromosomes.
434 D.J. Wolff
Fig. 17.18 Multiplex or multicolor FISH

(M-FISH) analysis for cancer. (a) Metaphase
from a leukemia patient with complex
rearrangements. (b) Analysis of a
hypodiploid colon cancer line with multiple
numerical and structural chromosome
abnormalities. The multicolor approach is
useful and successful for identifying both
rearrangements and aneuploidies. The origin
of the different chromosomes in the
rearrangements is noted on the karyotypes
Fiber Fish otides are incorporated during the reaction. This technol-
ogy has been used successfully with both repetitive and
Fiber FISH is a technique that is almost entirely used for single-copy probes. One of the more useful applications of
research. This technology allows the chromosomes to be this technology is that it can differentiate hybridization
stretched out and elongated [99]. The probes are applied and with the alpha satellite sequences for chromosomes 13 and
can be physically ordered on the fibers. This provides a much 21, something that cannot be done with traditional FISH.
higher spatial resolution and allows for correct orientation See Fig. 17.20.
and placement of probes and for precise mapping of probes.
Reverse FISH
Primed In Situ Labeling (PRINS)
Reverse FISH is used to identify material of unknown ori-
Primed in situ labeling (PRINS) is essentially PCR on a gin [100]. This unidentified material, such as a marker
slide. Primers of interest are hybridized on a slide and then chromosome or duplication, is flow sorted or microdis-
subjected to cycles of denaturation, reannealing, and elon- sected off of a slide after G-banding. The DNA from this
gation that are used to incorporate labeled nucleotides. material is extracted, PCR-amplified and labeled with a
The labels are then detected fluorescently, or labeled nucle- fluorochrome. This is then used as a probe and hybridized
Fig. 17.19 Multicolor banding. (a) Region-specific probes labeled with GTG-banding, the chromosome was initially diagnosed as a paracentric
different partial chromosome paints (PCP) and computer false color inversion of the long arm (left). mBAND analysis, however, suggests an
(MetaSystems’ mBAND) produces a definable number of colored bands isodicentric chromosome X (center). Note the identical band colors in
per chromosome, regardless of chromosome length. (b) This example both chromosome arms. An X centromere probe supports this interpreta-
shows an abnormal X chromosome (right homolog of each pair). Using tion (right) (Images provided by MetaSystems Group, Inc)
Fig. 17.20 Primed random in situ hybridization (PRINS). Metaphase is seen at the centromeres of these chromosomes. See text for details. Photo
chromosomes are subjected to PRINS with alpha satellite oligonucleotides courtesy of Drs. Steen Kolvraa and Lars Bolund, Aarhus University,
specific for chromosomes X, 11, and 17. Bright yellow fluorescein staining Sweden
436 D.J. Wolff
Fig. 17.21 Reverse FISH of a patient with an abnormal chromosome 8 typical DAPI staining. Arrows indicate both chromosomes 8 (d)
(a) G-banding suggested a duplication of bands 8p23.1 ® p23.3. Two Hybridization back to a metaphase from the patient, demonstrating that
pairs of chromosomes 8 are shown; arrows indicate the additional band. one chromosome 8 contains a duplication (arrow). The reverse FISH
This band was microdissected, and the DNA was amplified, labeled, results confirm the initial interpretation (G-banded images courtesy of
and used as a FISH probe (b, c) Hybridization to normal chromosomes. Lisa Plumley and Alma Ganezer. Reverse FISH was performed by Dr.
(b) The same metaphase is imaged with reverse DAPI to approximate Jingwei Yu)
G-banding patterns and identify the two chromosomes 8, and with (c)
to normal or patient metaphase chromosomes to identify labeled nucleic acid probes. Cold Spring Harb Symp Quant Biol.
1986;51:151–7.
the origin of the unknown material (Fig. 17.21a–d). This
4. Shaffer LG, Slovak ML, Campbell LJ, editors. ISCN 2009: an
procedure has been successfully used to identify a variety international system for human cytogenetic nomenclature. Basel: S.
of different chromosome abnormalities and is appropriate Karger; 2009.
when M-FISH would be excessive. As for many of the 5. Available from: www.acmg.net. Cited 20 Oct 2010.
6. Russel K, Enns RK, Dewald G, Barker PE, Rasmussen DJ,
specialized FISH techniques, copy number microarray
Watson M, Wood G, Wyatt PR. Fluorescence in situ hybridiza-
technology offers a better resolution and characterization tion (FISH) methods for medical genetics: approved guideline.
of chromosome abnormalities. Wayne: NCCLS;2004. http://www.clsi.org/source/orders/free/
mm7-a.pdf
7. Jauch A, Daumer C, Lichter P, Murken J, Schroeder-Kurth T,
Cremer T. Chromosomal in situ suppression hybridization of human
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Microarray-Based Cytogenetics
18
Lisa G. Shaffer
Introduction and losses that are indicative of chromosome abnormalities.

Depending on the software used, the diagnostician can quickly
The field of cytogenetics has experienced many revolutions discern the aberration’s position in the genome, allowing for
including hypotonic solution, banding, high-resolution prep- the identification of terminal and interstitial gains and losses of
aration and analysis, and fluorescence in situ hybridization the genome (Fig. 18.1). Because microarrays can only identify
(FISH). However, none of these advances resulted in the copy number alterations (CNAs) of the genome and cannot
rapid identification of novel cytogenetic aberrations that distinguish the molecular etiology, a chromosome rearrange-
microarray analysis has achieved. This chapter will review ment should be visualized by chromosome banding or FISH to
the various types of genomic microarrays available to iden- characterize it. This is especially important for identified gains
tify copy number gains and losses of the genome that result of the genome, which may represent a duplication, extra super-
in chromosomal abnormalities. As with any new technology, numerary marker chromosome, unbalanced translocation, or
there are advantages and challenges that accompany innova- insertion [1, 2].
tion. However, even with these challenges, the enormous Of the arrays that are available, two general types exist:
potential of microarray testing for uncovering the etiologies microarray-based comparative genomic hybridization
of intellectual, developmental, and physical disabilities is (aCGH) and single nucleotide polymorphism-based arrays
staggering. While cytogeneticists are accustomed to unusual (SNP arrays). Both types of arrays will detect copy number
findings in the laboratory, the amount of data and the inter- gains and losses. In addition, both aCGH and SNP arrays
pretive challenge of microarray data were not anticipated. will detect mosaicism with greater than about 20–30%
These challenges, as well as the advantages of microarrays, abnormal cells [3, 4]. However, there are some unique differ-
will be explored. ences between these two types of arrays, discussed in the
next section.
Types of Microarrays for Cytogenetic Analysis

Microarray-Based Comparative
Microarrays are constructed from various-sized targets rang- Genomic Hybridization
ing from bacterial artificial chromosomes (BACs), which are
80–200 kilobases in size, to synthetic oligonucleotides, which In aCGH, the patient sample and a control sample are each
are 25–85 base pairs in length. The targets, representing vari- labeled with a distinct fluorescent dye and hybridized in the
ous segments of the genome, can number in the thousands and same concentration to the same microarray, and the
up to more than a million targets on some commercially avail- fluorescent intensity of each dye is then captured by com-
able arrays. Depending on the genomic coverage (backbone) puter imaging. The amount of fluorescence, or dosage of the
and regions targeted on the array (overall termed the microar- dyes for a particular locus, is compared between the patient
ray content), microarrays can quickly identify genomic gains and a control, and the ratio of the two dyes is plotted on a
graph [5–10]. When the patient has a genomic gain or loss,
as compared to the control, the difference in the fluorescent
intensity of the dyes at this genomic location can be visual-
L.G. Shaffer, Ph.D. (*)
ized (Fig. 18.1). aCGH can be applied to both BAC-based
Signature Genomic Laboratories,
2820 North Astor, Spokane, WA 99207, USA arrays and oligonucleotide-based arrays (Figs. 18.2a, b and
e-mail: [email protected] 18.3a, b). Depending on the genomic coverage contained
442 L.G. Shaffer
Fig. 18.1 Examples of various deletions of 1p36.3. In each, probes are indicate deviation from a log2 ratio of zero. (a) ~2.5-Mb terminal dele-
ordered on the x-axis according to physical mapping positions, with the tion. (b) ~10.3-Mb interstitial deletion. (c) ~2.1-Mb interstitial deletion.
short arm oriented to the left and the long arm to the right. The y-axis Microarray analysis was performed at Signature Genomic Laboratories
indicates the log2 ratio of the patient:control fluorescence intensity. (Spokane, WA). Results were visualized using Genoglyphix® (Signature
A log2 ratio of zero indicates a normal (1:1 ratio) result. The blue spots Genomic Laboratories, Spokane, WA, USA)
within the targets on the array, in general, oligonucleotide of heterozygosity that can result from uniparental isodisomy
arrays have the potential for higher-resolution analysis, with (Fig. 18.4; see also Chap. 20). SNP arrays cannot distinguish
the ability to detect smaller alterations than can be identified heterodisomy from a normal result without comparison to
by BAC arrays. parental SNP results. Thus, in order to suggest uniparental
heterodisomy, significant stretches of homozygosity must be
present. In addition, absence of heterozygosity may indicate
Single Nucleotide Polymorphism-Based homozygosity in offspring from closely related parents (from
Microarrays consanguineous unions). Although aCGH can detect some
cases of triploidy based on the dosage over the sex chromo-
In contrast to aCGH, SNP-based arrays do not directly com- somes, SNP arrays readily detect triploidy because of the
pare a patient and a control specimen. SNP arrays compare separation of the various allele combinations [11].
the dosage of the patient at any given locus to a database of For cancer specimens, SNP arrays allow for the detection
control individuals. As with aCGH, gains and losses of the of loss of heterozygosity (LOH) or copy number neutral loss
genome are readily detectable using this method (Figs. 18.2c, (CNNL) that cannot be detected using CGH-based arrays.
d and 18.3c, d). FISH should also be used after SNP array These arrays also provide more information regarding ploidy
analysis to visualize a chromosome rearrangement. SNP changes, often associated with certain types of cancers. This
arrays have the added advantage of being able to detect DNA unique ability to detect single base changes will be important
base alterations, or genotyping, for any given SNP. The comas the understanding of the contribution of LOH/CNNL to
bination of multiple SNPs can identify regions with absence disease and disease progression increases. In addition,
18 Microarray-Based Cytogenetics 443
Fig. 18.2 Examples of microarray results for three different deletions normal dosage of two copies. The blue data points and shading indicate
of 10q21. In each, probes are ordered on the x-axis according to physical a loss of chromosome region 10q21.2q21.3, ~5.4 Mb in size. (c) SNP
mapping positions, with the short arm oriented to the left and the long array plot shows the B-allele frequencies. Deletions show either A or B
arm to the right. (a) Bacterial artificial chromosome (BAC) array. Two alleles, but no AB alleles, as indicated by the gap in the middle of the
experiments were performed, one shown as a pink line (patient:control) plot (arrow). (d) SNP array plot shows the dosage of the SNPs on chro-
and one shown as a blue line (control:patient). Regions of chromosome mosome 10. The depression in the probes over 10q21.1q21.2 indicates
10 in a normal dose of two copies plot at a log2 ratio of zero and the lines deletion and is ~6.4 Mb in size. Microarray analysis was performed at
come together. Region of deletion shows a deviation of the two lines, as Signature Genomic Laboratories (Spokane, WA). BAC and oligo results
the pink shading indicates. The deleted region in 10q21.2q21.3 is ~3 Mb were visualized using Genoglyphix (Signature Genomic Laboratories,
in size (b) Oligonucleotide aCGH. All of the data points at zero indicate Spokane, WA, USA)
because ploidy changes and clonal evolution can make the ability to detect copy number changes. The first genomic
interpretation of arrays challenging, the ability to sort out the microarrays were constructed from BACs and were targeted
various alleles (sometimes referred to as the B-allele fre- to known chromosomal syndromes, the subtelomeric regions,
quency) can aid in the identification of complex karyotypes which are known to be involved in deletion and unbalanced
that involve hyper- or hypodiploidy. translocations, and the pericentromeric regions, which are
usually retained in marker chromosomes [12]. In those early
years, the samples received by the laboratory were usually
Clinical Applications of Microarray Analysis from children whose phenotypes were suggestive of a chro-
mosome abnormality (typically complex presentations, with
The Use of Microarrays to Detect multiple systems involvement), yet no chromosomal diagno-
Cytogenetic Abnormalities in Children sis had been established through karyotyping. Thus, even
with these fairly limited, targeted arrays, the detection rate of
The genomic content of the microarray and the density of the chromosomal abnormalities was quite high, ~6% above that
location of the probes (resolution) will determine the array’s detected by chromosome analysis [12, 13]. Whole genome
444 L.G. Shaffer
Fig. 18.3 Three examples of trisomy 21. In each, probes are ordered on and the pink shading. (b) Oligonucleotide array. The pink data points and
the x-axis according to physical-mapping positions, with the proximal shading indicate a gain of chromosome 21. (c) SNP array. The plot shows
long arm oriented to the left and the distal long arm to the right. The log2 the B-allele frequencies for AAA, AAB, ABB, and BBB alleles, indicat-
ratio of the patient-labeled DNA fluorescence to the control-labeled DNA ing a trisomy. (d) SNP array. The plot shows the dosage of SNPs on
fluorescence is shown on the y-axis. A log2 ratio greater than zero indi- chromosome 21 with a log2 ratio of greater than zero indicating a gain
cates a gain. (a) Bacterial artificial chromosome (BAC) array. Two exper- across all probes. Microarray analysis was performed at Signature
iments were performed, one shown as a pink line (patient:control) and Genomic Laboratories (Spokane, WA). BAC and oligo results were visu-
one shown as a blue line (control:patient). The entire chromosome 21 alized using Genoglyphix (Signature Genomic Laboratories, Spokane,
shows a gain which is evident by the separation of the two experiments WA, USA)
BAC and oligonucleotide arrays, with more comprehensive rays an attractive alternative to karyotyping. Thus,
coverage of the genome, proved to have much higher detec- microarrays can detect the copy number gains and losses
tion rates (~15–20%) than the targeted arrays (reviewed in associated with deletions, duplications, and amplifications;
[14]), although the detection of results of unclear clinical unbalanced translocations and insertions; and marker and
significance also increased dramatically [15] (see sec- ring chromosomes. In addition to the clinical utility, there are
tion “Interpretation of Microarray Data”). Recently, physi- technical advantages to microarray testing. For example,
cians and diagnosticians recommended that microarray microarrays do not require tissue culture; rather, they can use
analysis be the first-tier test for children suspected of having DNA extracted directly from tissues, making this assay sub-
a chromosome disorder [14, 16]. Although microarrays can- stantially faster than karyotyping.
not detect balanced rearrangements because there is no net The resolution of the array is determined by the number
gain or loss of DNA, the ability of arrays to detect other of targets and the genomic coverage or density of probes
abnormalities that can be seen through the light microscope within and between the targets. The clinical utility of the
and those alterations that are submicroscopic, and the com- array is determined by the specific genomic coverage and the
prehensive nature of a whole genome assay, makes microar- potential pathogenicity of the particular loci targeted. Gains
Fig. 18.4 An example of uniparental isodisomy for chromosome 14 using as indicated by the large gap in the middle of the plot. The lower panel
a SNP array. The upper panel shows the B-allele frequencies. Complete shows normal dosage of SNPs on chromosome 14. Microarray analysis
isodisomy (homozygosity) shows either A or B alleles, but no AB alleles, was performed at Signature Genomic Laboratories (Spokane, WA)
and losses will be detected wherever there is a sequence rep- included known chromosome abnormalities in their prospec-
resented on the microarray. Thus, while microarrays detect tive study, and the selection of fetal samples, with the highest
copy gains and losses, they cannot discriminate between yield in those fetuses with abnormal ultrasound findings in
potentially pathogenic loci and segmental duplications, which these limited studies (reviewed in [26]). However, to date, less
can be found throughout the genome of normal individuals. than 1,000 prospective prenatal specimens tested by microar-
Consequently, if a repetitive region, such as a segmental ray have been reported in the literature. The adoption of array
duplication, is represented on the array, gains and losses of testing in the prenatal setting has been slower than that seen
this region can be detected and potentially misinterpreted. for postnatal testing. Recently, the American College of
Therefore, arrays for clinical use should be designed Obstetrics and Gynecology released a committee opinion for
specifically for particular applications by individuals knowl- aCGH, endorsing the use of arrays in pregnancies with abnor-
edgeable in the intended use and interpreted by experts in mal ultrasound findings [30]. This endorsement may raise
cytogenetics. awareness among obstetricians and result in an increased
usage of microarrays for prenatal testing (see also Chap. 12).
The Use of Microarrays to Detect

Fetal Chromosome Anomalies The Use of Microarrays in Oncology
Microarray analysis has been used to detect chromosome In cancer and related disorders, identifying chromosome and
anomalies in products of conception, terminated pregnancies, chromosomal region gains and losses is important for dis-
and ongoing pregnancies. Some of the first studies utilized ease detection and classification, providing prognostic infor-
products of conception to demonstrate the power of microar- mation and assessing disease progression, and can guide
ray testing, whereas other studies focused on retrospective therapeutic decisions [31–33].
analysis of terminated pregnancies with abnormal ultrasound Microarrays are well suited to provide this information, as
findings [17–22]. These studies showed abnormalities they readily detect copy number alterations. Copy number
detected by microarray analysis in about 8–16% of cases. assessment has been achieved for many hematologic malig-
More recently, microarray analysis has been incorporated nancies, including chronic lymphocytic leukemia (CLL), acute
into the diagnostic evaluation of fetuses in the prenatal setting. myelogenous leukemia (AML), myelodysplastic syndromes
Prenatal microarray testing can be performed on cultured and (MDS), plasma cell myeloma, follicular lymphoma, poly-
direct specimens from both amniotic fluid and chorionic villus cythemia vera, acute lymphoblastic leukemia (ALL) in child-
sampling (CVS). Several prospective studies have demon- hood, and adult ALL [31, 32, 34–55]. In all studies, the number
strated the diagnostic yield of chromosome abnormalities to of chromosomal aberrations identified was significantly
range from about one to approximately 9% [23–29]. The increased beyond that found with routine cytogenetics. This is
reported detection rates varied considerably depending on the especially evident in cases with normal karyotypes and submi-
genomic coverage of the array being used, whether the authors croscopic alterations found by microarray analysis [41, 52].
446 L.G. Shaffer
Microarrays have also been used to identify copy gains

and losses in solid tumors, including medulloblastoma, menin- Interpretation of Microarray Data
gioma, breast cancer, gastric cancers, and renal cell carci-
noma [56–62]. Given that many solid tumors show The ability of microarrays to interrogate thousands of loci
chromosome aberrations, arrays will continue to have an simultaneously has changed the practice of medical genetics,
impact on the diagnosis. However, because solid tumors are and although some copy number alterations can be clearly
quite often fixed by embedding in paraffin, the extraction of classified as pathogenic while others can be classified as
sufficient quality and quantity of DNA for microarray analy- benign, some loci are now known to confer susceptibility to
sis is challenging [63]. some abnormal phenotypes, while others remain of unclear
As discussed throughout this chapter, microarrays are clinical relevance. Copy number variations (CNVs) across
designed to detect genomic gains and losses; balanced trans- the genome have been demonstrated in normal individuals
locations cannot be routinely detected using either aCGH or and in some cases likely represent normal (benign) popula-
SNP-based arrays. A modification of aCGH analysis, called tion variation [66–68]. Cytogeneticists have been aware of
translocation-CGH, provides a new approach to the detec- genomic variation for decades, often encountering hetero-
tion of prognostically important translocations in neoplastic morphic acrocentric short arms and staining variability of
disorders [64, 65]. In this analysis, a linear amplification is certain pericentromeric regions. However, the identification
performed over the translocation breakpoint using primers in of multiple small genomic gains and losses in the normal
the vicinity of the junction. After labeling the reaction for an population has drawn much attention. These benign variants
aCGH experiment and hybridizing to a microarray with the are identified in both patient and control populations in
proper content over the translocation breakpoints, a peak can roughly equal frequencies.
be visualized at the site of the translocation breakpoint There are other regions of the genome that appear to con-
(Fig. 18.5). In theory, by using one of the known or potential fer susceptibility to certain phenotypes [68, 69]. These sus-
partner chromosomes, the recipient chromosome involved in ceptibility loci can be carried by an apparently unaffected
the translocation can be identified. Thus, a large pool of parent, are enriched in patient populations, and have a rela-
primers might be used in a multiplex assay to identify bal- tively low frequency in control populations. However, in
anced translocations and stratify patients into specific diag- some individuals who carry these copy number changes,
noses based on the rearrangements identified. abnormal phenotypes result. Recent examples include
Fig. 18.5 Microarray results showing a translocation between chro- translocation breakpoint. (b) Zoomed-in view of the breakpoint area of
mosomes 4 and 11. Probes are ordered on the x-axis according to 4q22.1 and the AFF1 gene. The Cancer Features track (gold) in the
physical mapping positions. (a) Zoomed-in view of chromosome 11 region shows the inclusion of the AFF1 gene. The pink shading indi-
plot at 11q23.3 showing the proximal 11q23.3 probes on the left and cates the breakpoint in AFF1. Microarray analysis was performed at
the more distal 11q23.3 probes on the right. The software displays a Signature Genomic Laboratories (Spokane, WA), and results were
number of features including the Cancer Features track (gold) showing visualized using Oncoglyphix™ (Signature Genomic Laboratories,
the genes in the region, including MLL. The pink shading indicates the Spokane, WA, USA)
deletions and duplications of 1q21, deletions and duplica- identified with microarrays, the addition of FISH may
tions of 16p11.2, and deletions of 16p13.1 [68–76]. increase the detection of additional cytogenetic changes,
In addition, CNVs of unclear clinical significance are often especially in leukemia and lymphoma in which certain trans-
found in patient populations that undergo microarray analysis locations are the hallmark of the disease.
[28, 77]. These alterations are considered unclear because of Hyperdiploidy, hypodiploidy, clonal evolution, and other
their very low frequency in patient populations, inheritance mechanisms leading to complex karyotypes may be difficult to
from a clinically normal parent, and absence in control popula- identify or interpret with microarray testing. The reason for this
tions. The interpretation of these CNVs as causative to the is that a mixture of complex genomic changes, multiple clones,
patient’s phenotype is challenging, and the study of parents is will all be extracted together into a single sample and may pro-
often not helpful because a rare, novel change, inherited from a duce a complex pattern on the array that may not be interpret-
normal parent, may represent a susceptibility locus, and this able. Therefore, the use of chromosome analysis and FISH
possibility cannot be excluded without further population stud- may be more appropriate for highly complex cancer genomes.
ies. Even the finding of a small, de novo change in a patient’s In addition to complex karyotypes, microarrays may not
DNA may not indicate a causal relationship between the altera- be sensitive enough to detect low-level clonal changes. Thus,
tion and the phenotype; however, it is generally accepted that changes in a very minor clone may not be apparent after
de novo changes are more likely to be causative than inherited microarray analysis. In this respect, minimal residual disease
CNVs. Even with these challenges in interpretation, most should not be assessed using microarrays but rather should
CNAs have clear clinical implications, and those that are deter- be evaluated using FISH or PCR (if possible) or cytogenetics
mined to be pathogenic do provide answers to families seeking to identify the presence of a low-level clone. At initial dis-
the reason for their child’s medical problems. ease presentation or relapse, microarray analysis is useful for
It is well worth the arduous endeavor of trying to interpret identifying genomic changes characteristic for a particular
these rare copy number changes because identifying the patient. These changes can be monitored using FISH or PCR
genetic etiology of disease allows for accurate genetic coun- during treatment or remission.
seling, reproductive management, and anticipation of poten- Quite often in cancer diagnosis, chromosomal changes are
tially serious medical problems in the child [78]. Databases observed, but the banding pattern is not sufficient to identify
are being established that may aid in the interpretation of the chromosomal origin of the structural change. Microarray
these alterations. Some examples include: DECIPHER,1 the analysis is very useful for the identification of the chromo-
Database of Genomic Variants,2 the database at the Children’s somal origin of derivative chromosomes, additional chromo-
Hospital of Philadelphia, the European Cytogeneticists somal material, and marker chromosomes. The challenge
Association Register of Unbalanced Chromosome Aberrations with marker chromosomes is that the presence of multiple
(ECARUCA),3 and the Signature Genomics Genoglyphix® marker chromosomes in a cell does not necessarily mean that
Chromosome Aberration Database [79, 80].4 they were all derived from a common chromosome source.
Thus, the microarray pattern may be complex, which may
indicate multiple origins or evolution of the markers. In addi-
Challenges with, and Advantages of, tion, the presence of a marker chromosome in a minority of
Microarray Testing cells may not be identified by microarray analysis due to loss
of sensitivity at levels below a certain threshold, typically less
Laboratory Challenges with Microarray Testing than ~20% of cells with that particular clone.
The choice of array used may impact the information gained.

Therefore, the first challenge for the diagnostician is choos- Advantages of Microarray Testing
ing the right array. The microarray should be designed with
a specific use in mind—including targets to particular regions The clear advantage of microarray analysis over conven-
of the genome. For example, in cancer, BAC arrays may not tional cytogenetics is the high resolution and genomic cover-
detect small, single gene deletions; this usually requires age that will uncover chromosome aberrations at detection
higher density and smaller targets and can be achieved by rates of 15–20% in children with intellectual and develop-
using oligonucleotide-based CGH or SNP arrays [52]. mental disabilities. In comparison to karyotyping, which will
Because balanced translocations cannot be routinely detect a chromosome abnormality in about 3–4% of children
with global developmental delay, an additional 6.4% of chil-
1
http://decipher.sanger.ac.uk/ dren with global delays had a pathogenic copy number alter-
2
http://projects.tcag.ca/variation/ ation by microarray analysis [81, 82]. There are other
3
http://agserver01.azn.nl:8080/ecaruca/ecaruca.jsp advantages in addition to the higher detection rate for chro-
4
http://www.genoglyphix.com mosome abnormalities.
448 L.G. Shaffer
The turnaround times can be exceptionally fast. The rate- Acknowledgments Microarray analysis for Figs. 18.1, 18.2, 18.3,
limiting step is the hybridization, which is usually 24–48 h 18.4, and 18.5 was performed at Signature Genomic Laboratories
(Spokane, WA). Results were visualized using Signature Genomic
with most arrays. Thus, if the DNA from the sample is Laboratories’ Genoglyphix® or Oncoglyphix™ software.
extracted on day one and the DNA is labeled and hybridized
to the array on the same day, the array can be washed, ana-
lyzed, interpreted, and reported on the morning of day three,
slightly more than 48 h from receipt of the specimen. References
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Part VI
Beyond Chromosomes
Fragile X—A Family of Disorders:
Changing Phenotype and Molecular 19
Genetics
Elaine B. Spector
Genetics of Fragile X Syndrome, Prior grandchildren, usually grandsons. Sherman, et al. observed
to the Availability of Molecular Analysis that the mothers of such males are much less likely to have
affected offspring than are their unaffected daughters [6, 7].
X-Linked Mental Retardation This became known as the Sherman paradox. The term
“transmitting male” (TM) was coined to describe such unaf-
In 1938, Penrose noted a higher incidence of mental retarda- fected carrier males. These TMs were thought to be the
tion (MR) in males and reports of families with only affected “missing” 20% of affected males described by Sherman et al.
males [1]. These observations were compatible with X-linked from 206 fraX families. Other unusual features of FXS are
inheritance, and numerous reports appeared in the literature [2]. that TMs have fewer mentally retarded daughters than do
Based on this early work, a clinically nonspecific X-linked unaffected carrier females, affected females occur more fre-
MR disorder was delineated and called Renpenning’s syn- quently (about one of three) than in other X-linked disorders,
drome, Martin-Bell syndrome, or nonspecific X-linked MR. and affected females have more affected offspring than do
In 1959, Lubs described the first family with cytogenetic unaffected carrier females.
expression of the “marker X,” which became the fragile X FXS is now known to be the most prevalent cause of
(fraX), and the heterogeneity of this nonspecific X-linked inheritable mental retardation, often presenting as an
MR disorder became apparent [3]. Numerous disorders have autism spectrum disorder, with a frequency of approximately
been delineated from this original subgroup of MR males. one in 4,000 males and one in 6,000 females (see also sec-
The fraX subgroup was unique because there was a diagnos- tion, “Epidemiology”).
tic laboratory test; the name Martin-Bell syndrome was
attached when this family, first described in 1943, was shown
to be positive for the fraX [4, 5]. However, the popular name Cytogenetic Expression of fraX
for this disorder became fragile X syndrome (FXS).
The fraX site (FRAXA) is located in band Xq27.3, one of six
fragile sites located on the X chromosome (Table 19.1).
Inheritance of fraX It can be visualized in both solid stained and banded prepara-
tions (Fig.. 19.1). However, banded preparations are required
Soon after the cytogenetic test became available, it became because other fragile sites and lesions can mimic fraX [8–
apparent that the inheritance pattern and penetrance of FXS 10]. Three other fragile sites have been found in bands Xq27-
were unlike those of any previously described X-linked dis- 28: FRAXD, FRAXE, and FRAXF [11–13]. The latter two
order, although it came closest to an X-linked dominant with sites cannot be cytogenetically distinguished from fraX.
reduced penetrance. It was determined that some males who FraX is not a chromosome abnormality. It is a chromo-
inherited the fraX were clinically normal, but passed the dis- somal “marker” that facilitated the diagnosis of FXS until
order to their normal daughters and frequently had affected better techniques were developed.
Cytogenetic Expression in Affected

E.B. Spector, Ph.D. (*) Males and Carrier Females
UCD DNA Diagnostic Laboratory, Department of Pediatrics,
In affected males, fraX expression varied from less than 49%
University of Colorado School of Medicine, Anschutz Medical
Campus, 12800 East 19th Avenue, Aurora, CO 80045, USA to 50%, with the low-expressing males comprising a minority
e-mail: [email protected] of the diagnosed cases. However, this group represents the
454 E.B. Spector
Table 19.1 Fragile sites on X chromosome

Name Location Type
FRAXA Xq27.3 Rare
FRAXB Xp22.31 Common
FRAXC Xq22.1 Common
FRAXD Xq27.3 Common
FRAXE Xq28 Rare
FRAXF Xq28 Rare
Fig. 19.1 Appearance of FRAXA. (a) Conventional stain (Giemsa) and (b) GTG-banded. The arrow indicates the location of the fraX site
false-negative males diagnosable with molecular techniques. Australasia at this time. England had the major experience
Why fraX does not express in >50% of metaphases is still not with fetal blood sampling. Worldwide experience with pre-
known. Cytogenetic testing of carrier (heterozygous) females natal diagnosis by cytogenetic analysis or cytogenetic anal-
was even more problematic. Among obligate carriers, only ysis plus DNA polymorphism analysis (see discussion later)
about 50% tested positive, and about one-third of these carriers exceeded 400 cases. The “state of the art” was summarized
were clearly affected to some degree. In general, fraX expres- at the Fourth International Workshop on Fragile X and
sion was easier to demonstrate (although lower than in males) X-Linked Mental Retardation. Fortunately, with the
in affected females than in those with normal intelligence. identification of the molecular defect in FXS, prenatal diag-
Guidelines were established for interpretation of these data nosis of the syndrome became much more accurate.
[14, 15].
Prenatal Diagnosis Molecular Aspects of Fragile X Syndrome

Prenatal testing was available on an experimental basis
beginning in 1981 using cytogenetic techniques. Testing Analysis Using Linked Polymorphisms
was done on fetal blood, amniocytes, or chorionic villus
cells with varying degrees of success. False-negative males From the mid-1980s through 1991, molecular (DNA)
were reported with all three tissue types. In the United analysis using linked polymorphisms was used in confirmed
States, amniocentesis was the major procedure, while chori- fraX families to help with prenatal diagnosis and carrier
onic villus sampling (CVS) was the standard in Europe and status. Although the gene for FXS had not been identified,
19 Fragile X—A Family of Disorders: Changing Phenotype and Molecular Genetics 455
its relative location on a linear map of the distal X long Table 19.2 Classification of trinucleotide repeat diseases
arm was known. Using genes and polymorphisms on both Class n Repeat Position of Repeat Examples
sides of fraX allowed molecular geneticists to track fraX 1 3 CGG 5¢ Untranslated region FXS
chromosomes through families. The risks of inheriting the FRAXE syndrome
fraX chromosome were expressed as probabilities. Success 1 CAG Spinocerebellar ataxia
with the method depended on the distance between the type 12 (SCA12)
tested polymorphism/gene and the FXS gene, the size of 2 2 CTG 3¢ Untranslated region Myotonic dystrophy (DM)
the family, and which polymorphism/genes were informa- 3 8 CAG Inside coding region Huntington’s disease
(HD)
tive. Regardless of these limitations, the combination of
Spinocerebellar ataxia
cytogenetic and linkage analysis allowed many families to type 1 (SCA1)
receive more reliable results than with chromosome anal- Kennedy’s disease
ysis alone. 4 1 GAA In first intron Friedreich Ataxia
Trinucleotide Repeats: Classification individuals have massive repeat sizes differing in lengths
(“mosaic”) in a pattern that is often conserved across tis-
The early 1990s marked the discovery of a new type of sues, resulting in FXS.
genetic mutation in humans: the trinucleotide or dynamic
repeat. The mechanism causing the FXS mutation was first
identified in 1991 and revealed that the mutation results from Instability of the CGG Repeat
the expansion of a trinucleotide repeat located in or near an
expressed sequence [16–19]. For the fragile X syndrome, the Through observational studies of families with FXS, several
trinucleotide repeat is cytosine-guanine-guanine or CGG. factors involved in CGG repeat instability have been pro-
This revelation was soon followed by the discovery that a posed, including the sex of the transmitting parent, the size
similar mechanism causes myotonic dystrophy (DM) and and structure of the CGG repeat, and other yet-to-be-
spinocerebellar ataxia type 1 (SCA1). To date, more than 30 identified factors. With the resolution of the Sherman para-
human diseases known to be associated with the expansion dox, it is now known that a premutation-sized repeat has the
of a trinucleotide repeat [20–22]. propensity to expand when passed through a female germ
Trinucleotide repeat disorders can be categorized in one line, and the size of the resulting expansion is positively cor-
of two ways: (1) according to the specific trinucleotide related with the maternal repeat size [26–30]. In contrast,
sequence or (2) according to the location of the expansion in when passed through a male germ line, the premutation does
relation to the coding sequence. Here, we have chosen to not dramatically change in repeat size and often remains the
describe trinucleotide repeat disorders based on the position same or even contracts [29–31].
of the expansion in relation to the coding sequence. The In addition to the sex of the transmitting parent, the size
repeats may be located in the 5¢ untranslated region, in an and structure of the CGG repeat play a role in instability.
intron, in an exon, or in the 3¢ untranslated region. The list of Sequencing of the CGG repeat revealed that the repeat is not
disorders continues to grow. One characteristic of these dis- pure and is interspersed with one to three AGGs (adenine-
orders, that each generation shows an earlier age of onset and guanine-guanine sequences) every 9–10 CGGs in the general
increasing severity, is known as anticipation. All the disor- population. Among families with FXS, premutation-sized
ders are either X-linked or autosomal dominant except repeats usually have one AGG at the most proximal end of
Friedreich ataxia, which is autosomal recessive [22]. the repeat or none at all [32–34]. Transmission studies of
The CGG trinucleotide repeats are located at folate- families with premutation- or intermediate-sized repeats
sensitive fragile sites, and their characteristics are sum- demonstrate that these are unstable if >34 repeats at the 3¢
marized in Table 19.2 [23, 24]. Based on the trinucleotide end of the repeat structure are uninterrupted by an AGG [29,
repeat size in FRAXA, an individual’s status can be 32, 34]. To date, all known expansions have occurred at the
classified as normal (5–44 CGG repeats), indeterminate 3¢ end of the repeat. This polarity of expansion further dem-
or gray zone (45–54 CGG repeats), premutation (55–200 onstrates the importance of the 3¢ end of the repeat in the
CGG repeats), or full mutation (>200 CGG repeats) [25]. expansion process. While the role of the AGG interruption
An individual with a normal repeat size is characterized has only been minimally defined by experimental studies,
by stability of the repeat length and normal intelligence, these observational and population studies suggest that the
while an individual with a premutation repeat size shows AGG sequence acts as an anchor during DNA replication to
instability of the repeat length from generation to genera- prevent expansions or deletions that are the result of slips or
tion but normal intelligence. In contrast, full mutation misalignments of the repeat sequence during replication
456 E.B. Spector
[35–38]. Despite the identification of these factors, the exact 17 exons [46]. The polymorphic CGG repeat exists in the 5¢
mechanism of the formation of the repeat expansion has not untranslated region (UTR) of FMR1. Among the general popu-
been completely elucidated. lation, the CGG repeat ranges from 6 to 55 repeats and usually
Many models have been proposed to explain the expansion the size does not change in size when passed from parent to
of trinucleotide repeats. One of the first proposed mechanisms offspring [26]. The most common forms of the repeat sizes
involved in repeat instability at the molecular level was slip- found in human populations studied are 21 and 28–30 CGG
page of the replication fork during DNA synthesis. Unpaired repeats [47–50]. Although the CGG repeat has no known func-
bases form loops, which result in expansions or contractions in tion, it is found in all species of mammals investigated [51, 52].
the next round of replication, depending on whether the looped The common CGG-repeat sizes have not proven to be
repeats are located in the newly synthesized or template strand associated with a disease phenotype; however, the conse-
[39]. However, slippage alone cannot explain all aspects of quence of an expanded CGG repeat (>200 repeats) in FMR1
repeat expansions, especially large expansions and contrac- is the fragile X syndrome. The hyperexpanded CGG repeat
tions. It is clear that other yet-to-be-identified factors are signals the hypermethylation and deacetylation of the FMR1
involved in the expansion process. Experimental support has promoter, the CGG repeat, and a nearby CpG island, which
come from studying a yeast model deficient in rad27 [40]. The transcriptionally silences the gene [17, 53–56]. Recent
Rad27 protein is involved in removing DNA loops, such as in vitro experiments demonstrated that it is methylation and
those arising during displacement synthesis of the Okazaki chromatic modification triggered by the expansion that are
fragments. Propagation of a CGG repeat in rad27 null yeast responsible for the transcriptional silencing of FMR1 rather
results in a highly significant increase of repeat expansions than the CGG repeat expansion itself [57, 58] (see also sec-
[40]. The human homolog of this gene is FEN1. tion, “Epigenetic Changes in the FMR1 Gene”).
Recognition of the unusual structural properties of trinu- Because FXS is essentially caused by the loss of the
cleotide repeats yielded new insights. Disease-causing FMR1 gene product, there is much interest in gathering
repeats are almost exclusively formed by (CNG)n triplets. information on the normal expression patterns of the gene
Single-stranded (CNG)n can form hairpin-like structures and its product’s function for the development of interven-
that can include both Watson-Crick and mismatched base tions or therapies. The FMR1 transcript is approximately
pairs. Due to their different sequences, the leading and lag- 4.4 kb in size and is alternatively spliced at the 3¢ end, giving
ging strands have different tendencies to form hairpins. The rise to various isoforms [46, 59]. Expression studies in human
secondary structures are likely to affect recognition and sub- and mouse tissues demonstrated that FMR1 is widely
sequent repair or recombination of the structure [41, 42]. expressed, with the highest levels localized to the brain, tes-
Unusual DNA structures may stall DNA polymerase, leading tes, ovaries, esophageal epithelium, thymus, spleen, and eye
to instability. A complex model based on replication fork [60–62]. High expression of FMR1 in regions of the brain
stalling and restarting has been described in detail [43]. such as the neurons of the hippocampus and the granular
Unlike other trinucleotide repeat disorders, there is layer of the cerebellum is consistent with the mental retarda-
absence of repeat instability in somatic cells in FXS. tion phenotype typical of FXS [63, 64] (see also section,
Methylation may stabilize the CGG repeats in full mutations “Clinical Aspects of Fragile X Syndrome”). Identification of
(see section, “Epigenetic Changes in the FMR1 Gene”). other mutations (e.g., deletions and point mutations in
Repeat expansion from pre- to full mutation occurs exclu- patients with FXS) has confirmed that FMR1 is the only gene
sively in females due to sex-specific factors. It has long been involved in the pathogenesis of the disorder and that the loss
known that sperm from full-mutation male patients possess of the FMR1 product causes the syndrome.
only premutation alleles [44]. This must result from reduc- A search for genes similar to FMR1 within the human
tion of repeat instability occurring during a limited time in genome found two identified autosomal homologs, fragile
early development. Both prezygotic and postzygotic models, X-related (FXR) genes 1 and 2, located at 3q28 and
directly after separation of the germ cells, have been pro- 17p13.1, respectively [65, 66]. Analysis of mouse and
posed [45]. Material is not available from premutation human genomic sequences demonstrates similarities in
females to support either hypothesis. gene structure among FMR1, FXR1, and FXR2, suggesting
that the three genes have an ancestral gene in common [67].
The functions of FXR1 and FXR2 are presently unclear;
The Fragile X Gene and Its Product: neither gene has been shown to be associated with human
FMR1 and FMRP disease. Many investigators have postulated that, because
of their similarity to FMR1, the FXR genes are somewhat
In 1991, the responsible gene was identified by positional clon- redundant, but although there are similarities, significant
ing and named the fragile X mental retardation-1 (FMR1) gene differences have been noted [68]. Furthermore, FXR1 and
[17–19]. FMR1 encompasses 38 kb of Xq27.3 and consists of FXR2 are not overexpressed in cells from persons with
FXS, suggesting that neither gene product compensates for support the electric signal transmission, and increase the
the loss of the FMR1 gene product [69, 70]. number of possible contacts between neurons [84].
The full-length protein product of FMR1 is 69 kilodaltons On their surface, the dendritic spines express glutamate
in size and is known as the fragile X mental retardation pro- receptors (GluR) of two types: the ionotropic receptors
tein, or FMRP [71]. At the protein level, FMRP is highly alpha-amino-3-hydroxy-t-methyl-4-isoxazolepropionic acid
conserved across humans, mice, the African clawed frog (AMPA) and N-methyl-d aspartic acid (NMDA), and the
(Xenopus laevis), and chickens [18, 59, 72, 73]. Although metabotropic receptors (mGluRs). A broad variety of pro-
not as highly conserved as among vertebrates, a homolog for teins mediate the signaling from the GluRs [85].
the FMR1 coding sequence has also been identified in Cognitive function, motivation, learning, and memory are
Drosophila melanogaster [74]. based on spine plasticity. After the formation of numerous
Much has been accomplished in elucidating the function dendritic spines during fetal cortical neurogenesis, the spines
of FMRP and how its absence leads to the development of need to mature or be pruned; immature spines show a
the FXS phenotype. Several properties of FMRP were the significant impairment in signal transduction. Abnormalities
first clues to its function. First, FMRP contains two ribonu- in spine formation have been observed in FXS, which can be
cleoprotein K homology domains (KH domains) and clusters directly correlated to the cognitive impairment. It has been
of arginine and glycine residues (RGG boxes), features typi- postulated that the loss of FMRP in FXS leads to an exces-
cal of RNA-binding proteins [71, 75]. Second, FMRP con- sive expression of mRNA near synapses, making it impossi-
tains both a nuclear localization signal (NLS) and a nuclear ble to regulate protein synthesis adequately, thus increasing
export signal (NES) [76]. Two coiled coils and a G-quartet- long-term depression due to receptor loss. FMRP is consid-
binding structure have been identified (Fig. 19.2) [77]. FMRP ered to be a repressor of specific mRNA translation and
is primarily a cytosolic protein, but its presence in the nucleus numerous proteins are upregulated, particularly in the hip-
has been reported by nuclear staining experiments [63, 78]. pocampus, when FMRP is absent [86].
Furthermore, FMRP has been detected in the nuclear pore FMRP colocalizes primarily with polyribosomes and
[79]. Taken together, current evidence suggests that FMRP ribosomes at/in the endoplasmic reticulum membrane and
shuttles between the nucleus and the cytoplasm affecting appears to play an important role in the regulation of transla-
protein synthesis in dendrites (dendritic spines) and synapses tion of specific target mRNAs. A subset of mRNAs contain-
[76, 80–82]. ing a G-quartet (a nucleic acid structure in which four guanine
Dendritic spines are small membranous extensions on residues are arranged in a planar configuration) has been
neuronal dendrites [83]. They serve as synaptic storage sites, identified that are potential targets for FMRP, including
Fig. 19.2 The FMR1 gene-coding exons (numbered boxes) and protein cates the untranslated CGG repeat alleles, <45 = normal range, 45–54 = gray
domains. NLS nuclear localization signal, KH1/KH2 RNA-binding domains, zone, 55–200 = fragile X premutation, >200 = fragile X full mutation
NES nuclear export signal, RGG RGG box RNA binding. The triangle indi- (Modified from Schneider et al. [77]; reprinted with permission)
458 E.B. Spector
important neuronal proteins like microtubule-associated memory consolidation and learning [84, 98–103]. Altered
protein 1B (MAP1B) and semaphorin 3F [87–89]. FMRP spine morphology (long and thin dendritic spines) has been
also can bind to mRNAs that do not contain a G-quartet. observed in postmortem brains of fragile X patients and in
In addition, experimental evidence suggests that FMRP is Fmr1 KO mice [84, 98–102]. The presence of the protein
involved in suppression of translational activities. FMRP machinery near synaptic connections allows neurons to rap-
forms complexes with messenger ribonuclear particles idly respond to signals at particular synapses through local
(mRNP) and is associated with translating ribosomes [76, translation of specific mRNAs in the vicinity of the synapse,
90, 91]. Because RNPs are formed in the nucleus, this obser- and FMRP plays a crucial role. The response is mediated
vation further supports the hypothesis that FMRP shuttles through the action of mGluR activation [77, 104, 105].
between the nucleus and the cytoplasm. Recent experiments It has been proposed that FMRP located at the synapse
suggest that FMRP may play a role in regulation of transla- represses translation of mRNAs encoding proteins that regu-
tion for certain messages. Laggerbauer et al. demonstrated late endocytic events involving the AMPA receptor. Upon
that FMRP suppresses translation by preventing the assem- synaptic stimulation, FMRP may dissociate from these
bly of the 80S subunit of the ribosome on the target RNAs mRNA targets to allow translation and facilitation of
[92]. New evidence suggests that translational control may AMPA receptor internalization. The model predicts that in
be mediated through the RNA interference (RNAi) and/or the absence of FMRP, the upregulated translation of a sub-
microRNA (miRNA) pathways [93, 94]. set of mRNAs would result in the perturbation of AMPA
The two major activities identified for FMRP, cytoplasm- receptor internalization dynamics (Fig. 19.3) (see Oostra and
nucleus shuttling and translational regulation, imply that Willemsen [44] for further discussion).
FMRP is a facilitator for the expression and localization of Protein kinases are crucial for the regulation of neuronal
several messages and proteins. The search for FMRP’s part- development and synaptic transmission upon response to extra-
ners has identified at least seven such proteins, one of which cellular or intracellular signals. The mGluR theory is in line
includes FMRP itself [68]. In contrast, very few specific with the translation control pathways within the dendritic spines;
mRNAs that bind FMRP have been identified. a simplified version is depicted in Fig. 19.4 [44]. Strong evi-
FMRP was shown to bind its own mRNA and also approx- dence supports the postsynaptic FMRP signaling model. Data
imately 4% of fetal brain mRNAs [71]. Nearly a decade suggest that dephosphorylation of FMRP may regulate FMRP
would pass before the identity of the specific mRNAs (other and that the release of FMRP-induced translational suppression
than the FMR1 transcript) binding to FMRP would be may involve a dephosphorylation signal. Rapid dephosphoryla-
identified [91–93]. These mRNAs contain a G-quartet struc- tion of FMRP allows target mRNAs to be translated, whereas
ture that facilitates binding to FMRP. FMRP can be phos- rephosphorylation represses translation. Several proteins
phorylated, a mechanism that possibly affects the binding of involved in this process have been identified [106, 107].
specific mRNAs [94]. Further research is needed to characterize the cascade of
The ability of FMRP to bind RNA and suppress translation signaling upon mGluR activation and the mechanism
has definite clinical relevance. As an RNA-binding protein, whereby FMRP phosphorylation regulates translation of tar-
FMRP is found to form a messenger ribonucleoprotein get mRNAs.
(mRNP) complex that associates with translating polyribo-
somes [95]. FMRP is also known to be involved in transla-
tional control and could suppress translation both in vitro and Epigenetic Changes in the FMR1 Gene
in vivo [86]. In 1987, Davis et al. demonstrated that the mRNAs
were transported into dendrites of cultured hippocampal neu- Methylation of the CGG repeat, which occurs in the pro-
rons [96]. Since then, a large number of dendritic localized moter region of the FMR1 gene, takes place early in embry-
mRNAs have been identified, and it is suggested that the trans- onic development and is a dynamic process. In early germ
lation of those mRNAs can be regulated in a spatially restricted cells from female full-mutation fetuses, the FMR1 repeat is
manner in response to stimulation [97]. At the cellular level, fully expanded and unmethylated [108]. In chorionic villus
abnormal dendritic spines are found in the brains of both samples from full-mutation fetuses, the expanded repeat is
human patients with FXS and Fmr1 knockout (KO) mice, methylated to an increasing degree as development pro-
implying that synaptic plasticity is affected in the absence of gresses [109]. Almost all FXS patients carry a fully methy-
FMRP. Based on these observations, it has been proposed that lated full expansion.
FMRP is involved in synaptic plasticity via regulation of Infrequently, individuals are identified who carry full
mRNA transport and local protein synthesis of specific mutations that are not methylated. These patients often do
mRNAs at synapses. Transport and regulated translation of not show the full spectrum of the fragile X syndrome, dem-
mRNAs in dendrites are important for neuronal function, onstrating that methylation and not repeat elongation alone
including modulation of synaptic plasticity. This is essential in causes the phenotype [110, 111].
Fig. 19.3 The CGG repeat in the FMR1 gene. Schematic representation. Methylation due to extensive elongation of the CGG repeat in the
tion of normal, PM (premutation), and FM (full mutation) alleles of the 5¢-UTR of the FMR1 gene is depicted as a lock (Modified from Oostra
FMR1 gene and the effect of the expansion on transcription and transla- and Willemsen [45]; reprinted with permission)
Fig. 19.4 The mGluR theory: in the absence of FMRP, as in fragile X features of the disease (Reprinted with permission from Willemsen R,
syndrome, the balance between FMRP and Gp1 mGluRs is lost, and Levenga J, Oostra BA. CGG repeat in the FMR1 gene: size matters.
unchecked protein synthesis at the synapse leads to the characteristic Clin Genet. 2011;80(3):214–5)
each allelic type have evolved over time and may change
FMR1-Associated Disorders with increased empirical data and research.
• Normal Alleles: Normal alleles have a range of 5–44
Allelic Forms of the FMR1 Gene repeats. The most common repeat lengths are 29 and 30
CGG repeats.
There are four allelic forms of the gene: normal, intermedi- • Intermediate (Gray Zone, Inconclusive, Borderline): The
ate, premutation, and affected. The associated numbers of range from 45 to 54 repeats is intermediate. Alleles in this
CGGs for each are defined here; however, the cutoffs for range can be considered normal in the sense that such
460 E.B. Spector
alleles have not been observed to expand to a full muta- tonia, and perseveration in speech and behavior. Social
tion in a single generation. Moreover, there is no observed anxiety and avoidance are prominent features of FXS in
increased risk for the specific premutation-associated dis- both sexes.
orders, although data are limited. Hagerman reviewed in detail the physical and behavioral
• Premutation: Premutation alleles range from 55 to ~200 phenotype of FXS [113]. The variability of expression makes
repeats. These alleles are long repeat tracks that are unsta- clinical diagnosis difficult. Therefore, FXS should be con-
bly transmitted from female parent to child. Premutations sidered in the differential diagnosis of all mentally retarded
are not associated with somatic variation, are not hyper- individuals.
methylated, and are not associated with classic features of
fragile X syndrome. Women with alleles in this range are Cognitive Phenotype
at risk to have affected children, although all known moth- In males, preliminary evidence suggests that there are specific
ers of affected children have alleles of 59 repeats or higher deficits in arithmetic, visual motor, and spatial skills; short-
[112]. Female members of families with CGG repeats in term auditory, visual, and working memory; executive func-
this range benefit from genetic counseling and prenatal tion; visuospatial-processing abilities; processing of
diagnosis. sequential information; and sustained attention. Approxi-
• Full Mutations: Full mutations associated with the fragile mately 85% of males and 25–30% of females with the full
X phenotype exhibit more than 200 CGG repeats and mutation have an intelligence quotient (IQ) less than 70. The
typically several hundred to several thousand repeats. severity of intellectual impairment is related to FMRP
There is usually broad somatic variation within each deficiency [77, 114, 115]. Some individuals with only a mild
patient. Hypermethylation is typically present on most or decrease of FMRP may present with a normal or borderline
all copies. IQ with or without learning disabilities (LD). LD with a nor-
mal or borderline IQ is a typical presentation in females with
FXS. IQ decreases with age, although the reason for this lon-
Clinical Aspects of Fragile X Syndrome gitudinal decline is unclear [116]. Adult males with FXS
function within the moderate to severely retarded range. IQ
Several disorders are now associated with mutations in the is not correlated with the size of the CGG repeat. However, it
FMR1 gene and are reviewed as follows. does appear to be correlated with the mosaic status of the
male. Affected males with both somatic full mutation and
premutation size repeats or those who are methylation mosa-
Full-Mutation Phenotypes ics have higher IQs than the affected males who are nonmo-
saic or fully methylated. On occasion, such males will test in
Physical Phenotype the normal/low normal range [117].
In males, the classic features of FXS are X-linked mental In females, with FXS cognitive studies indicate specific
retardation, macroorchidism, and minor dysmorphic facial weaknesses in arithmetic as well as short-term auditory mem-
features including a long, oblong face with a large mandible ory and visual-spatial tasks. They also have significant deficits
and large and/or prominent ears. Pectus excavatum, mitral in executive function. Full-mutation females have mean IQs
valve prolapse, and strabismus have also been described in in the low-average range (74–91), and, as in males, the IQ is
males with FXS. At least 80% of affected males have one or not correlated with CGG-repeat size. Most studies have found
more of these features, but expression varies with age. Other a relationship between IQ and X-inactivation ratios.
frequent features are a high-arched palate, hyperextensible
finger joints, velvet-like skin, and flat feet. Females with a Aging in FXS
full mutation may express these same features of FXS, There is an ever-increasing group of patients with fragile X
depending upon X-inactivation status [22]. syndrome. Until recently, there were no studies on the behav-
ior and cognitive problems in aging for those with FXS. Utari
Behavioral Phenotype et al. studied a group of individuals (44 males and 18 females)
The behavior of males with the FXS can be quite variable. with the syndrome who were over 40 years of age [118]. The
They show distinct behavioral features in the areas of atten- most frequent difficulties faced by these patients were neuro-
tion, hyperarousal, social function, anxiety, and aggression logical problems (38.7%), gastrointestinal problems (30.6%),
[113, 114]. Additionally, they are often diagnosed with obesity (29.8%), and heart problems (24.2%), which include
autism because they exhibit poor eye contact, hand flapping, mitral valve prolapse (MVP), cardiac conduction abnormali-
and social deficits that are the most prominent features of ties, heart attack, and heart rhythm disorder. Males had a
autism. They also exhibit various degrees of speech delay. significantly higher percentage of neurologic problems com-
Other complicating features can include irritability, hypo- pared with females. However, only movement disorders,
including Parkinson’s disease, were significantly different in and FMRP in premutation male carriers and found that
prevalence between males and females; some of these may be carriers with 100–200 CGG repeats had a fivefold increase
the result of long-term treatment with antipsychotics. An in FMR1 mRNA levels while carriers with 55–100 repeats
increase in seizures was observed in the older patients as had a twofold increase compared with controls [132, 133].
well. It is not known if this represents a second peak in sei- Moreover, these high-end premutation carriers (100–200
zure onset or if seizures are a complication of aging. repeats) had reduced levels of FMRP compared with con-
trols [132]. Additional experiments suggest that the elevated
Other Clinical Aspects level of FMR1 mRNA is correlated with CGG-repeat size
Several recent reviews explore the neurologic and pathologic and is not simply a response to decreased levels of FMRP
findings in FXS and related disorders [77, 113, 119]. Medical [133, 134].
follow-up, pharmacotherapy, treatment of emotional and An RNA gain of function mechanism has been suggested
behavioral problems, and intervention approaches for FXS for FXTAS based on the aforementioned observation of ele-
have also been reviewed and are ever expanding [120]. vated levels of CGG containing FMR1 mRNA, along with
either no detectable change in FMRP or slightly reduced
FMRP levels, observed in peripheral blood leukocytes and
Premutation Carrier Phenotypes brain regions [132, 134–138] of premutation carriers.
Many conflicting reports exist in the literature concerning
Unlike the full mutation, the existence of a phenotypic conse- cognitive, behavioral, and physical phenotypes among
quence of the premutation in males was controversial for female premutation carriers; these reports have been reviewed
some time. However, a specific phenotype has been identified [139, 140]. For reports on cognitive ability, studies of vary-
and is referred to as fragile X tremor ataxia syndrome (FXTAS) ing designs have shown that the prevalence of mental retar-
[121]. Approximately 40% of older males with the premuta- dation, the range of cognitive ability, and the range of IQ
tion will eventually develop FXTAS. The features of this dis- scores among adult female premutation carriers did not dif-
order include an intention tremor, ataxia, Parkinsonism, fer compared with control groups [141, 142]. However, at
neuropathy, cognitive deficits (particularly executive function least two studies have suggested differences among female
deficits with eventual cognitive decline to dementia in some), carriers compared with controls in specific subsets of IQ
and autonomic dysfunction including hypertension, impo- scores. In terms of a behavioral phenotype related to the pre-
tence, and eventual bladder and bowel incontinence [118, mutation, several studies suggest a difference based on
122–126]. Although some patients with FXTAS have a rapid specific behavioral or psychological measures among women
decline over 5–6 years, others are stable for a decade or two. with premutations compared with controls [123, 143].
More rapid decline typically occurs when the features of However, many of these suggested differences were not rep-
FXTAS are combined with another disorder. FXTAS occurs licated in other studies. Lastly, for physical or anthropomet-
in approximately 8% of female carriers [127]. ric measures, two studies suggest that female premutation
The neuroanatomical hallmark of FXTAS is intranuclear carriers do not have the same facial dysmorphic features
eosinophilic inclusions in neurons and astrocytes throughout typically observed in patients with the full mutation, while
the brain, with highest numbers in the hippocampus and lim- two studies suggest otherwise [138, 139].
bic system [128]. These inclusions have also been found in While the existence of a cognitive, behavioral, or physical
Leydig and myotubular cells of the testicles and in peripheral phenotype among premutation females remains controver-
nerve ganglia throughout the body [126]. They contain the sial, one consequence is consistently associated with the pre-
excess mRNA and also a number of proteins including lamin mutation: Fragile X primary ovarian insufficiency (FXPOI),
A/C and MbP that are dysregulated by the elevated mRNA referred to as premature ovarian insufficiency (POI) in older
[129]. The inclusions are probably not pathognomonic; they literature. FXPOI is defined as the cessation of menses before
are a marker for RNA toxicity. the age of 40 years. In contrast, the mean age of menopause
While cognitive or behavioral deficits have not been in the general population is 51 years. The first reports of
definitively attributed to the premutation in males, a molecu- female carriers of the fragile X mutation having FXPOI were
lar phenotype related to this repeat size range has emerged. anecdotally noted at the first International Fragile X
Early on, investigators examined levels of FMR1 mRNA and Conference (1987) in Denver, Colorado [144–149]. Schwartz
FMRP from the lymphocytes of carriers of premutation et al. were the first to report an association between the frag-
alleles and found that the levels were not significantly differ- ile X premutation and POI in a multicenter study [145]. The
ent compared with controls [55, 63]. Recent changes in tech- relationship between the fragile X premutation and POI was
nology, however, have made measurements of FMR1 mRNA eventually confirmed by a large, multicenter study, which
more sensitive and accurate [130, 131]. Using this technol- demonstrated that 16% of premutation carriers experienced
ogy, Tassone et al. reexamined the levels of FMR1 mRNA POI, while only 0.4% of noncarriers and none of the
462 E.B. Spector
full-mutation carriers experienced POI [146]. Results from transmitted unstably from parent to offspring [29]. Intermediate
this collaborative effort conclusively demonstrated that the alleles, like premutation alleles, do not cause hypermethyla-
premutation form of the CGG repeat, not the full mutation, is tion of the CpG island near FMR1 and are not thought to affect
associated with POI. Also, these data, combined with addi- cognitive or behavioral development. However, a recent study
tional reports from other sites, suggest that the rate of POI from Wessex, United Kingdom, found that boys placed in spe-
among premutation carriers is 21% (95% confidence inter- cial education had a higher frequency of alleles in the interme-
val: 15–27%) [147]. Overall, approximately 14% of idio- diate and premutation range compared with controls [159].
pathic familial POI and 2% of sporadic POI in the general The results from these data suggested, for the first time, that
population can be attributed to the fragile X premutation large CGG repeats smaller than premutations were somehow
allele [147]. responsible for the child’s placement in special education
The cause of POI among premutation carriers is related to [49, 159]. Although an excess of intermediate and premuta-
excess mRNA produced by the cells. Many models have tion alleles has not been observed in other special education
been proposed to explain the role of the premutation allele populations, new cognitive and molecular data warrant fur-
(as opposed to the full-mutation allele) in the development of ther research to identify and define a phenotypic consequence
POI among many (but not all) premutation carriers, but of intermediate-repeat alleles of FMR1, if one exists [132,
recent studies have yielded few clues to lend support to any 160, 161].
one model. Regardless of the cause, the occurrence of POI is
one of the factors that can limit the usefulness of preimplan-
tation genetic testing (PGT) as a reproductive option for car- Timing of the Premutation Expansion
rier females [150–152]. In fact, recent hormonal studies
suggest that female premutation carriers may unknowingly One of the yet unsolved questions is when in development the
be experiencing ovarian dysfunction at an early age and may expansion from premutation to full mutation occurs.
be facing a poorer prognosis for future pregnancy much ear- Expansion could occur during oögenesis (meiotic) or after
lier than expected [153]. The objective of PGD for FXS is to fertilization (mitotic). Reyniers et al. showed that full-muta-
utilize only those embryos that receive the normal X chro- tion or mosaic full/premutation males produce only premuta-
mosome from the mother. Donor egg, where available, is tion sperm and therefore premutation daughters, since repeat
another reproductive option that allows carrier females, even expansion occurs only in females [26, 162]. Testicular selec-
those with POI, to have unaffected children [154]. tion against full-mutation sperm is unlikely, since male Fmr1
It is important to recognize that there are other medical and knockout mice show fertility [163]. These data support a
psychiatric problems that can occur in some carriers, and these model of expansion only in somatic cells and protection of
are not necessarily part of FXTAS or FXPOI but may never- the premutation in the germ line cells. However, Malter et al.
theless be related to mRNA toxicity. Neuropathy is relatively showed that, in full mutation fetuses, only full-mutation
common in older carriers and can occur without other symp- alleles (in the unmethylated state) were found in oöcytes from
toms of FXTAS. Hypertension is seen in the majority of older intact ovaries or in immature testes from 13-week fetuses, but
carriers and may be secondary to the autonomic dysfunction that both full and premutation alleles were found in the germ
related to RNA toxicity [127]. Autoimmune problems may be cells of a 17-week male fetus [164]. They hypothesize that the
more frequent in female premutation carriers [127]. full mutation contracts in the fetal testes, with subsequent
Psychopathology that is more common in those with the selection for the premutation sperm. In females, the expan-
premutation includes anxiety, depression, and obsessive sion could occur during maternal oögenesis or very early in
compulsive behavior; these problems are clinically significant embryogenesis prior to general methylation. The answer
for 25–40% of carriers [155, 156]. In addition, there is newer requires analysis of oöcytes from premutation females.
evidence that the premutation has a neurodevelopmental
component in some children, especially boys, causing a
higher incidence of ADHD, shyness, and social deficits Current Genetic Aspects of Fragile X Syndrome
including autism spectrum disorder [123, 157, 158]. Further
studies of premutation carriers identified during newborn Epidemiology
screening may further delineate the percentage of carriers
with these problems. Crawford et al. provided an extensive review of the literature
and indicated a prevalence of FXS ranging from 1 in 3,717 to
Intermediate Carriers 1 in 8,198 in Caucasian males in the general population
Intermediate alleles, also known as “gray-zone” alleles, range [165]. The female prevalence rate is presumed to be approxi-
from 45 to 54 CGG repeats and are classified differently than mately one-half of the male rate. In another study carried out
premutation or common alleles in that they may or may not be over 4 years in metropolitan Atlanta, Crawford et al. determined
the prevalence of the FXS to be 1 in 2,545 African-American The rules of inheritance, as currently understood, include the
males and 1 in 3,717 Caucasian males [166]. However, the following [174]:
prevalence estimate for Caucasian males, determined from 1. Every affected individual has a carrier mother with an
this and from other studies, fell within the 95% confidence observable expansion. No new mutation has gone directly
interval for African-American males. The prevalence of the from normal to full. Full-mutation males do not pass a full
fragile X mutation in an Afro-Caribbean population in mutation to their daughters.
the French West Indies was similar (1 in 2,539) to that in the 2. Affected females have a full mutation, and unaffected
African-American population in Atlanta [167]. Falik et al. females may have premutations or nonpenetrant full
have suggested that the Tunisian Jewish population is the mutations. As a result, a female with a full mutation has
only other ethnic group to have a higher prevalence of FXS an obligate carrier mother, but a female with a premuta-
than the Caucasian population [168]. However, these studies tion could have received that X chromosome from either
were not supported by the data of Tolodano-Alheder et al. parent.
[169]. Further studies are required to determine if the fre- 3. The risk that a female carrier will have a child with a full
quency of FXS differs in ethnic populations. A recent sys- mutation is directly related to the size of her expansion.
tematic review of population screening for fragile X syndrome A repeat size of 99 appears to be the point of significance,
summarized the data and suggested that if population screen- as nearly all premutations with ³99 repeats become full
ing is to be instituted more psychosocial support will be mutations in subsequent offspring [173].
required [170]. 4. Premutations appear to be inherited silently for many gen-
The premutation is common in the general population erations. No family has been documented in which a nor-
with a prevalence of 1 in 130–260 females and 1 in 250–810 mal allele expanded to a premutation allele. Thus, many
males [171]. There is variability in the prevalence figures present families may have the same ancestral premutation,
depending on where the study was done and the ethnic or but this cannot be traced reliably. Using polymorphism
racial background of the patients. The study recently reported analysis, Smits et al. showed one family with five living
by Cronister et al. in 2008 has shown that the premutation is males with FXS who share an X chromosome to be related
less common in those of Chinese background and more com- through their last common ancestor six or more genera-
mon in the Middle East, particularly in Israel, as reported by tions in the past [175].
others [169, 172].
The premutation form of the CGG repeat is the precursor
to the full mutation in that the repeat is very unstable when Diagnostic Laboratory Testing for Fragile X
transmitted from parent to offspring, eventually expanding to Syndrome
the full-mutation form when passed through a female germ
line [27]. Using this definition, premutations can range from Cytogenetic Testing
50 to <200 repeats. The absolute lower boundary of the pre-
mutation repeat size that is at risk for expanding to the full From 1977 to 1992, the standard laboratory test for diagnosis
mutation in a single generation is still under debate [25, 173]. of FXS was cytogenetic scoring for expression of the fraX in
Studies of premutations among families with a member metaphase cells (Fig. 19.1).
reported affected by FXS suggest that the smallest premuta- Compared to routine chromosome analysis, fraX testing
tion to expand to the full mutation in a single generation is 59 was fraught with technical difficulties as well as biological
repeats [173]. However, small premutation alleles (~50–65 limitations. Culture conditions had to be altered to facilitate
repeats) ascertained from the general population have proven expression of the fragile site via folate stress (see Chap. 14).
to be more stable than those ascertained from families with fraX expression was variable (between 1 and 50%), with
FXS [173]. Given the uncertainty in the lower boundary of females usually having fewer positive cells than males, and
the premutation, the prevalence of the premutation varies obligate carriers often tested negative. Expression of the
from study to study, depending on the ranges of CGG repeats marker tended to be easier to appreciate in unbanded (or
that are considered premutations. under-banded) cells, which in turn created the potential for
uncertainty of X chromosome identification. As a result,
many cells had to be scored. Also, the presence of the other
Molecular Rules of Inheritance three fragile sites on Xq reduced the reliability of the assay.
Lastly, lower expression in cell types other than lymphocytes
DNA analysis of the FMR1 allele can detect all stages of made prenatal diagnosis difficult if not impossible.
the trinucleotide repeat expansion. Reduced penetrance, One significant advantage, however, was that the test was
the Sherman paradox, and other unusual characteristics combined with routine chromosome analysis, and as a result,
of FXS were explained by the silent premutation state. chromosome abnormalities could be diagnosed as well.
464 E.B. Spector
Molecular Testing of methylation [175, 176]. A genotype classification method

using a methylation-specific triple PCR method that distin-
By the time DNA-based diagnosis of FXS became available, guishes all normal and premutation males and females and
the problems with cytogenetic testing had become apparent all full-mutation males and females has been described [177,
[176]. DNA-based testing has solved these problems, and 178]. This method may provide a suitable alternative for
therefore cytogenetic fraX testing has been retired. In fact, the Southern blot analysis and yield estimates of allele sizes
reimbursement (CPT) code for such testing has been deleted. similar to other PCR-based methods.
The objective of all DNA-based methods for FXS is to Recently, a PCR-based screening method has been reported
identify a piece of DNA containing the CGG repeat and for detection of carrier females and affected newborns. These
determine its length and methylation status in order to clas- assays use CGG-repeat primed PCR and automated capillary
sify it as normal, premutation, or full mutation. electrophoresis and detect the presence or absence of an
expanded FMR1 allele with high sensitivity and specificity,
DNA-Based Methods minimizing the need for Southern blot analysis [177–179].
The two DNA-based methods available for FMR1 testing Importantly, the use of triplet primers allows females who are
are Southern blot, with or without methylation, and PCR homozygous for a single allele to be distinguished from
(polymerase chain reaction). PCR is more sensitive for pre- females with a normal allele and an expanded allele. This
mutations or carrier testing, and the results are usually screening method can also detect the expanded allele in
expressed as total repeat number. PCR is followed by capil- affected males. The technique uses two gene-specific primers
lary electrophoresis (CE) or polyacrylamide gel electropho- (forward and reverse) and a CGG-repeat primer in a single
resis (PAGE) for size resolution for the detection of up to tube. After amplification, the products, which include the
100–150 CGG repeats. For the last 20 years, Southern blots full-length amplicon that completely encompasses the triplet
have been the preferred method for detecting full mutations repeat region and a multiplicity of CGG-repeat primed prod-
and, if double digestion is utilized, the methylation status can ucts, are resolved by capillary electrophoresis. The resulting
be determined. The results are expressed as D kb (delta kb, electropherogram supports quantification of the number of
defined as the difference between the patient and a normal CGG repeats, the determination of the allele zygosity, and the
reference). Both DNA-based methods are considered diag- sequence context of any AGG spacer elements [178].
nostic and are 99% sensitive and 100% specific. Detailed Commercially available reference materials have been
descriptions and illustrations of these techniques are pro- characterized [180, 181]. These reference materials are avail-
vided in the American College of Medical Genetics Standards able as DNA isolated from cell lines or the cell lines them-
and Guidelines for fragile X testing [174]. The most current selves for normal, premutation, and full mutations from the
version can be obtained from www.acmg.net [174]. Coriell Repository [182]. The genotypes of these cell lines
are listed at the Centers for Disease Control (CDC) Get-RM
Latest PCR-Based Methods Web site [183].
Standard PCR of GC-rich regions is difficult and special An important caveat for DNA-based methods is the fact that
amplification conditions are required. The difficulty increases a small percentage (<1%) of patients with FXS have a normal
with increasing numbers of CGG repeats; therefore, in the CGG-repeat size. To date, numerous deletions have been
past, many PCR strategies did not attempt to detect large reported in the literature [180]. Also, two other types of muta-
alleles. It is not possible to use standard PCR to distinguish tions have been reported: a two-base-pair substitution that alters
between a female who is homozygous for a normal allele and splicing and leads to altered levels of FMRP, and a missense
a female who has one normal allele and a second, large mutation that leads to dysfunctional FMRP [181, 184]. Prior
nonamplifiable allele. Similarly, patients who are mosaic for et al. reported a case of germ line mosaicism, an important
premutations and full mutations will appear to have only pre- issue when counseling deletion families [185].
mutations. Even though a standard strategy can detect alleles
in the premutation range, amplification usually favors the Protein/mRNA-Based Diagnosis
smaller allele, and mosaicism may be missed. Thus, because Monoclonal antibodies against FMRP have been used with
of disproportionate amplification, standard PCR is not reli- success to diagnose affected males and some affected females
able for determining the ratio of different allele species in a [186]. This earlier method is more rapid than DNA-based
mosaic individual. testing but cannot be used for premutation testing. It has been
PCR amplification of the relevant portion of the FMR1 successfully utilized for prenatal diagnosis and may also be
gene is not affected by methylation. Although PCR-based used for a patient with the physical and mental features of the
tests that are specifically modified to detect methylation sta- FXS without evidence for an expanded CGG repeat [80]. The
tus have been described, the common PCR strategies that protein test can be performed on a variety of samples, includ-
have been in use for many years are completely independent ing blood and hair root [187]. Iwahashi et al. introduced a
quantitative sandwich ELISA assay for the FMRP1 protein. Carrier testing could be elected by any individual whether
This assay can be used in peripheral blood lymphocytes, is he or she has a positive family history or not, especially
quantitative and scalable, is specific for the FMRP, and could since the frequency of premutation carriers in human popu-
potentially be used for diagnosis of various FMR1-associated lations appears to be high. Family members whose carrier
clinical phenotypes as well as newborn screening [188]. status was determined by DNA linkage should be tested to
confirm the result. Likewise, DNA testing is recommended
for low-expressing family members who were diagnosed
FRAXE Syndrome cytogenetically.
Cytogenetically, FRAXE was described in 1992 [12]. The

gene (AFF2, formerly FMR2) is located 600 kb distal to Prenatal Diagnosis (See Also Chapter 12)
FMR1, and the repeat sizes in normal, premutation, and full-
mutation individuals are similar (Table 19.3). FRAXE expan- Prenatal DNA testing is indicated in families where the
sion can decrease or increase in both males and females, and mother is a known carrier of a premutation/full-mutation
two deletions have been identified [189]. No point mutations CGG repeat. This is the only situation in which the offspring
within AFF2 have been reported. The phenotype of FRAXE is at risk to inherit a full mutation. Specimens from either
syndrome appears to be mild MR (IQ = 60–80); however, the amniocentesis or CVS (direct or tissue culture) can be used
collection of cognitive and behavioral data from FRAXE to determine the allele size of the fetus. Timing and avail-
families may further differentiate this phenotype from that of ability are issues that help determine the procedure selected.
FRAXA [190, 191]. A knockout mouse model for AFF2 CVS is done early in pregnancy and, if sufficient tissue is
(fmr2) exhibiting impaired learning and memory may also obtained, testing can be performed on uncultured cells. In
help to further define the mild phenotype of FRAXE observed CVS tissue, full mutations are not always methylated, so
in humans [192]. Preliminary work suggests that AFF2’s interpretation must be based on the size of the allele, not its
gene product acts as a transcription activator, but its function methylation pattern. Maternal cell contamination, if present,
in relation to the phenotype remains largely unknown [192]. can be detected via fetal to maternal comparison.
FRAXE expansions are not common in human populations Interpretation of results of testing is usually unremarkable,
(approximately 1 in 23,500 individuals), and, although avail- except in the case of full-mutation females. The severity of
able, DNA analysis for the FRAXE expansion is not widely the disorder cannot be predicted in an individual female, but
utilized [193]. is based on the risk probabilities developed in family studies
of such females.
Indications for Prenatal Diagnosis

and Carrier Testing Genetic Counseling
Carrier Testing Genetic counseling is a vital part of a multidisciplinary

approach to helping families adjust to and cope with the
Women who have affected children are obligate carriers. stresses of FXS and its impact on the family (see the excel-
Determining DNA status for these women is indicated if future lent review by Gane and Cronister [194]). Genetic counsel-
pregnancies are planned. Other family members who could ing covers a multitude of areas such as diagnosis, prognosis,
share an X chromosome with an obligate carrier are at risk and recurrence risks, family planning options, management, and
should be referred for counseling and possible testing. psychosocial issues, to name a few. It provides the family
Table 19.3 Characteristics of the cloned folate-sensitive fragile sites

Copy Number
Symbol Location Disease Normal Premutation Full mutation
FRAXA Xq27.3 FXS 6–54 61–200 230 to >1,000
FRAXE Xq28 Fragile XE syndrome 6–25 ?50–200 200 to > 800
FRAXF Xq28 None 6–29 ? 300–1,000
FRA16A 16p13.1 None 16–50 ?50–200 ?1,000–2,000
FRA11B 11q23.3 Offspring predisposed 11 85–100 100–1,000
to Jacobsen syndrome
Adapted from Howard-Peebles [32]
466 E.B. Spector
with educational and emotional support so they can adjust to international workshop on the fragile X and X-linked mental retar-
and cope with present as well as future circumstances. dation. Am J Med Genet. 1991;38:158–72.
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Howard-Peebles, author of the chapter in previous editions, for providing A, Reiner O, Richards S, Victoria MF, Zhang F, Eussen BE, van
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Identification of a gene (FMR-1) containing a CGG repeat coinci-
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Genomic Imprinting and Uniparental
Disomy 20
Jin-Chen C. Wang
in mice, the maternal genome is essential for embryogenesis,

Introduction while the paternal genome is essential for placental
development.
Genomic imprinting refers to the process of differential The human equivalents to these observations in mice are
modification and expression of parental alleles; the parental the ovarian teratoma and the complete hydatidiform mole
origin of the allele dictates whether it is transcribed. It is an and the two types of triploidy, namely, digynic triploidy and
epigenetic form of gene regulation that allows expression of diandric partial hydatidiform mole (see Chap. 8). Ovarian
only one parental allele. As a result, the same gene functions teratoma is an embryonal tumor that contains tissues pre-
differently depending on whether it is maternally or pater- dominantly derived from ectodermal but also mesodermal
nally derived. This concept is contrary to that of the tradi- and endodermal germ layers. The ovarian teratoma has been
tional Mendelian inheritance in which genetic information shown to be parthenogenetic and contains two sets of the
contributed by either parent is assumed to be equivalent. maternal genome and no paternal genome [8]. The complete
The term “imprinting” was coined by Crouse to describe mole, on the other hand, is androgenetic and contains two
the modification and the selective elimination of paternal X sets of the paternal genome and no maternal genome [9, 10].
chromosomes from somatic and germline cells of the fly Studies of the parental origin of the extra haploid set of
Sciara, in which the “imprint” a chromosome bears is deter- chromosomes in triploids reveal that this is maternal (digy-
mined only by the sex of the parent through which the chronic triploidy) when severe intrauterine growth restriction
mosome has been inherited. It has since been used in many and abnormally small placentas are seen, while it is paternal
other species, including man [1, 2]. (diandric triploidy) in partial hydatidiform moles, in which
Evidence for the existence of genomic imprinting is man- the placenta is abnormally large [11–13]. Intercross experi-
ifold. Initial experimental approaches included studies in ments in mice between either Robertsonian or reciprocal
mouse embryos using nuclear-transplantation techniques translocation carriers further demonstrate that maternal
[3–7]. These experiments involved the removal and reintro- duplication/paternal deficiency or maternal deficiency/
duction of pronuclei into zygotes, thus creating embryos that paternal duplication of certain mouse chromosomes or
had either only the maternal or paternal genome. In parthe- regions of chromosomes results in different phenotypic
nogenetic eggs—i.e., eggs that contain two maternal pronu- abnormalities [14].
clei and no paternal pronucleus—fetal development was Observations of X-chromosome inactivation in different
relatively good, but extraembryonic tissue development was species and different tissues provide further evidence of
poor. In contrast, in androgenetic eggs—i.e., eggs containing imprinting. Although inactivation of the X chromosome in
two paternal pronuclei and no maternal pronucleus—the females of placental mammals is in general random in
development of extraembryonic tissue was good, but fetal somatic cells, studies in interspecies crosses between marsu-
development was poor. In either case, the embryos failed to pials reveal that the paternally derived X chromosome is
reach term. Thus, both maternal and paternal genomes are preferentially inactivated in female kangaroo somatic tissues
required for normal development, and it appears that, at least [15, 16]. In extraembryonic tissues, the paternally derived X
chromosome is preferentially inactivated in mice [17–19].
Further, the paternal X in mice is imprinted to become inactive
J.-C.C. Wang, M.D. (*)
early during embryonic development, perhaps as early as the
Cytogenetics, Integrated Genetics, 655 East Huntington Drive,
Monrovia, CA 91016, USA two-cell stage. Although apparently incomplete, this early
e-mail: [email protected] form of inactivation insures dosage compensation throughout
474 J.-C.C. Wang
development. Silencing of the paternal X chromosome per- [38]. These observations suggest that DNA methylation may
sists in cells of extraembryonic tissues, but it is erased and exert its effect on gene transcription by altering interactions
followed by random X inactivation in cells of the embryo between DNA and nuclear proteins.
proper [20]. Earlier studies on the pattern of X inactivation in The involvement of methylation in the initiation and/or
trophoblastic cells of human placenta yielded conflicting maintenance of genomic imprinting has been examined
results; both preferential inactivation of the paternal X and extensively. Experiments with transgenic mice, in which a
random X inactivation have been reported [21–25]. Those foreign gene was inserted into the mouse genome by microin-
studies analyzed only one or two X-linked loci to infer the jection, have demonstrated that some transgenes show differ-
activity of the entire X chromosome. By analyzing allele-specific ent states of methylation specific to the parent of origin and
expression of 22 X-linked genes, a recent study concluded that the methylation pattern of those transgenes changes from
that X inactivation is random in the human placenta and that generation to generation depending upon the sex of the trans-
the placenta is arranged in relatively large patches of cells with mitting parent [39–41]. In most cases, a paternally inherited
either maternal or paternal inactive X chromosome [26]. transgene is less methylated than one that is maternally inher-
Direct evidence that genomic imprinting exists in man is ited. In a study of transgene-bearing elements of the Rous
provided by the observation of a variety of human conditions sarcoma virus (RSV) and a fused c-myc gene, the paternally
or diseases such as Prader-Willi syndrome (PWS) and inherited transgene was undermethylated in all tissues and
Angelman syndrome (AS), certain types of cancer, and was expressed only in the heart [41]. This observation sug-
uniparental disomy. These are discussed in detail later. gests that methylation status alone does not determine the
Not all chromosomes or all regions of one chromosome expression of a transgene and that undermethylation may be
are involved in genomic imprinting [27]. It is estimated that necessary, but not sufficient, for gene expression. In this same
<1% of the mammalian genome is imprinted [28, 29]. In the study, the somatic organs of a male animal with a maternally
mouse, approximately 100 genes undergo genomic imprint- inherited transgene exhibited a methylated transgene pattern,
ing and hundreds more are predicted by examining specific but in the testes the transgene was undermethylated, suggest-
epigenetic features rather than local sequence features [30]. ing that the maternally derived methylation pattern is elimi-
Approximately 70 imprinted genes have been identi fi ed nated in the testes of male offspring during gametogenesis.
in humans (on chromosomes 1, 2, 4, 6, 7, 8, 10, 11, 14, 15, The role of DNA methylation in genomic imprinting is
16, 18, 19, 20), and many more possibly imprinted genes are further demonstrated by observations made in three imprinted
waiting to be identified and confirmed [31, 32]. endogenous genes in mice: insulin-like growth factor 2
(Igf2), H19 (these two genes are closely linked on mouse
chromosome 7) and the Igf2 receptor gene (Igf2r, on mouse
Mechanism chromosome 17).
Studies of mouse H19 showed that it is subject to tran-
Imprinting is a phenomenon that is reversible from generation scriptional regulation by genomic imprinting, with the mater-
to generation. The process must therefore involve the estab- nal allele expressed and the paternal allele silent [42]. By
lishment of the imprint during gametogenesis, the maintenance comparing CpG methylation and nuclease sensitivity of
of the imprint through embryogenesis and in adult somatic chromatin in mouse embryos, Ferguson-Smith et al. showed
tissues, and then the erasure of the imprint in the germline that hypermethylation and chromatin compaction in the
[33–35] (see Fig. 20.1). Thus, stable and differential region of the H19 promoter are associated with repression of
modification of chromatin is required. Differential methyla- the paternally inherited copy of the gene [43]. This normally
tion, controlled by DNA methyltransferase enzymes, of the silent paternal H19 allele is activated in DNA methyl-
cytosine residues of DNA on maternal and paternal chromo- transferase-deficient embryos, providing in vivo evidence
somes appears, at least in part, to fulfill this role. that a direct correlation is present between DNA methy-
DNA methylation is involved in human X-chromosome lation and gene activity [44].
inactivation. Using 5-azacytidine, which causes hypomethy- Studies of the mouse Igf2 gene showed that, contrary to
lation of DNA, Mohandas et al. were able to achieve reacti- H19, the paternal allele is expressed in embryos, while the
vation of an inactive human X chromosome [36]. Yen et al. maternal allele is silent, but both parental alleles are tran-
showed that the human HPRT (hypoxanthine phosphoribo- scriptionally active in the choroid plexus and leptomeninges
syltransferase) gene is hypomethylated on the active X chro- [45]. Therefore, imprinting of Igf2 may also be tissue specific.
mosome relative to the inactive X [37]. Furthermore, DNA In addition, studies using mouse embryos with maternal
methylation has been shown, in experiments involving gene duplication and paternal deficiency of the region of chromo-
insertion into mouse L cells, to render these sequences insen- some 7, which encompasses Igf2, showed that the chromatin
sitive to both DNase I and restriction endonucleases, by of the 5¢ region of the repressed maternal Igf2 allele is poten-
directing DNA into an inactive supranucleosome structure tially active for transcription; i.e., it is hypomethylated and
20 Genomic Imprinting and Uniparental Disomy 475
contains DNase I hypersensitive sites [46]. Recently, a region 11 at band 11p15.5 [56–58]. In the case of IGF2, although it
of paternal-specific methylation between H19 and Igf2 has is the paternal allele that is active, the maternal allele is
been postulated to function as the imprint control region. hypomethylated while the paternal allele is methylated at the
This imprint control region, when unmethylated, acts as a 5¢ portion of exon 9, similar to the findings in mouse studies.
chromatin boundary or insulator that blocks the interaction Unlike this gene in mice, the human IGF2R gene is not
of Igf2 with its enhancer, thus resulting in silencing of the imprinted [59].
Igf2 gene, as is observed on the maternal chromosome. On The differentially methylated domains (DMDs) of
the paternal chromosome, this region is methylated, resulting imprinted genes contain CpG-rich imperfect tandem repeats
in the loss of enhancer-blocking activity and allowing the expres- with similar predicted secondary structures. It is this repeat-
sion of Igf2 [47, 48]. A deletion within this imprint control related DNA structure, not the sequence, that is implicated in
region results in loss of imprinting of both H19 and Igf2. the imprinting mechanism—the establishment and mainte-
Studies of the mouse Igf2r gene indicated that the mater- nance of parent of origin-specific methylation patterns. It is
nal allele is expressed and the paternal allele is silent [49]. suggested that a structural feature or features of these tandem
The parental-origin-specific difference in methylation for repeats are the conserved DMD imprinting signal (reviewed
this gene has been demonstrated in two distinct CpG islands in [35]).
[50]. Here, while the promoter is methylated on the inactive In summary, epigenetic modifications by methylation of
paternal allele, an intronic CpG island is methylated only on alleles of each imprinted gene are established during oögen-
the expressed maternal allele, suggesting that methylation of esis and spermatogenesis. This imprint pattern is maintained
the latter site is necessary for expression of the Igf2r gene. throughout embryogenesis and in adult somatic tissues. In
In humans, the methylation patterns of the parental alleles fetal gonads, global demethylation of the progenitor germ
have been determined for several imprinted loci on chro- cells occurs and the inherited imprinting pattern is erased.
mosome 15 at bands 15q11.2-q13. These include the This is followed by reestablishment of methylation of
MKRN3 gene (D15S9) studied in PWS and AS patients and in imprinted genes during gametogenesis, depending on the sex
complete hydatidiform moles, the small nuclear ribonucleo- of the fetus. This reprogramming in germ cells ensures that
protein polypeptide N (SNRPN) gene, and the DNA sequence sex-specific genomic imprinting is initiated and that an accu-
PW71 (D15S63) [51–55]. Distinct differences in methyla- rate imprinting cycle is achieved through each generation
tion of the parental alleles are observed in all instances. This (reviewed in [60]) (refer to Fig. 20.1).
is also true for some of the other known imprinted genes in A difference in DNA replication timing of maternal and
humans: H19 (maternal allele active) and IGF2 (paternal paternal alleles of imprinted genes has also been observed
allele active), both located on the short arm of chromosome [61–65]. Cell-cycle replication timing has been shown to
Fig. 20.1 Diagrammatic representation of

the imprinting process. Light-colored circles
represent undermethylated genes, dark-
colored circles represent hypermethylated
genes. m maternally derived chromosome,
pink. p paternally derived chromosome, blue.
See text for details
476 J.-C.C. Wang
correlate with gene activity: genes that are expressed generally known as AHO without multiple hormone resistance, OMIM
replicate earlier [66, 67]. Furthermore, most genes on homol- #612463). AHO is caused by heterozygous deactivating
ogous chromosomes replicate synchronously [68]. This is mutation in the GNAS gene located at 20q13. GNAS is
not the case for imprinted genes. Using fluorescence in situ imprinted (paternal allele inactive); the imprinting appears to
hybridization (FISH; see Chap. 17) on interphase nuclei and be tissue specific, with maternal expression only in certain
scoring for the stage of the two alleles in S phase, Kitsberg cells, such as cells of the proximal renal tubule [75]. In fami-
et al. showed that the imprinted genes H19, Igf2, Igf2r, and lies with AHO, the strongest predictor of the endocrine phe-
Snrpn in mice and their corresponding positions in the human notype is the parent of origin. PHP1A occurs only after
genome all replicate asynchronously, with the paternal allele maternal inheritance of the molecular defect, either GNAS
replicating early [61]. Studies of genes in the 15q11.2-q13 mutation or GNAS imprinting defects, whereas PPHP occurs
region in humans demonstrated that most show a paternal- only after paternal inheritance of the molecular defect [76].
early/maternal-late pattern, with some exhibiting the oppo- These observations indicate involvement of imprinting in
site pattern [62, 63]. Therefore, it appears that imprinted disease phenotype.
genes are embedded in DNA domains with differential repli-
cation patterns, which may provide a structural imprint for
parental identity [62]. This asynchronous replication timing Chromosome Deletion/Duplication Syndromes
is established in the gametes during late gametogenesis and
maintained throughout development [65]. Prader-Willi Syndrome/Angelman Syndrome
Thus, the process of genomic imprinting is very complex, The best-studied examples of genomic imprinting in human
and while DNA methylation plays a critical role in genomic disease are the Prader-Willi and Angelman syndromes. These
imprinting, the process is much more complex than simply are clinically distinct disorders; both map to the chromosome
inactivating a gene by methylation. It may involve an interac- 15q11.2-q13 region, but they involve different genes [77–81].
tion between DNA methylation, histone modification includ- The etiologies of these disorders include the absence of a
ing acetylation and methylation, chromatin compaction, DNA parent-specific contribution of this region due to either deletion
replication timing, and potentially other mechanisms [69, 70]. or uniparental disomy (UPD), disruptions in the imprinting
process, and mutations within the gene [82–96].
The clinical phenotype of PWS has been well character-
Genomic Imprinting and Human Diseases ized [97, 98]. Briefly, it includes hypotonia during infancy,
obesity, hyperphagia, hypogonadism, characteristic facies,
Genomic imprinting provides an explanation for the obser- small hands and feet, hypopigmentation, and mental
vation that the transmission of certain genetic diseases can- deficiency. Approximately 70–75% of cases have an intersti-
not be explained by traditional Mendelian inheritance, but tial deletion of a 4 Mb sequence at 15q11.2-q13 on the pater-
that rather the phenotype depends upon whether the gene nally derived chromosome 15 [69]. Approximately 20–25%
involved is maternally or paternally inherited. Conversely, of cases are due to maternal uniparental disomy for chromo-
the existence of such diseases provides evidence that genomic some 15 and 1% or so as a result of an abnormality of the
imprinting occurs in man. Human conditions that fall into imprinting process, causing a maternal methylation imprint
this category include certain deletion/duplication syndromes, on the paternal chromosome 15 [86, 89, 93, 94] (Table 20.1).
a number of cancers, and many disorders arising from unipa- Many paternally expressed transcripts have been identified
rental disomy. In addition, imprinted genes may also contrib- in a cluster in the proximal part of the 15q11.2-q13 region.
ute to modification of disease phenotype, such as is observed These include MKRN3, MAGEL2, NDN, PWRN1, NPAP1,
in Albright hereditary osteodystrophy, language develop- SNURF-SNRPN, a number of C/D box small nucleolar RNA
ment, and some psychiatric disorders and complex behav- (snoRNA) genes, and other additional transcripts (reviewed
ioral phenotypes, including bipolar affective disorder and in references [99–103]). This clustering of paternally
catatonic schizophrenia [71–74]. expressed transcripts suggests strong regional control of the
Albright hereditary osteodystrophy (AHO) is character- imprinting process [103]. It has been recently demonstrated
ized by short stature, obesity, brachydactyly, mild-to-moderate that deficiency of SNORD116 (previously HBII-85) snoRNAs
mental handicap, and subcutaneous ossifications. A propor- causes the key characteristics of the PWS phenotype
tion of patients with AHO have associated end-organ [104, 105]. Other imprinted genes in the 15q11.2-q13 region,
resistance to parathyroid hormone (PTH), known as “pseudo- such as MAGEL2 and NDN, probably also contribute to the
hypoparathyroidism type I” (PHP Ia, also known as AHO PWS phenotype.
with multiple hormone resistance, OMIM #103580). The clinical phenotype of AS patients is distinct from
Individuals with AHO and normal endocrine responsive- that of PWS [106, 107]. Briefly, it includes microcephaly,
ness have “pseudo-pseudohypoparathyroidism” (PPHP, also ataxia, characteristic gait, spontaneous laughter, seizures, severe
Table 20.1 Etiology and recurrence risk of Prader-Willi syndrome and Angelman syndrome
Etiology PWS AS Recurrence risk
Deletion ~70–75% Paternal chr 15 ~70% Maternal chr 15 <1%
UPD ~20–25% Maternal UPD ~6% Paternal UPD <1%
IC abnormality ~1% ~3–6% 50%
(PWS: when present in father)
(AS: when present in mother)
Gene mutation – ~10% UBE3A 50%
(AS: when present in mother)
Unknown – ~10% –
mental retardation, and hypopigmentation. Approximately These observations in PWS and AS indicate that the PWS
70% of AS patients have a deletion of the same 4 Mb genes are active only on the paternal chromosome 15 and the
sequence at 15q11.2-q13 on the maternally derived chromo- AS gene is active only on the maternal chromosome 15.
some 15 [83–85]. Approximately 6% are due to paternal These two syndromes serve as classical examples of genomic
uniparental disomy for chromosome 15; 3–6% as a result of imprinting in humans.
an abnormality of the imprinting process, causing a paternal Deletion, UPD, or IC disruption can all result in an abnor-
methylation imprint on the maternal chromosome 15; and mal methylation pattern of the PWS/AS parental alleles.
approximately 10% as a result of a mutation within the AS Therefore, the most cost-effective approach to laboratory
gene (reviewed in Refs. [81, 87–93, 95, 96, 108, 109]) diagnosis of PWS/AS is to perform DNA methylation stud-
(Table 20.1). In contrast to PWS, mutation of a single gene, ies first. This will detect virtually all cases of PWS and
the gene for E6-associated protein (E6-AP) ubiquitin-protein approximately 80% of the cases of AS. If the result is abnor-
ligase (UBE3A) (maternal allele active) has been identified mal, fluorescence in situ hybridization (FISH) to detect
in some AS families and is considered the candidate gene for 15q11.2-q13 microdeletion, followed by UPD studies, should
AS [81, 96]. The imprinting of UBE3A is tissue specific, be performed to determine the exact etiology. In the case of
being restricted to the brain [110–112]. More recently, AS, UBE3A mutation analysis can be considered when the
another imprinted gene ATP10A, mapped within 250 kb telo- methylation study is normal (Fig. 20.2).
meric to UBE3A, has also been shown to be expressed only
on the maternal allele [113]. It is speculated that ATP10A Beckwith-Wiedemann Syndrome
may be involved in phospholipid transport and may also con- Beckwith-Wiedemann syndrome (BWS) is an overgrowth
tribute to the AS phenotype. Both UBE3A and ATP10A are disorder associated with neonatal hypoglycemia, abdominal
located at the distal part of the 15q11.2-q13 region. wall defects, macroglossia, visceromegaly, gigantism, mid-
In both PWS and AS patients with abnormalities of the face hypoplasia, and a predisposition to embryonal tumors
imprinting process, Buiting et al. identified inherited (seen in 7.5–10% of patients) including Wilms tumor (most
microdeletions in the 15q11.2-q13 region [114]. They pro- common, see next section), rhabdomyosarcoma, and hepato-
posed that these deletions probably affect a single genetic blastoma [118, 119] (see next section). Most cases (85%)
element that they called an “imprinting center (IC).” This are sporadic. BWS is a multigenic disorder resulting from
AS/PWS-IC has been shown to have a bipartite structure and dysregulation of a number of imprinted genes at the chromo-
overlaps the SNRPN promoter with the AS-IC being only some 11p15.5 region and is caused by several molecular
35–40 kb upstream of the PWS-IC [115–117]. Mutations or mechanisms. These include:
disruptions of the imprinting center impair the imprinting • Paternal UPD for the p15 region of chromosome 11 in
process. These mutations can be transmitted silently through approximately 20% of sporadic cases [120, 121].
the germline of one parent, the one in whom the gene is nor- • Cytogenetic abnormalities involving 11p15, present in a
mally silent, but appear to block the resetting of the imprint small number (~1%) of all BWS patients. These include
in the germline of the opposite sex. Thus, a female with a duplication of the paternal 11p15 region as a result of
PWS-IC mutation will not have affected children. Her sons, either a de novo rearrangement or a familial translocation/
however, if they inherit the mutation and are therefore unable inversion and maternally inherited balanced rearrange-
to reactivate the cluster of PWS genes in their germ cells, ments involving 11p15 [122–124].
will be at risk of having PWS children, both male and female. • IC mutation in the gene cluster IGF2/H19 or KCNQ1/
The opposite is true for AS; i.e., a male with an AS-IC muta- KCNQ1OT1 [125] (see later). In familial cases, the segre-
tion will not have affected children, but his daughters, if they gation appears to be autosomal dominant with incomplete
inherit the mutation, will be at risk of having AS children. penetrance [119]. Furthermore, penetrance appears to be
478 J.-C.C. Wang
Fig. 20.2 Laboratory diagnostic approaches for Prader-Willi syndrome/Angelman syndrome
more complete with maternal inheritance; i.e., there is an usually born to carrier mothers in familial cases. The same
excess of transmitting females [126, 127]. explanation can be applied to the observation that in BWS
• Mutation in the maternally active CDKN1C (p57KIP2) patients with balanced rearrangements involving 11p15, the
gene [128]. CDKN1C , a cyclin-dependent kinase inhibi- rearrangements are usually maternally inherited. A disrup-
tor, is a negative regulator of cell proliferation; its overex- tion/mutation of the IC has occurred in the rearrangement
pression arrests cells in G1. Germline CDKN1C mutations process, preventing the resetting of imprinting in the mater-
have been found in 40% of familial and 5% of sporadic nal germline, and H19/CDKN1C/KCNQ1 remains inactive
BWS cases [129]. on the maternal allele.
Linkage studies confirm that BWS maps to 11p15.5 [130, 131]. In addition to these abnormalities involving 11 p15, other
Imprinted genes in this region have been shown to consist not yet well-defined mechanisms or genetic loci may also
of two domains separated by non-imprinted genes [132, cause the BWS phenotype.
133]. The proximal centromeric domain contains CDKN1C Laboratory diagnostic approaches for BWS include cyto-
(p57KIP2) (maternal allele active), KCNQ1 (maternal allele genetic analysis to rule out an 11p15 abnormality, UPD study
active), and KCNQ1OT1 (LIT1 or KvDMR1) (paternal allele for the 11p15 region, mutation analysis of the CDKN1C gene,
active) [125, 134–136]. The distal telomeric domain contains and methylation studies of H19/IGF2 and KCNQ1OT1. One
H19 (maternal allele active) and IGF2 (paternal allele active, study reported that by analyzing the methylation status of the
located approximately 130 kb centromeric to H19). The H19 and KCNQ1OT genes in leukocytes, more than 70% of
paternally expressed genes are growth promoter genes, while the 97 patients could be diagnosed [138]. Of all cases with
the maternally expressed genes are growth suppressor genes. abnormal methylation, 80% involved the promoter region of
Functional imbalance between the growth promoter and the KCNQ1OT gene and 20% the H19 gene.
growth suppressor genes causes the phenotype seen in BWS.
In some BWS patients who inherited an 11p15.5 allele from
both parents, an altered pattern of allelic methylation of H19 Imprinting Disorders and Assisted Reproductive
and IGF2 has been reported [121, 137]. In these patients, a Technology
paternal imprint pattern is seen on the maternal allele, which
results in the non-expression of H19, while IGF2 is expressed Assisted reproductive technology (ART), including in vitro
from both parental alleles. This switching from normally fertilization (IVF) and intracytoplasmic sperm injection
monoallelic expression to biallelic expression is known as (ICSI), accounts for 1–3% of all births in developed coun-
loss of imprinting (LOI) and is caused by imprinting center tries (see Chap. 11). A possible link between ICSI and
abnormalities. As in PWS/AS, an IC abnormality prevents Angelman syndrome was first reported in 2002 [139]. Two
the resetting of imprinting in the maternal germline and unrelated children conceived by ICSI developed AS. In nor-
explains the observation that the affected individuals are mal individuals, the maternal SNRPN allele is methylated,
and the paternal allele is unmethylated. In these two patients, absolute risk of BWS after ART is estimated to be <1%;
hypomethylation of the SNRPN promoter region was detected; thus, routine screening of BWS in children born after ART
only an unmethylated band was present by methylation- does not appear to be warranted [142].
specific PCR studies, and the normally methylated maternal
band was absent. Microdeletion, uniparental disomy, and
AS-IC mutation were all excluded as an etiology. The authors Cancer
suggested that hypomethylation of the SNRPN locus in these
two patients resulted from a sporadic imprinting defect on the Normal epigenetic modifications of DNA involve three types
maternal chromosome. Subsequently, three additional ART- of changes: chromatin modifications, DNA methylation, and
conceived cases of AS were reported: two by ICSI and one genomic imprinting. These are altered in cancer cells. The
using ovarian hyperstimulation alone [140, 141]. These epigenetic dysregulation in cancer cells includes global
findings are suggestive but not conclusive for an association genome hypomethylation, regional hypo- and hypermethyla-
between ART and AS due to an imprinting defect. tion, histone modification, and disturbed genomic imprinting
An association between ART and another imprinting dis- [147, 148]. Thus, an altered genomic imprinting process is a
order, Beckwith-Wiedemann syndrome (see previous sec- common mechanism for cancer development.
tion), was reported in 2003. Three case series from BWS
registries reported an estimated sixfold increase of BWS Paraganglioma
cases among children born after ART (reviewed in [142, A type of non-childhood tumor, paraganglioma (PGL) of the
143]). More than 60 such BWS patients have been reported. head and neck (glomus tumor), has been mapped to chromo-
In the vast majority of these BWS patients, loss of methyla- some 11 at two distinct loci, 11q23 and 11q13.1, by linkage
tion at the KCNQ1OT1 gene at 11p15 is observed. Since analysis [149, 150]. Approximately 30% of cases are famil-
KCNQ1OT1 is normally maternally methylated/paternally ial. Mutation in SDHD (succinate dehydrogenase subunit D),
unmethylated, this finding indicates that hypomethylation of a gene mapped to 11q23 that encodes a mitochondrial respi-
the allele on the maternal chromosome is the cause of BWS ratory chain protein, has recently been reported in families
in these patients. BWS remains the imprinting disorder with with PGL [151–153]. Inheritance of PGL is autosomal domi-
the strongest evidence for an association with ART. nant with both males and females affected. However, trans-
A novel imprinting syndrome resulting from maternal mission is almost exclusively through the father, and only
hypomethylation at multiple loci was proposed in 2006 [144]. male gene carriers will have affected offspring. The disease
In a cohort of 12 patients with transient neonatal diabetes is only very rarely observed in the offspring of affected
resulting from loss of maternal methylation at the transient females [149, 154–157]. These observations suggest genomic
neonatal diabetes locus, the authors found that six patients imprinting. However, expression of SDHD is biallelic (i.e., it
had hypomethylation at other loci. None of the patients were is expressed from both maternal and paternal alleles) in all
conceived following ART, although one was born following a tissues studied to date (lymphoblastoid cell lines, adult brain,
period of subfertility. Similarly, studies of BWS patients with fetal brain, and kidney) [151]. Therefore, the mechanism for
loss of maternal methylation at KCNQ1OT1 showed that the observed genomic imprinting inheritance pattern of this
approximately 25% had hypomethylation at multiple, addi- tumor is as yet uncertain. It remains possible that imprinting
tional maternally imprinted loci [145, 146]. Again, only a of SDHD is tissue specific and may be restricted to the carotid
proportion of patients were conceived following ART. These body, the most common tumor site of PGL, and other para-
observations suggest that ART is not specifically associated ganglionic cells.
with maternal hypomethylation syndromes.
An association between ART and other imprinting disor- Wilms Tumor/Rhabdomyosarcoma
ders such as Silver-Russell syndrome and retinoblastoma has In a number of embryonal tumors, loss of heterozygosity
also been suggested but not established. (LOH) of a specific parental allele has been observed. In all
The association of imprinting disorders and ART appears cases studied, the maternal allele is preferentially lost. This
to be related to subfertility, ovulation induction, and/or suggests that duplication of some paternal alleles results in
embryo culture. The exact mechanism for the association is enhanced cell proliferation, while duplication of certain
not clear. In vitro embryo culture might predispose to loss of maternal alleles may inhibit cell proliferation.
methylation. Alternatively, imprinting defects and subfertil- In Wilms tumor and rhabdomyosarcoma, LOH involves
ity might have a common cause, and treatment for infertility chromosome 11 [158–160]. LOH does not involve markers
with ovarian hyperstimulation may further increase the risk for 11p13, the proposed Wilms tumor locus, but only mark-
of imprinting defects [141]. ers on 11p15.5 [159]. Known imprinted genes in the 11p15.5
To date, the imprinting disorder that has the strongest evi- region include H19, IGF2, and CDKN1C (p57KIP2) (see pre-
dence for an association with ART is BWS. Nevertheless, the vious). The expression of CDKN1C is reduced in Wilms
480 J.-C.C. Wang
tumor [135]. In addition, by using several overlapping neuroblastoma [171]. Preferential amplification of the paternal
subchromosomal transferable fragments from 11p15 distinct MYCN allele in neuroblastoma tumor tissues has been
from H19 and IGF2, Koi et al. were able to obtain in vitro reported [172]. In tumors with MYCN amplification, loss of
growth arrest of rhabdomyosarcoma cells [161]. These parental 1p alleles was found to be random [172, 173]. In
observations suggest that CDKN1C, which is normally active tumors without MYCN amplification, loss of 1p was previ-
on the maternal allele only, may be a candidate for a tumor ously reported to be preferentially maternal (16 of 17 cases)
suppressor gene. Loss of the active CDKN1C allele on the but random in a later study that suggested no imprinted gene
maternal chromosome results in tumor development. Besides in this region [173, 174]. On the other hand, the TP73 tumor
LOH, another possible mechanism, loss of imprinting (LOI; suppressor gene located at 1p36.32 has been shown to be
see BWS), has been proposed. Ogawa et al. reported biallelic imprinted (maternal active) [32].
IGF2 RNA synthesis in 4 of 30 Wilms tumors they studied An imprinted gene cluster at 14q32.2 that includes two
[162]. Thus, “relaxation” of IGF2 gene imprinting on the closely linked but reciprocally imprinted genes, DLK1
maternal allele has occurred, resulting in its expression. This (paternal active) and MEG3 (also known as GTL2) (maternal
would be equivalent to having two copies of an active IGF2 active), has been identified [175]. These two genes have sim-
gene, as would occur with a paternal duplication or with ilarities to IGF2 and H19, genes involved in BWS (see previ-
paternal UPD. A similar biallelic expression of IGF2 was ous section), respectively. Both MEG3 and H19 (gross
reported in 30% of breast cancer patients studied [163]. suppressors) are maternally expressed, and both DLK1 and
Disruption of the imprinting mechanism (i.e., LOI) may IGF2 (growth promoters) are paternally expressed.
therefore also play a role in tumorigenesis. A third possible Hypermethylation of the MEG3 promoter differentially
mechanism has also been proposed in a proportion of Wilms methylated region was associated with MEG3 transcriptional
tumor patients. In some patients, LOI was observed in both repression and was detected in 5 of 20 (25%) neuroblasto-
the Wilms tumor tissue and the normal adjacent kidney tis- mas tumors [176]. Therefore, loss of MEG3 expression may
sue, but IGF2 expression was significantly higher in tumor also contribute to tumorigenesis in a subset of human
tissue. The overexpression in tumor tissue was accompanied cancers.
by activation of all four IGF2 promoters [164]. These studies
indicate that while genomic imprinting plays an important
role in tumorigenesis, a single mechanism does not account Uniparental Disomy
for all cases.
The term uniparental disomy (UPD) was introduced by Engel
Retinoblastoma/Osteosarcoma in 1980 [177]. It describes a phenomenon in which both
In retinoblastoma and osteosarcoma, loss of both functional homologs or homologous segments of a chromosome pair
copies of the retinoblastoma gene (RB1) on chromosome 13 are derived from a single parent. An example of the latter is
at band q14, usually by mutation or deletion, has been the paternal UPD for 11p15 in BWS described previously.
observed [165]. In familial cases, a mutation in one of the Discussion here will be restricted to uniparental disomies for
alleles is present in the germline. De novo mutations in the entire chromosomes, of which there are two types. Uniparental
germline occur preferentially in the paternal chromosome, isodisomy describes a state in which both copies of a chro-
consistent with the general observation that new germline mosome are not only derived from one parent but also repre-
mutations arise predominantly during spermatogenesis [166, sent the same homolog (i.e., two copies of the same exact
167]. In sporadic, nonfamilial tumors, loss of function of both chromosome). Uniparental heterodisomy refers to both of
alleles occurs somatically. In sporadic osteosarcomas, the ini- one parent’s homologs being represented (i.e., both chromo-
tial mutation occurs preferentially on the paternal chromosomes of the pair from the same parent). The type of UPD
some 13, suggesting that genomic imprinting may be involved present is not always readily apparent, and it should be noted
[168]. In sporadic retinoblastoma, epigenetic change with that, because of the recombination that takes place during
hypermethylation of the RB1 gene was reported in 9% of meiosis, UPD along the length of an involved chromosome
cases [169]. RB1 is a tumor suppressor gene, and it has been pair can be iso- for certain loci and hetero- for others.
recently shown to be imprinted [170]. These observations UPD for an entire chromosome can occur as a result of
suggest a role of genomic imprinting in retinoblastoma. gamete complementation, as suggested by Engel [177]. Since
aneuploidy is relatively frequent in gametes, the chance
Neuroblastoma union of two gametes, one hypo-, the other hyper-haploid for
Neuroblastoma is the most common extracranial tumor of the same chromosome, will result in a diploid zygote with
childhood. Deletions of chromosome 1p and amplification of UPD for that chromosome. Structural rearrangements, such
the MYCN gene on chromosome 2p are frequently seen in as Robertsonian or reciprocal translocations (see Chap. 9),
increase the chance of meiotic malsegregation and thus may trisomic conceptus is by forming a smaller marker chro-
predispose to UPD. This is best illustrated by the case mosome from one of the trisomic chromosomes after los-
reported by Wang et al., in which UPD for chromosome 14 ing most of its active genetic material. If the one
was observed in a child with a paternal (13;14) Robertsonian chromosome that rearranged and became the marker chro-
translocation and a maternal (1;14) reciprocal translocation mosome is the single chromosome contributed by one par-
[177] (see Fig. 20.3 and Chaps. 3 and 9). Studies in animals ent, the remaining two of the trisomic chromosomes will
also support this concept. Maternal or paternal disomies are be from the same parent and thus represent UPD for this
readily produced in mice with intercrosses between either chromosome pair.
Robertsonian or reciprocal translocation carriers [14]. A third possible mechanism for the occurrence of UPD is
Another mechanism for the occurrence of UPD is by “monosomy rescue,” the duplication of the single chromo-
“trisomy rescue” [178]. The vast majority of trisomic con- some in monosomic conceptuses [179]. In this case, uniparen-
ceptuses are nonviable; they may survive to term only if tal isodisomy for the entire chromosome would be observed.
one of the trisomic chromosomes is postzygotically lost. Two mechanisms contribute to the phenotypic effects of
In one-third of these cases, such loss will result in UPD in UPD. Unmasking of a recessive gene can occur as a result of
the now disomic cells (Fig. 20.4). Since the loss occurs uniparental isodisomy, in which the disomic chromosomes
postzygotically, mosaicism in such conceptuses is often are homozygous. This was illustrated initially in an individ-
observed, with the trisomic cell line sometimes confined to ual with cystic fibrosis who had maternal uniparental isodis-
the placenta (see Chap. 12). Another way of “rescuing” a omy for chromosome 7 and later in many other patients with
Fig. 20.3 An example of paternal UPD formation by gamete comple- der(13;14)(q10;q10). The patient inherited both chromosomes 14
mentation. Malsegregation involving chromosome 14 occurred in both from the father and neither from the mother. Segregation is normal for
parents as the result of structural rearrangements. Mother: reciprocal chromosome 13 in the mother and for chromosome 1 in the father.
translocation t(1;14)(q32;q32). Father: Robertsonian translocation Chromosomes are Q-banded
482 J.-C.C. Wang
Fig. 20.4 A diagrammatic

representation of maternal UPD
formation by “trisomy rescue.” A
trisomic zygote resulting from
maternal meiosis I
nondisjunction is depicted here.
Loss of one of the trisomic
chromosomes through either
mitotic nondisjunction or
anaphase lag results in euploidy.
Uniparental disomy occurs in
one-third of these cases. m1 and
m2 maternally derived
chromosomes, p paternally
derived chromosome
recessive disorders and UPD (see later in chapter) [179]. The the peroxisome biogenesis genes [183]. A boy had autism
second mechanism is the effect caused by imprinted genes whose mother and brother also had autistic features; the
on the involved chromosome. This is best illustrated by brother did not have UPD [184]. Another case involved a
PWS/AS patients who have no deletion of 15q11.2, but rather patient with insulin-dependent diabetes mellitus. Maternal
have UPD, as discussed previously. In addition to these two UPD for chromosome 1 was accidentally discovered during
mechanisms, in cases where UPD arises as a result of “tri- a family linkage study [185]. This patient was developmen-
somy rescue,” the presence of a mosaic trisomic cell line in tally and mentally normal at age 23. Therefore, maternal
the placenta and/or fetus may modify the phenotype. UPD for chromosome 1 does not appear to have an imprint-
Of the 47 possible types of UPD of whole chromosomes, ing effect.
36 have been reported to date. Some provide clear evidence
for imprinting and some seem to suggest no such effect,
while others will require accumulation of additional data upd(1)pat
before their status in this regard can be determined.
At least seven cases of paternal UPD for chromosome 1
have been reported. A 7-year-old boy presented with pyc-
upd(1)mat nodysostosis as a result of a homozygous mutation of the
cathepsin K gene, for which the father was a heterozygote
At least seven cases of maternal UPD for chromosome 1 and the mother was normal [186]. The child was otherwise
have been reported. One patient had lethal autosomal reces- developmentally normal. Five additional patients, one with
sive Herlitz-type junctional epidermolysis bullosa as a result congenital insensitivity to pain with anhidrosis, two with
of homozygosity for a nonsense mutation in the LAMB3 gene Herlitz junctional epidermolysis bullosa, one with Leber
on chromosome 1 [180]. The mother was a heterozygous congenital amaurosis, and one with hemolytic-uremic syn-
carrier for the mutation and the father had two normal LAMB3 drome, were reported [187–191]. All five had paternal iso-
alleles. The patient died at 2 months of age. Autopsy was not disomy for chromosome 1. None had any overt
performed but weight and length were reportedly normal, dysmorphisms or malformations. Their phenotype resulted
and no overt dysmorphisms or malformations were noted. from having two copies of the mutated recessive genes,
Another child with Chediak-Higashi syndrome (CHS) was both inherited from their fathers. Another patient was a
found to be homozygous for a nonsense mutation in the LYST 43-year-old female with short stature, ptosis, micro-/ret-
gene for CHS on chromosome 1 [181]. The mother was a rognathia, scoliosis, hearing loss, myopathy, and infertility.
carrier of the mutation, while the father had two normal LYST She has isochromosomes for the short arm and long arm of
alleles. Two additional unrelated patients had lethal trifunc- chromosome 1 [i(1)(p10),i(1)(q10); see Chaps. 3 and 9]
tional protein deficiency due to homozygous alpha-subunit [192]. It was not clear whether the abnormal phenotype in
mutations. In both patients, the mothers were heterozygous this woman resulted from an imprinting effect or from
for the mutation and the fathers did not have the mutation homozygosity for some undetected recessive alleles. These
[182]. One patient had Zellweger syndrome due to homozy- observations provide no clear evidence for an imprinting
gosity for a maternally inherited mutation in PEX10, one of effect of paternal UPD 1.
upd(2)mat phenotypically normal. Paternal UPD 2 therefore does not

appear to have an imprinting effect.
Maternal UPD for chromosome 2 has been reported in at
least ten cases. Four cases were associated with confined pla-
cental mosaicism (CPM) for trisomy 2. Two cases, one with upd(3)mat
and one without phenotypic abnormalities, resulted from
de novo isochromosome formation of the short arm and long At least two cases of maternal isodisomy for chromosome
arm of chromosome 2 [i(2)(p10),i(2)(q10); see Chaps. 3 and 3 have been reported. Both cases presented with autosomal
9]. One case with no phenotypic abnormalities was discov- recessive disorder as a result of homozygous mutations:
ered at age 3 during paternity testing. Three cases had an one dystrophic epidermolysis bullosa with no additional
autosomal recessive disorder due to a maternally inherited phenotypic abnormalities and one Fanconi-Bickel syn-
homozygous mutation; two of these were unrelated patients drome, a rare disorder with growth failure, hepatomegaly,
with lethal mitochondrial trifunctional protein deficiency as renal Fanconi syndrome, and abnormal glucose homeosta-
a result of mutations in the HADHA gene, and one patient sis caused by mutations in SLC2A2 (previously GLUT2) at
had infantile-onset ascending spastic paralysis caused by 3q26.1-3q26.3 [204, 205]. Another case with confined pla-
mutations in the gene ALS2 [182, 193–200]. No phenotypic cental mosaicism (CPM, see Chap. 12) for trisomy 3
abnormalities were reported other than those associated with detected by chorionic villous biopsy and a marker of chro-
the specific autosomal recessive disorders. A common phe- mosome 3 origin detected by a subsequent amniocentesis
notype was observed in the four cases associated with CPM was found to have maternal uniparental disomy for the two
and one of the two cases with isochromosomes. This includes chromosomes 3 [206]. The fetus had IUGR and microceph-
intrauterine growth restriction (IUGR), oligohydramnios, aly that could be attributed to CPM and the marker chromo-
pulmonary hypoplasia, hypospadias (in two patients), and some. There is no clear evidence that maternal UPD 3 has
normal development in the four surviving patients at ages 6, an imprinting effect.
20, 31 months, and 8 years, respectively. IUGR, oligohy-
dramnios, and pulmonary hypoplasia can be explained by
placental dysfunction as a result of trisomy 2 mosaicism. upd(3)pat
However, these same features were also present in one of the
two cases with isochromosomes, suggesting a possible A case of paternal UPD for chromosome 3 was detected ser-
imprinting effect of maternal UPD 2 [198]. In another case endipitously during a whole genome linkage study [207]. No
reported recently, UPD for maternal 2q and paternal 2p was apparent phenotypic abnormality was observed.
detected in a 36-year-old woman with normal physical and
mental development [201]. Therefore, it is not clear whether
maternal UPD 2 confers an imprinting effect. upd(4)mat
A case of maternal UPD for chromosome 4 as a result of

upd(2)pat isochromosome formation of the short arm and long arm of
chromosome 4 [i(4)(p10),i(4)(q10); see Chaps. 3 and 9] was
At least three cases of paternal isodisomy for the entire reported in an abstract [208]. Cytogenetic studies were per-
chromosome 2 have been reported. A 34-year-old woman formed because of multiple early miscarriages. The patient
diagnosed with retinitis pigmentosa was found to have a was otherwise phenotypically normal. Two additional cases
homozygous MERTK mutation [190]. She was otherwise of maternal isodisomy have been reported: one patient had
phenotypically normal. The patient’s father was heterozy- afibrinogenemia as a result of a maternally inherited homozy-
gous for the mutation and the mother did not carry the gous mutation of the fibrinogen alpha-chain gene at 4q28;
mutation. Two other cases, one with Crigler-Najjar syn- the other adult female patient had a history of major depres-
drome type I due to a paternally inherited homozygous sive disorder and multiple suicide attempts but normal fertil-
UGT1A1 mutation at 2q37 and one with Donnai-Barrow ity and no major medical complaints [209, 210]. Another
syndrome (faciooculoacousticorenal syndrome) due to case with confined placental mosaicism for trisomy 4 in a
homozygous mutation in the LRP2 gene at chromosome fetus with intrauterine growth restriction and oligohydram-
2q31.1, had no phenotypic abnormalities other than the nios followed by intrauterine fetal death at 30 weeks of ges-
disease-related findings [202, 203]. These cases further tation was determined to have maternal UPD 4 [211]. No
illustrate unmasking of autosomal recessive disorders as a external malformations were detected in this stillborn. There
result of uniparental disomy. An additional case with isodi- is no clear evidence to date that maternal UPD for chromo-
somy for paternal 2p as described under upd(2)mat was some 4 confers an imprinting effect.
484 J.-C.C. Wang
upd(5)pat Three regions on chromosome 7 have been shown to

contain imprinted genes [231, 232]. One region at 7p12 con-
Paternal UPD for chromosome 5 was reported in a child with tains an imprinted gene GRB10 (growth factor receptor-
autosomal recessive spinal muscular atrophy [212]. The binding protein 10), a known growth suppressor that is
child had no other developmental abnormalities. Spinal mus- expressed on the maternal allele and is therefore one of the
cular atrophy in this case can be explained by the paternal candidate genes for SRS [233–235]. A second region at
transmission of two copies of the defective gene. There is no 7q32.2 contains a number of other imprinted genes including
evidence for an imprinting effect. CPA4, MEST (also known as PEG1), and COPG2IT1. CPA4
was proposed to be a candidate for SRS [236]. The role of the
other genes is not yet clear [236–239]. A third region at 7q21.3
upd(6)mat contains three imprinted genes SGCE (epsilon-sarcoglycan,
maternally imprinted), PEG10, and PPP1R9A. Mutation of
Maternal uniparental isodisomy for chromosome 6 was first the SGCE gene causes an autosomal dominant movement
identified in a renal transplant patient in the process of HLA disorder myoclonus-dystonia. A 36-year-old man with both
typing [213]. Another patient with congenital adrenal hyper- SRS and myoclonus-dystonia has been reported [232].
plasia resulting from unmasking of the maternally inherited SRS is also the first human disorder with imprinting dis-
mutation in the 21-hydroxylase gene had intrauterine growth turbances that affect two different chromosomes: chromo-
restriction but good catch-up growth [214]. There is no clear somes 7 and 11 [240]. Approximately 40% of SRS show
evidence for an imprinting effect. hypomethylation in the IC region upstream of H19 at
11p15.5, which results in overexpression of the growth sup-
pressor H19. Hypermethylation of the same IC region, on
upd(6)pat the other hand, is associated with the overgrowth disorder,
BWS (see section “Chromosome Deletion/Duplication
More than 15 cases of paternal uniparental disomy for chro- Syndromes”). These two developmental syndromes, SRS
mosome 6 have been reported (reviewed in references [215– and BWS, are therefore clinically and epigenetically oppo-
219]). All except one were isodisomy. Many patients had site diseases (reviewed in [241]).
transient neonatal diabetes mellitus (TNDM) associated with Maternal UPD 7 clearly has an imprinting effect.
very low birth weight. Two genes, an imprinted cell cycle
control gene PLAGL1 (also known as ZAC) and hydatidi-
form mole associated and imprinted gene HYMAI (untrans- upd(7)pat
lated with unknown function) at 6q24 with differential
methylation of parental alleles, have been identified [220– At least four cases of paternal isodisomy for the entire chromo-
222]. These genes are expressed only from the paternal allele some 7 have been reported. One patient had recessive congeni-
and are potential candidate genes for TNDM. Increased tal chloride wasting diarrhea with normal growth and
expression of this gene by paternal UPD appears to result in development [242]. One patient had cystic fibrosis as a result of
the diabetic phenotype. It was estimated that paternal UPD 6 inheriting two copies of the DF508 mutation from his father.
accounts for approximately 15–20% of cases of TNDM This patient also had complete situs inversus and immotile cilia
[221, 223]. Paternal UPD 6 clearly has an imprinting effect. with growth retardation and significant respiratory disease
[243]. The other two patients also had cystic fibrosis. One of
them had normal growth and the other had overgrowth and
upd(7)mat developmental delay [244, 245]. In addition, two patients had
paternal isodisomy 7p and maternal isodisomy 7q [246, 247].
More than 60 patients with maternal UPD for chromosome 7 These two patients had similar phenotypes that resembled the
have been reported in the literature [27, 224–227]. This was phenotype seen in maternal UPD 7, and their growth retardation
the first documented UPD in humans, identified initially in was considered to be a result of maternal isodisomy for 7q. It is
two individuals with cystic fibrosis and short stature [179, not clear whether paternal UPD 7 confers an imprinting effect.
228]. Approximately 10% of patients with Silver-Russell
syndrome (SRS) are noted to have maternal UPD 7 [225,
226, 229, 230]. SRS is a heterogeneous disorder. The clinical upd(8)mat
phenotype includes intrauterine growth restriction and per-
sistent postnatal growth delay, body asymmetry, triangular One case of maternal isodisomy for the entire chromosome 8
face, prominent forehead, decreased subcutaneous tissue, has been reported [248]. The patient was a 39-year-old male
delayed bone age, and usually normal intelligence. with normal appearance, stature, and intelligence. He had
early-onset ileal carcinoid, slight thoracic scoliosis, and 10 [256]. Two other cases of maternal UPD 10 reported were
numerous pigmented nevi. More cases are needed before a associated with either a marker chromosome 10 or a trisomy
conclusion can be drawn as to whether maternal UPD 8 has 10 cell line, and the abnormal phenotypes were attributed to
an imprinting effect. the karyotypic abnormalities. There is no evidence to date
that this UPD confers an imprinting effect.
upd(8)pat
upd(11)pat
A single case of paternal uniparental isodisomy for chromo-
some 8 has been reported [249]. This five-and-half-year-old Paternal UPD for the entire chromosome 11 has been reported
girl had normal development and lipoprotein lipase (LPL) in at least three cases. One patient had hemihypertrophy,
deficiency due to a mutation of the LPL gene. The patient congenital adrenal carcinoma, and Wilms tumor [257]. The
was ascertained due to a diagnosis of chylomicronemia. The second had associated confined placental mosaicism (see Chap.
father was a heterozygous carrier for the same mutation. It 12) for trisomy 11, and intrauterine death occurred between 19
appears that normal development can occur in paternal UPD and 20 weeks gestation. This fetus had growth restriction, aber-
8 and that an imprinting effect of this UPD may not exist. rant intestinal rotation, and hypospadias [258]. The third patient
had possible mosaic paternal isodisomy along the entire chro-
mosome 11. The clinical findings in this patient did not differ
upd(9)mat from that of other BWS patients [259]. In addition, many cases
of paternal segmental UPD for distal 11p associated with BWS
At least seven cases of maternal UPD for chromosome 9 have been observed (see previous section). The existence of an
have been reported. Two patients had recessive cartilage-hair imprinting effect due to paternal UPD 11 is clear.
hypoplasia, a disorder that maps to the short arm of chromo-
some 9 [250]. Two homozygotic female twins had Leigh
syndrome as a result of inheriting two copies of the mutated upd(12)mat
SURF1 gene from their mother [251]. Both twins died of
respiratory failure at age 3. No gross dysmorphic features or A case of maternal UPD for chromosome 12 was reported
malformations were noted apart from Leigh syndrome. One [260]. The infant had normal somatic and psychomotor
case involved a fetus associated with confined placental development with no congenital anomalies or dysmorphic
mosaicism (see Chap. 12) for trisomy 9 [252]. Pathological features at 6 weeks of age. Chromosome analysis demon-
examination of the abortus was not possible. One case of strated mosaicism with the presence in some cells of a small
syndromic congenital hypothyroidism, characterized by thy- marker chromosome consisted of chromosome 12 centro-
roid dysgenesis, cleft palate, spiky hair, and choanal atresia meric heterochromatin with no euchromatic material. This
and bifid epiglottis who was homozygous for a maternally suggests that the mechanism for the occurrence of UPD in
inherited FOXE1 (9q22) mutation, has been reported recently this case is by trisomy rescue. It appears that maternal UPD
[253]. Another 34-year-old healthy woman with recurrent 12 may not have an imprinting effect.
spontaneous abortions had isochromosomes of the short and
long arms of chromosome 9 [i(9)(p10),i(9)(q10); see Chaps.
3 and 9]. Molecular analysis demonstrated maternal isodis- upd(13)mat
omy [254]. The available data indicate that maternal UPD 9
may not have an imprinting effect. At least two cases of maternal UPD for chromosome 13 have
been reported [261, 262]. In both cases, a normal phenotype
was associated with the presence of an isochromosome for
upd(10)mat the long arm of chromosome 13. These indicate that an
imprinting effect due to maternal UPD 13 is very unlikely.
A case of prenatally diagnosed maternal UPD for chromosome
10 associated with confined placental mosaicism (see Chap.
12) has been reported [255]. The infant was phenotypically upd(13)pat
and developmentally normal at 8 months of age. Another case
of familial hemophagocytic lymphohistiocytosis characterized At list six cases of paternal UPD for chromosome 13 have
by lethal primary immunodeficiency was reported in a patient been reported. One was the mother of one of the maternal
who was homozygous for the perforin gene (PRF1) at 10q22 UPD 13 patients described previously [261]. This phenotypically
as a result of maternal uniparental disomy for chromosome normal individual presumably received the isochromosome
486 J.-C.C. Wang
13q from her father, who was not available for study, but translocations involving chromosome 14; cases with a
DNA polymorphism studies of her mother revealed the normal karyotype have also been observed [285]. A similar
absence of maternal chromosome 13 alleles in this patient phenotype is present in these patients and includes polyhy-
[263]. Four cases had de novo der(13;13)(q10;q10) translo- dramnios, low birth weight, hirsute forehead, blepharophi-
cations (see Chaps. 3 and 9) [264–266]. Three of them were mosis/short palpebral fissures, protruding philtrum, small
complete isodisomies, and one exhibited evidence of recom- ears, small thorax, abnormal ribs, simian creases, and joint
bination with proximal isodisomy and distal heterodisomy. contractures. Severe mental retardation was seen in a patient
All four patients were phenotypically normal. Another case who was beyond 20 months of age at the time of reporting
of maternal UPD 13 was reported in a patient with hearing [279]. These observations indicate that an imprinting effect
loss as a result of unmasking of the recessive gap junction due to paternal UPD 14 exists.
protein GJB2 gene at 13q11-q12 [266]. Therefore, paternal Studies comparing maternal and paternal UPD cases with
UPD 13 does not appear to have an imprinting effect. cases of partial trisomy and partial monosomy of various
segments of 14q have suggested that 14q23-q32 may be the
region where the imprinted genes on chromosome 14 reside
upd(14)mat [286, 287]. Further studies of segmental and full paternal
isodisomy for chromosome 14 indicated imprinted genes at
Maternal UPD for chromosome 14 has been reported in 14q32 as the critical components of the phenotype observed
many cases (reviewed in [267–275]). Although many are in upd(14)pat [288]. This is consistent with the observation
associated with Robertsonian translocations involving chro- that overexpression of the paternally active gene RTL1 plays
mosome 14, cases with a normal karyotype have also been a major role in the upd(14)pat phenotype [277] (see also pre-
observed. A distinct clinical phenotype is present and con- vious section discussing maternal UPD 14).
sists of intrauterine growth restriction, mild-to-moderate Human chromosome 14 has significant homology to
motor and/or mental developmental delay, hypotonia, short mouse chromosomes 12 and 14 [289]. Mouse chromosome
stature, and precocious puberty. Less frequent findings 12 is imprinted, and both maternal and paternal disomies
include hydrocephalus, dysmorphic features (prominent cause early embryonic death [290]. Thus, the observation of
forehead, supraorbital ridge, short philtrum, downturned cor- imprinting effects for both maternal and paternal UPD 14 in
ner of mouth), small hands, hyperextensible joints, scoliosis, humans is not unexpected.
and recurrent otitis media.
Chromosome 14 contains a cluster of imprinted genes at
14q32.2 including RTL1 and DLK1 (both paternal active) upd(15)mat
and MEG3 (also known as GTL2, maternal active) (see also
previous “Neuroblastoma” section). They are regulated by a More than 100 cases of maternal UPD for chromosome 15
differentially methylated region (DMR) between RTL1/ have been reported in the literature in association with
DLK1 and MEG3 genes (intergenic DMR). A number of Prader-Willi syndrome [86, 89, 291, 292] (see section “Prader-
patients with biparental inheritance of chromosome 14 but Willi Syndrome/Angelman Syndrome in Genomic Imprinting
with a clinical phenotype similar to that of maternal unipa- and Human Diseases”). As previously discussed, UPD(15)
rental disomy have been found to have various deletions in mat accounts for approximately 20–25% of patients with
the imprinted region or hypomethylation of the intergenic PWS. Many patients had associated trisomy 15 mosaicism,
and the MEG3 DMRs [273–278]. These findings indicate which was confined to the placenta in most cases. Comparison
that abnormal methylation patterns of the imprinted genes of the phenotypes of PWS patients with different etiologies
are associated with the maternal UPD 14 phenotype. has shown that advanced maternal age was present in moth-
Therefore, methylation analysis of the imprinted gene MEG3 ers of patients with maternal UPD, while a higher frequency
can be performed to detect upd(14)mat and to determine the of hypopigmentation is seen in patients due to deletion of
molecular basis in patients with a phenotype similar to paternal 15q11.2-q13 [291–293]. Advanced maternal age
upd(14)mat but who do not have UPD [273, 274]. can be expected in UPDs that result from “trisomy rescue,”
Evidence for an imprinting effect due to maternal UPD 14 as advanced maternal age is associated with meiotic nondis-
is clear. junction. Hypopigmentation results from mutation/deletion
of the OCA2 gene (mouse homolog pink-eyed dilution p
gene) located at 15q11.2-q12 [294–296]. The human OCA2
upd(14)pat gene is not imprinted, and both copies are functional in UPD
patients. Hypopigmentation is therefore more prominent in
Many cases of paternal UPD for chromosome 14 have been PWS patients due to deletion. Differences in other clinical
reported [279–284]. Most are associated with Robertsonian features between these two groups are less clear-cut. While
there may not be a significant difference in the overall severity, apparently characteristic facial dysmorphisms (slightly
female UPD patients were found to be less severely affected upslanted palpebral fissures, almond-shaped eyes, broad
than female deletion patients [291]. UPD patients were found nasal root, upturned nares, long philtrum, thin upper lip,
to be less likely to have “typical” facial appearance, some- prominent ears, and triangular face) may exist [307, 309,
what higher IQ, and milder behavior problems and more 311]. In addition, in a later study, statistical analysis per-
likely to have psychosis and autism spectrum disorders [292, formed on a large series of mosaic trisomy 16 cases with
297, 298]. Deletion patients have a higher frequency of sleep molecular determination of UPD status indicated that upd(16)
disturbance, hypopigmentation, and speech articulation mat was associated with fetal growth restriction and with
defects [299]. These differences can again be attributed, at increased risk of major malformation [313]. One protein
least partially, to the presence of two copies of non-imprinted coding gene, ZNF597 at 16p13.3, has been found to be
genes in UPD cases, whereas there is haploinsufficiency of imprinted (maternal allele active) [32]. Although not yet cer-
these genes in the deletion cases. tain, the existence of an imprinting effect due to maternal
UPD 16 is a distinct possibility.
upd(15)pat
upd(16)pat
Many cases of paternal UPD for chromosome 15 associated
with Angelman syndrome have been reported in the litera- A single case of paternal UPD for chromosome 16 has been
ture [88, 90, 300–302]. Paternal UPD 15 accounts for reported [314]. This case was associated with confined
approximately 6% of AS patients. AS patients with paternal placental mosaicism (see Chap. 12). Paternal isodisomy for
UPD may have a milder phenotype than those with a mater- chromosome 16 was prenatally diagnosed and confirmed
nal deletion of 15q11.2-q13; UPD patients have better physi- after birth. Intrauterine growth restriction was present with
cal growth, fewer movement abnormalities, less ataxia, and a catch-up growth observed at 13 months of age. Minor physi-
lower prevalence of seizures [90, 300–303]. One possible cal abnormalities included bilateral pes calcaneus and addi-
mechanism for the milder phenotype in UPD patients may be tional rudimentary mandibular dental arch. Psychomotor
the presence of many non-imprinted genes in the 15q11.2-q13 development was normal. It is not clear whether paternal
region in these patients, whereas these are absent in deletion UPD 16 has an imprinting effect.
patients. Alternatively, as proposed by Bottani et al., it may
be due to the “leaky” expression of the imprinted paternal
genes, where two copies of the allele will result in an expres- upd(17)mat
sion higher than in deletion cases, in which only one
imprinted paternal allele is present [90]. A case of maternal UPD involving the entire chromosome 17
Both maternal and paternal UPD 15 clearly confer was reported in a 2-year-old boy with trisomy 17 confined
imprinting effects. placental mosaicism (see Chap. 12) [315]. His growth and
psychomotor development was normal. Another case with
infantile nephropathic cystinosis as a result of a homozygous
upd(16)mat 57-kb deletion encompassing the CTNS gene at 17p13 was
recently reported [316]. The mother was heterozygous for
More than 20 cases of maternal UPD for chromosome 16 the deletion and the father did not carry the deletion. The
have been described, and potentially many more cases are child had maternal UPD for chromosome 17, and the abnor-
not reported [304–312]. Again, associated trisomy 16 mosa- mal phenotype was resulted from unmasking of the recessive
icism, usually confined to the placenta, is present in most gene. There is no evidence that maternal UPD 17 confers an
cases. A clinical phenotype of maternal UPD 16 has not been imprinting effect.
clearly defined; the possibility of the presence of an unde-
tected trisomy 16 cell line complicates the comparison
among reported cases. IUGR is a frequent finding. IUGR upd(20)mat
may result from the presence of trisomy 16 cells in the pla-
centa; however, no catch-up growth was observed in these At least three cases of maternal UPD 20 have been reported
patients [304]. Development has been normal in all cases, the [317–319]. One of them was associated with a mosaic cell
oldest reported at 4 years of age. Imperforate anus has been line containing a small marker chromosome consisting of the
reported in three cases, hypospadias in two, and congenital pericentromeric region of chromosome 20, and another was
cardiac anomalies were observed in five cases, with an A-V associated with confined placental mosaicism (see Chap. 12)
canal defect in one and ASD and VSD in four. Subtle but for trisomy 20. The common features in these three patients at
488 J.-C.C. Wang
ages 4 years, 35, and 17 months, respectively, are pre- and upd(22)mat
postnatal growth retardation. Isolated findings included mild
facial dysmorphism, strabismus, microcephaly, macroceph- Maternal UPD for chromosome 22 not associated with
aly, developmental delay, and hyperactivity. A further case mosaic trisomy 22 has been reported in three cases [327–
involved a live-born girl with maternal uniparental isodisomy 329]. All three phenotypically normal individuals were
of chromosome 20 in the diploid cell line who had moderate ascertained via history of multiple spontaneous abortions
psychomotor retardation, central hypotonia with peripheral and were found to have balanced (22;22) Robertsonian trans-
hypertonia, multiple minor dysmorphism, and marked kypho- locations (see Chaps. 3 and 9). Two other cases of maternal
sis [320]. However, the clinical phenotype in this patient was uniparental heterodisomy for chromosome 22 associated
complicated by the presence of a trisomy 20 cell line in both with prenatally diagnosed mosaic marker chromosome 22
blood and urine specimens. Imprinted genes have been found were reported: one male infant had no dysmorphic features
on chromosome 20; available clinical information in the lit- at birth and reportedly normal development at age 6 months
erature unfortunately does not unequivocally support the pos- and the other, a girl, had normal development at 18 months
sibility that maternal UPD 20 has an imprinting effect [32]. of age [330, 331]. Additionally, a prenatally diagnosed case
with nonmosaic trisomy 22 in placental tissue and apparently
nonmosaic normal 46,XY cells in newborn blood had severe
upd(20)pat intrauterine growth restriction, first-degree hypospadias, and
other features attributed to prematurity [332]. There is no
No pure paternal UPD involving the entire chromosome 20 evidence that maternal UPD 22 has an imprinting effect.
has been reported. One case had a structurally abnormal chro-
mosome 20 derived from a terminal rearrangement that joined
two chromosomes 20 at band p13 [45,XY,psu dic(20;20) upd(22)pat
(p13;p13)] [321]. DNA polymorphism studies indicated that
the two chromosomes 20 in this terminal rearrangement were A single case of paternal UPD for chromosome 22 was
derived from one paternal chromosome, thereby representing reported in an abstract [333]. It was observed in a phenotypi-
paternal isodisomy. The patient had multiple anomalies cally normal individual with a balanced (22;22) Robertsonian
including microtia/anotia, micrencephaly, congenital heart translocation (see Chaps. 3 and 9). Paternal UPD 22 is not
disease, neuronal subependymal heterotopias, and colonic likely to have imprinting effect.
agangliosis. However, this case was complicated by the pres-
ence of trisomy 20 cells in skin. Therefore, although an
imprinting effect is possible for paternal UPD 20, a definitive upd(X)mat
conclusion cannot be drawn without further case reports.
Maternal UPD for the two X chromosomes in females has
been reported in three cases [334, 335]. The first two cases
upd(21)mat were detected by screening a normal population of 117 indi-
viduals. The third patient had Duchenne muscular dystrophy
Maternal UPD for chromosome 21 has been reported in at due to homozygosity of a maternally inherited deletion of
least two patients [322, 323]. Both had a balanced de novo exon 50 of the dystrophin gene. These observations indicate
(21;21) Robertsonian translocation (see Chaps. 3 and 9) and that maternal UPD for the X chromosome may not have an
were phenotypically normal. Although maternal UPD 21 has imprinting effect. To date, no imprinted genes on the X chro-
been reported in early abortus specimens, it has not been mosome have been identified in humans.
possible to clearly attribute embryonal death to UPD [324].
Therefore, maternal UPD 21 may be considered at this time
to have no imprinting effect. upd(X)pat
A single case of paternal UPD for the two X chromosomes in

upd(21)pat the 46,XX cell line of a 14-year-old girl with 45,X/46,XX
mosaicism (see Chap. 10) has been reported [336]. This
Two cases of paternal UPD for chromosome 21 have been patient had impaired gonadal function and short stature. The
reported [325, 326]. In both cases, UPD resulted from de novo presence of a 45,X cell line makes it difficult to determine if
formation of a Robertsonian translocation (see Chaps. 3 and the observed clinical features in this patient can be attributed
9). Both individuals were phenotypically normal. Paternal to paternal UPD for the X chromosome. Therefore, it is
UPD 21 does not appear to have an imprinting effect. unknown at this time if paternal UPD X has an imprinting
effect. However, no imprinted genes on the X chromosome 4. McGrath J, Solter D. Completion of mouse embryogenesis
have been identified in humans. requires both the maternal and paternal genomes. Cell.
1984;37:179–83.
5. Surani MAH, Barton SC, Norris ML. Development of reconsti-
tuted mouse eggs suggests imprinting of the genome during game-
upd(XY)pat togenesis. Nature. 1984;308:548–50.
6. Barton SC, Surani MAH, Norris ML. Role of paternal and maternal
genomes in mouse development. Nature. 1984;311:374–6.
A single case of paternal contribution of both the X and Y 7. Surani MAH, Barton SC, Norris ML. Nuclear transplantation in
chromosomes in a male patient was reported in an abstract the mouse: heritable differences between parental genomes after
[337]. This patient was ascertained because he had hemo- activation of the embryonic genome. Cell. 1986;45:127–36.
philia A, which was transmitted from his father. No abnor- 8. Linder D, McCaw BK, Hecht F. Parthenogenic origin of benign
ovarian teratomas. N Engl J Med. 1975;292:63–6.
malities other than hemophilia were described. Paternal UPD 9. Kajii T, Ohama K. Androgenetic origin of hydatidiform mole.
for XY may therefore not have an imprinting effect. Nature. 1977;268:633–4.
10. Lawler SD, Povey S, Fisher RA, Pickthal VJ. Genetic studies on
hydatidiform moles. II. The origin of complete moles. Ann Hum
Genet. 1982;46:209–22.
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Genetic Counseling
21
Sarah Hutchings Clark
Introduction Genetic counselors work in a variety of settings. There

are genetic counselors who work primarily in the areas of
Genetic counseling, in the traditional sense, has been defined prenatal, pediatric, adult, and cancer genetics, as well as
as a communication process, whereby individuals and public health, administration, research, and molecular and
families are educated about the genetic conditions in their fam- cytogenetic testing. Genetic counselors can be found in a
ilies and about those for which they could be at risk. Genetic variety of public and private medical settings, in state and
counseling, as its name implies, also involves addressing the federal offices, in research and diagnostic laboratories,
psychosocial issues that accompany the diagnosis, or possi- and in health insurance companies. Some genetic counselors
ble diagnosis, of such a condition. The counselor supports are certified by the American Board of Genetic Counseling
the family in learning about the diagnosis and in decision (ABGC), the organization that is also responsible for the
making about issues surrounding the diagnosis or potential accreditation of genetic counseling graduate programs.
diagnosis. However, genetic counseling is still a rapidly The term genetic counseling was first coined by Sheldon
evolving field, with many counselors becoming increasingly Reed in 1947. He was also vital in establishing the respect
involved in “nontraditional” roles. for counselees that is a cornerstone of the field of genetic
In 1983, the National Society of Genetic Counselors counseling. Dr. Reed had a deep concern for the feelings of
(NSGC), the field’s professional membership organization, his patients, and he cared about how genetic conditions
defined the role of genetic counselors: influenced their lives [2]. However, the roots of the field can
Genetic counselors are health professionals with specialized be traced back to the early 1900s. At that time, people were
graduate degrees and experience in the areas of medical genetics not only concerned about elucidating the genetic mecha-
and counseling. Most enter the field from a variety of disciplines, nisms behind hereditary conditions but were also interested
including biology, genetics, nursing, psychology, public health in eugenics. This interest facilitated tragic consequences,
and social work.
Genetic counselors work as members of a health care team, pro- including the killing of thousands of people with genetic
viding information and support to families who have members conditions, along with individuals of Jewish descent, in the
with birth defects or genetic disorders and to families who may Holocaust. Additionally, individuals with hereditary condi-
be at risk for a variety of inherited conditions. They identify fami- tions or mental retardation were encouraged or forced not to
lies at risk, investigate the problem present in the family, interpret
information about the disorder, analyze inheritance patterns and reproduce. The field of genetics later rejected eugenics and
risks of recurrence and review available options with the family. moved away from this unfortunate past [3].
Genetic counselors also provide supportive counseling to fami- Formally speaking, genetic counseling is a relatively new
lies, serve as patient advocates and refer individuals and families field. The first class with a master’s degree in genetic counsel-
to community or state support services. They serve as educators
and resource people for other health care professionals and for ing graduated from Sarah Lawrence College in 1971. In 1975,
the general public. Some counselors also work in administrative a formal definition of genetic counseling was proposed and
capacities. Many engage in research activities related to the field adopted by the American Society of Human Genetics. In
of medical genetics and genetic counseling [1]. 1979, genetic counselors formed a professional society, the
National Society of Genetic Counselors, which has played a
critical role in establishing and furthering the profession [4].
Genetic counseling is based on the principles of nondirec-
S.H. Clark, M.S., C.G.C. (*)
tiveness and a client-centered approach. The principle of
Department of Advanced Obstetrics and Gynecology,
Stamford Hospital, 30 Shelburne Road, Stamford, CT 06904, USA nondirectiveness states that genetic counselors are to provide
e-mail: [email protected] information in a way that does not encourage, or discourage,
500 S.H. Clark
a certain course of action. In other words, genetic counselors As is likely apparent at this point, one major goal of a
do not tell their clients what decisions to make. That is not to genetic counseling session is to provide information. Genetic
say that genetic counseling should be devoid of guidance, counselors seek to convey relevant information in a manner
particularly in complex situations, but that the counselor that is clear and understandable to each individual patient or
should provide guidance within the framework of the family member. Information is provided about the clinical
patient’s beliefs and values. The counselor assists and sup- features, natural history, and potential variability of the
ports the individual and family as they process the informa- particular condition. Additionally, the genetic basis of
tion provided during the counseling session and as they the condition and mechanism by which it occurs, recurrence
attempt to reach a decision regarding the course of action risks, available options for research and clinical testing, test
that is the most appropriate for them. To effectively and results, evaluation, and treatment are discussed [3].
responsibly accomplish this, the counselor must have some The presence of a genetic condition or birth defect in a
understanding of the patient’s “social, cultural, educational, family can have a significant impact on family relationships
economic, emotional, and experiential circumstances” [3]. and on the way that the patient and family interact with
This is by no means a simple task, particularly in light of the society as a whole. Individuals and families facing a genetic
complex and powerful emotions that genetic conditions often condition are often in an emotionally vulnerable state. The
evoke. By maintaining a client-centered approach, genetic emotions experienced by the individual and family can vary
counselors seek to empower their patients and to support and widely and can be extremely powerful. Feelings of guilt,
encourage them in their ability to make the best decisions for stigmatization, and altered self-esteem are relatively com-
themselves in their own unique circumstances [3]. mon, whether the diagnosis of a genetic condition is made
prenatally, during childhood, adolescence, or adulthood.
Therefore, the counselor seeks to support the patient and
Components of a Genetic Counseling Session family emotionally in an empathic manner and to advocate
and the Role of the Genetic Counselor for them. In keeping with this goal, the potential impacts of
the condition, including positive and negative economic,
The components of a genetic counseling session can vary widely psychological, and social effects, and available resources to
depending upon the reason for the referral and the specific needs assist in dealing with the condition are presented to the
of the patient and/or family. However, several components are individual and/or family [3]. It is important to realize that
frequently part of the counseling session, particularly if it is the different individuals may have unique perceptions of and
first time that the counselor and patient are meeting. reactions to the information discussed during a genetic coun-
The first step is to elicit the patient’s understanding of seling session. Genetic counselors are trained to be sensitive
why he or she has been referred and to clarify the reason for to this fact and to remain nonjudgmental in the face of it.
referral, if necessary. The counselor also seeks to establish a
mutually acceptable set of goals for the session and to under-
stand the concerns of the patient and/or family. This is General Indications for Referral to a Genetic
referred to as contracting. Counselor
During the majority of sessions, the genetic counselor
obtains a detailed family, medical, and pregnancy history in There are many indications for an appropriate referral to a
the form of a pedigree (Fig. 21.1). In medical genetics, a genetic counselor. Several of the more common reasons for
pedigree is the accepted, standardized method of document- referral are addressed here. The indications that are specifically
ing the family history in the form of a diagram, which indi- related to cytogenetic issues are introduced and are then
cates the family members, their relationships to one another, discussed in additional detail in the following section.
their status with regard to the genetic condition or trait in
question, and any other relevant medical issues. In addition
to providing valuable information about the medical aspects Family History or Clinical Suspicion of a Genetic
of the family history, obtaining the information for the pedi- Syndrome or Chromosome Abnormality
gree allows the genetic counselor to gain useful information
about the dynamics of the family in general and in relation to The presence of certain birth defects (also known as con-
the condition in question [5, 6]. The pedigree also often genital anomalies), mental retardation, and/or other charac-
allows the counselor to begin to establish a relationship with teristic features can raise the level of suspicion that an
the patient. Pedigrees, in varying forms, have been a part of individual is affected with a genetic syndrome or chromo-
genetics since the early days of the field. Interestingly, the some abnormality. When possible, the identification of a
history of the pedigree provides valuable insights into the cause for the congenital anomalies and/or mental retardation
evolution of the field of genetics [7]. in an individual not only allows for genetic counseling
21 Genetic Counseling 501
Fig. 21.1 Pedigree of a family carrying a balanced translocation involving the long arm of chromosome 7 and the short arm of chromosome 10.
See key for interpretation of symbols
regarding recurrence risk but can also be important, psycho- diagnosis, assist in discussing test results, and support the
logically and practically, for the individual and family. The individual and/or family emotionally.
evaluation of an individual to rule out the presence of a Although beyond the scope of this book, it is important to
genetic condition often involves the evaluation of that indi- recognize that genetic counselors routinely interact with
vidual by a medical geneticist. Certain biochemical, molecu- individuals who have a personal or family history of a genetic
lar, cytogenetic, and physiologic tests may also be helpful. syndrome. It is, therefore, also important to be acquainted
The genetic counselor can be an important part of the health- with the more common patterns of inheritance:
care team that evaluates and cares for the patient. The coun- In genetic syndromes that follow an autosomal recessive
selor can aid the geneticist in his or her clinical evaluation of pattern of inheritance, a carrier has one copy of a genetic
the patient, help to coordinate further testing, and help to alteration, or mutation, and, as a general rule, does not exhibit
keep the patient and/or family apprised of the need for such symptoms of that syndrome. If both members of a couple are
testing. The counselor can also help to keep the family carriers of an autosomal recessive disorder, there is a 25%
informed of the possible conditions in the differential chance for them to have an affected child in each pregnancy.
502 S.H. Clark
Examples of autosomal recessive conditions include cystic cancer significantly increases the chance to develop the con-
fibrosis, which results in thickened mucus primarily affecting dition. Hallmarks of hereditary cancer families include rela-
the lungs, digestive tract, and male reproductive tract; and tively early-onset cancer as compared to the general
Tay-Sachs disease, a fatal neurodegenerative disorder that is population, bilateral or multiorgan cancer, multiple affected
more common in the Ashkenazi Jewish, Cajun, and French- family members (usually following an autosomal dominant
Canadian populations. pattern of inheritance), and unusual cancer or the presence of
In autosomal dominant inheritance, there is a 50% chance certain characteristic clinical features. When an individual is
for an affected individual to transmit the disease-causing referred for cancer genetic counseling, the genetic counselor
mutation to each of his or her offspring. Depending upon the educates the counselee about the genetics of cancer predis-
particular condition, inheriting the mutation might or might not position. Based on personal and family history information,
mean that an individual will show features of that condition, a the counselor also provides a risk assessment for cancer or
phenomenon known as incomplete or reduced penetrance. for a hereditary cancer predisposition. The risks, benefits,
Additionally, there can be a wide range of clinical severity, even and limitations of appropriate, available molecular testing
within a family; this is known as variable expressivity. Examples options and research opportunities are discussed, as are the
of autosomal dominant conditions include Huntington’s potential results and their possible psychosocial and practi-
disease, an adult-onset neurodegenerative condition, and cal implications. Options for cancer risk reduction, such as
Marfan syndrome, a condition that affects connective tissue. prophylactic surgery, chemoprevention, and cancer screen-
In X-linked recessive inheritance, there is a 50% chance ing, are also likely to be reviewed.
for each son of a female carrier to be affected and a 50% As discussed in Chap. 15, certain translocations are char-
chance for each daughter of a female carrier to be a carrier acteristic of certain cancers. For example, the (9;22) translo-
herself. Under certain uncommon circumstances, females cation, which results in the “Philadelphia chromosome” and
can be affected with X-linked recessive conditions. As in the fusion of two genes, BCR and ABL1, is associated with
autosomal recessive inheritance, carriers have one mutation, chronic myelogenous leukemia (CML). Similarly, Burkitt
except in this case on one X chromosome instead of on an lymphoma is associated with an (8;14) translocation. The
autosome, and generally do not exhibit features of the condi- identification of cytogenetic abnormalities in a cancer patient
tion. Examples of conditions that follow an X-linked recessive can have important diagnostic and prognostic implications
pattern of inheritance include fragile X syndrome, which is and can also play a role in designing a treatment strategy
the most common inherited form of mental retardation (see [8, 9]. Occasionally, when chromosome analysis is per-
Chap. 19), and hemophilia, a bleeding disorder. formed for the indication of a hematological abnormality, a
In X-linked dominant inheritance, there is a 50% chance chromosome abnormality that may be constitutional is
for each child of an affected woman to inherit the disease- identified. In such a situation, this should be verified, and, if
causing mutation. Affected females tend to be more common true, the patient should be counseled about the finding and
and are often less severely affected than are affected males; the associated implications, not only for him- or herself, but
X-linked dominant conditions, particularly those that are for other family members as well [9].
rare, can be prenatally lethal in affected males. Incontinentia
pigmenti type 2, which affects the skin, skin derivatives, and
central nervous system, is an X-linked dominant condition Consanguinity
that is frequently lethal in affected males [8].
In multifactorial inheritance, a genetic predisposition When both members of a couple share at least one common
increases the chance that an individual will develop a particular ancestor, they may be referred to a genetic counselor to discuss
condition. Certain environmental factors, such as diet and exer- the possibility for an increased risk of birth defects and/or
cise, also have a role in determining if the individual will be genetic conditions in their offspring. Using information about
affected. Examples of multifactorial conditions are diabetes, the degree of relationship between the members of the couple,
heart disease, and neural tube defects. Generally speaking, the their ethnicities, and family history, the counselor discusses the
more distant the degree of relationship between the individual in potential for increased risk, if any, and offers any appropriate
question and the affected relative, the lower the recurrence risk, options for carrier and/or prenatal testing [10]. Although in
until such risk approximates that of the general population. some cultures consanguinity is accepted and even common,
in other cultures, it carries a social stigma. Not only might a
consanguineous couple be dealing with an increased risk of
Personal or Family History of Cancer abnormalities in their offspring, but they might also be facing
criticism from their family and society. In these situations, the
In the majority of cases, cancer is sporadic in an individual. genetic counselor can provide emotional support and referral
However, in some families, a genetic predisposition to to an appropriate support organization.
Advanced Maternal Age Advanced Paternal Age
The chance of having a pregnancy or child affected with a Advanced paternal age, frequently defined as 40 or older at
chromosome abnormality increases with advancing maternal the time of conception, is an acceptable, although infre-
age (Table 21.1) [11, 12, 14]. While previous standard of quent, reason for a referral to a genetic counselor. Studies
care required that prenatal diagnosis (see Chap. 12) via have shown an increased risk for genetic defects associated
chorionic villus sampling (CVS) or amniocentesis be offered with advanced paternal age. These genetic defects include
to all pregnant women who will be 35 or older at their esti- sporadic, dominant single gene mutations, most commonly
mated date of delivery (EDD), it is now recommended that Pfeiffer syndrome, Crouzon syndrome, Apert syndrome,
such diagnostic testing be offered to all women, regardless of achondroplasia, thanatophoric dysplasia, and MEN2A and
age [14–16]. MEN2B. The risk for a sporadic, autosomal dominant
genetic syndrome in the offspring of men over the age of 40
is presently felt to be less than 0.3–0.5%. Studies also indi-
Table 21.1 Risks for chromosome abnormalities at term by maternal cate that advanced paternal age may be associated with an
age [14]
increased risk of complex conditions, including some birth
Risk for trisomy Risk for any chromosome defects, schizophrenia, autism spectrum disorders, and
Maternal age at term 21b [12] abnormalityb, c [11]
some cancers. There does not, however, appear to be an
15a 1:1,578 1:454
increased risk of chromosome abnormalities associated with
16a 1:1,572 1:475
advanced paternal age with the possible exception of tri-
17a 1:1,565 1:499
somy 21 and Klinefelter syndrome. Most of the paternal
18a 1:1,556 1:525
age-related birth defects cannot be reliably detected by pre-
19a 1:1,544 1:555
20 1:1,480 1:525
natal diagnosis [17].
21 1:1,460 1:525
22 1:1,440 1:499 Abnormal Prenatal Screen
23 1:1,420 1:499
24 1:1,380 1:475 Screening can be used, along with maternal age, to estimate
25 1:1,340 1:475 the possibility that a fetus is affected with Down syndrome
26 1:1,290 1:475
or trisomy 18. Such aneuploidy screening can be performed
27 1:1,220 1:454
through the utilization of ultrasound, maternal serum, or,
28 1:1,140 1:434
frequently, a combination of the two.
29 1:1,050 1:416
30 1:940 1:384
31 1:820 1:384 Teratogen Exposure
32 1:700 1:322
33 1:570 1:285 The term “teratogen” applies to any medication, chemical, or
34 1:456 1:243 environmental agent that has the potential to cause adverse
35 1:353 1:178 effects, such as birth defects, on a developing fetus. When
36 1:267 1:148
the mother or father of a current or future pregnancy has been
37 1:199 1:122
exposed to an agent that could have a detrimental effect on
38 1:148 1:104
that pregnancy, a referral to a genetic counselor is appropriate.
39 1:111 1:80
Of note, certain maternal conditions, such as phenylketonuria
40 1:85 1:62
41 1:67 1:48
(PKU), which is an inherited metabolic disorder, diabetes,
42 1:54 1:38 and seizure disorders increase the risk for birth defects in a
43 1:45 1:30 pregnancy. The counselor will consult current resources and
44 1:39 1:23 discuss with the exposed individual or couple the potential
45 1:35 1:18 adverse effects associated with the exposure in question. Any
46 1:31 1:14 available options for minimizing these potential adverse
47 1:29 1:10 effects or for identifying them prenatally are also discussed.
48 1:27 1:8
49 1:26 1:6
50 1:25 Data not available
Infertility
a
Reference [13]
b
Risks based on maternal age at term. Term risks do not include
Certain chromosome abnormalities and genetic conditions
chromosomally abnormal fetuses spontaneously lost before term result in varying degrees of infertility (see Chap. 11).
c
Includes risk for trisomy 21. Does not include 47,XXX Therefore, when an individual or couple experiences infertility,
504 S.H. Clark
it is appropriate to rule out the possible genetic and cyto- educating the patient or family about the clinical features
genetic causes. If such a cause is identified, a genetic coun- of the condition, recurrence risks, and available supportive
selor can be important in educating the individual about the treatments. Although the identification of a cause for the
condition. The genetic counselor can also assist the physi- phenotypic abnormalities in an individual can be an empow-
cian in discussing the available options that could allow for ering event for the patient and family, it can also induce
reproduction. In addition, if the individual is able to repro- significant stress. The genetic counselor, acting as a member
duce using his or her own gametes, the possible recurrence of the team caring for the individual, often plays an impor-
risks for future offspring should be addressed. tant role in helping the family to cope with the diagnosis
both practically and emotionally.
Recurrent Spontaneous Abortion Autosomal Trisomies

Down Syndrome
Miscarriage is more common than many people recognize. Down syndrome, which is caused by non-mosaic trisomy 21
In fact, it is estimated that 10–15% of all recognized preg- in approximately 94% of cases, is the most common human
nancies end in miscarriage [18]. There are many possible chromosome abnormality, affecting approximately 1 in 800
causes of miscarriage, including a chromosomally abnormal individuals [8, 20, 21]. Individuals with Down syndrome fre-
conceptus. Approximately 50% of recognized first trimester quently have a characteristic facial appearance and frequently
miscarriages are chromosomally abnormal [8, 18, 19]. In resemble one another more than they resemble their family
some individuals, pregnancy loss is recurrent. In addition to members. Certain health conditions and birth defects are more
having the potential to cause significant psychological dis- common in individuals with Down syndrome, including con-
tress, recurrent miscarriage warrants a complete evaluation, genital heart defects, gastrointestinal problems, leukemia,
which could include genetic, cytogenetic, and endocrinology Alzheimer disease, immune dysfunction, thyroid dysfunction,
studies, in an attempt to identify the cause. As discussed and problems with hearing and vision. Poor muscle tone and
later, some causes of recurrent miscarriage confer increased delayed growth are also frequent findings. In 1997, the median
reproductive risks for the patient, as well as his or her family age at death was noted to be 49 years with congenital heart
members. defects presenting a major cause of early mortality. Everyone
with Down syndrome has some degree of mental retardation.
While the general IQ range is usually said to be 25–50, a
Cytogenetic Indications for Genetic Counseling range of mental capability exists. Children with Down syn-
drome often benefit from early programs aimed at stimula-
Family History or Clinical Suspicion tion, developmental enrichment, and education [20–22].
of a Chromosome Abnormality
Trisomy 13
As previously mentioned, congenital anomalies, mental Trisomy 13 results in severe mental retardation and multiple
retardation, developmental delay, or certain characteristic birth defects. The abnormalities most commonly noted in
features are all examples of indications for chromosome this condition involve the heart (congenital defect in 80%),
analysis. Several chromosome abnormalities are detectable brain, eyes, ears, lip, and palate (cleft lip and/or cleft palate),
through conventional chromosome analysis, while others, hands and feet (such as polydactyly or extra digits), and gen-
such as microdeletion syndromes, require specialized analy- italia. This condition is frequently fatal early in infancy with
sis, such as fluorescence in situ hybridization (FISH) (see only 5–9% of affected individuals surviving the first year of
Chap. 17) or microarray (see Chap. 18). The following is a life with a median survival of 7–10 days [8, 21, 22].
brief introduction to several of the more common chromo-
some abnormalities encountered in genetic counseling. The Trisomy 18
style of genetic counseling associated with the identification Like trisomy 13, trisomy 18 results in severe mental retarda-
of a chromosome abnormality often varies depending upon tion and birth defects. Congenital heart defects and abnor-
the age of the affected individual. Although the clinical malities of the hands and feet (clenched hands with
information is unlikely to be significantly different, the tone overlapping fingers) defects are common, as is growth
of the discussion often varies depending on whether the deficiency. Several other congenital anomalies, including
diagnosis is made prenatally, when termination of the preg- those involving the kidneys, central nervous system, skeletal
nancy might be an option, or during childhood, adolescence, system, gastrointestinal system, and genitalia, are also asso-
or adulthood. As previously mentioned, regardless of ciated with this condition. Approximately 5–10% of babies
whether a chromosome abnormality is diagnosed prenatally affected with trisomy 18 survive the first year of life with a
or postnatally, the genetic counselor often plays a role in median survival of 10–14 days [8, 21, 22].
Some cases of Down syndrome, trisomy 13, or trisomy 18 Prader-Willi Syndrome

are the result of unbalanced translocations. If such a translo- Approximately 70–75% of cases of Prader-Willi syndrome
cation is carried, in a balanced form, by one of the parents, result from deletion on the paternally derived copy of chro-
recurrence risks are generally greater than they would be if mosome 15 [del(15)(q11.2q13)]. Other potential causes are
simple trisomy 13, 18, or 21 was present in the affected indi- maternal uniparental disomy for chromosome 15 and an
vidual. It should also be noted that mosaic chromosome imprinting mutation. Imprinting refers to certain genes
abnormalities, with a chromosomally normal cell line, can be being active on only the maternally or paternally derived
associated with a less severe mental and physical phenotype, copy of a particular chromosome (see Chap. 20). Affected
although the severity of the condition cannot be predicted individuals usually have low muscle tone and feeding
from the karyotype. difficulties during infancy. Later in childhood, however,
For more comprehensive coverage of trisomy, refer to obsessive eating and obesity develop. Other features com-
Chap. 8. monly seen in individuals with this condition include short
stature, mental retardation, small hands and feet, small
Unbalanced Chromosome Rearrangements underdeveloped genitals, characteristic facial features, and
A family history of birth defects and/or mental retardation, decreased sensitivity to pain. Behavior problems, such as
sometimes accompanied by a history of recurrent pregnancy skin picking, stubbornness, temper tantrums, obsessive-
loss, can result from the segregation of a familial chromo- compulsiveness, and, in some, psychosis, can also be pres-
some rearrangement, such as a translocation or inversion ent [21, 24]. See also Chap. 9.
(Fig. 21.1; see also Chap. 9).
Angelman Syndrome
Microdeletion Syndromes Approximately 70–75% of cases of Angelman syndrome are
Microdeletion syndromes, as their name implies, are the caused by the same microdeletion found in the majority of
result of relatively small chromosomal deletions that may be cases of Prader-Willi syndrome, except that the deletion
undetectable via conventional cytogenetic analysis. When a occurs on the maternally derived copy of chromosome 15,
clinician suspects that an individual is affected with one of and there are in fact differences at the molecular (DNA)
these conditions, FISH or microarray techniques are gener- level. The clinical features most commonly found in affected
ally employed to confirm, or rule out, the diagnosis. individuals include severe mental retardation, spontaneous,
Occasionally, certain ultrasound findings raise the possibil- excessive fits of laughter, “jerky” limb movements, charac-
ity of a particular microdeletion syndrome in the fetus, as teristic facial features, sleep abnormalities, and seizures [21,
can be the case with 22q11.2 deletion syndrome when a 25]. Imprinting also plays an important causative role in this
heart defect is noted on prenatal ultrasound. In these cases, disorder (see Chaps. 9 and 20).
analysis can be performed on the material obtained from a
chorionic villus sampling (CVS) or amniocentesis. Several Williams Syndrome
of these microdeletion syndromes occasionally result from Williams syndrome is the result of a microdeletion on chro-
the unbalanced segregation of a familial chromosome rear- mosome 7 at the q11.23 locus and involves, among others,
rangement. See Chaps. 12 and 17. the elastin (ELN) gene. The condition is usually sporadic, but
as with the 22q microdeletion syndrome, can also follow an
22q11.2 Deletion Syndrome (Including DiGeorge and autosomal dominant pattern of inheritance. As infants,
Velocardiofacial Syndromes) affected individuals tend to experience failure to thrive, gas-
This syndrome results from an interstitial deletion of the long trointestinal complications, delayed milestones, and delayed
arm of chromosome 22. One interesting feature of this condi- speech. The rate of growth is slow, and mental retardation,
tion is the potential for wide clinical variability within and characteristic facial features, cardiovascular defects, urinary
between families. At times, subsequent to the diagnosis of a tract abnormalities, and joint problems are often present.
child, one of the parents is found to be affected, although usu- One of the most interesting features of Williams syndrome is
ally more mildly. The microdeletion is frequently sporadic the unique, characteristic personality. Affected individuals
(approximately 93% of cases) but can also be inherited in an tend to be extremely friendly and talkative. Certain behavior
autosomal dominant manner. A variety of features in multiple problems, such as a generalized anxiety and sleep difficulties,
organ systems have been reported in individuals with can be encountered [21, 26]. See Chap. 9.
DiGeorge syndrome. Some of the more common features
include learning disabilities, heart defects, cleft palate, short Smith-Magenis Syndrome
stature, immune deficiency, low muscle tone in infancy, Smith-Magenis syndrome, which is the result of a deletion
hypernasal speech, low calcium levels, renal abnormalities, involving the short arm of chromosome 17 [del(17)
psychiatric illness, and characteristic facial features [21, 23]. (p11.2p11.2)], is almost always sporadic. In infancy, individuals
506 S.H. Clark
with Smith-Magenis syndrome tend to have feeding problems Infertility

and low muscle tone. Language and motor skills are delayed, At times, when one of the members of a couple is a carrier of
and mental retardation is a feature of the condition. Other a structural chromosome rearrangement (see Chap. 9), the
features include short stature, severe sleep disturbances, unbalanced segregation of that rearrangement can result in
characteristic facial features that become more evident with miscarriage before the couple is aware of the pregnancy. This
age, and behavioral problems. The behavioral problems can cause the couple and their physicians to suspect infertil-
often include self-injury, attention deficit, and temper ity. True infertility is also a frequent feature of certain sex
tantrums [18, 21, 27]. chromosome abnormalities, and, therefore, the clinician and
genetic counselor must also consider the possibility of a sex
Miller-Dieker Syndrome chromosome disorder when faced with an infertile couple.
Miller-Dieker syndrome is also the result of an interstitial dele- See also Chaps. 10 and 11.
tion involving the short arm of chromosome 17 [del(17)
(p13.3p13.3)], more distal than that seen in Smith-Magenis Sex Chromosome Abnormalities
syndrome. The abnormalities associated with this condition It has been estimated that, overall, approximately one in 400
involve the central nervous system, with lissencephaly, or a infants have some form of sex chromosome aneuploidy [32].
smooth brain, being a characteristic feature. This results in A thorough discussion of sex chromosomes and sex chromo-
severe mental retardation, seizures, low muscle tone, and a some abnormalities can be found in Chap. 10. A potentially
small head size. Certain characteristic facial features are also challenging situation that genetic counselors face regarding
associated with Miller-Dieker syndrome. The majority of the diagnosis of a sex chromosome abnormality is that the
affected individuals die within the first two years of life. patient is often an adolescent. It is imperative for the coun-
Approximately 80% of affected individuals have a sporadic selor to discuss this finding and its implications on the
deletion. However, the remaining 20% inherit the deletion from patient’s level of understanding. Additionally, he or she must
a parent with a balanced chromosome rearrangement [21, 28]. appreciate that the diagnosis may create for a young adult a
potentially unique and more delicate set of psychosocial
Subtelomere Rearrangements issues, as this diagnosis may come at a time when the indi-
Cryptic microdeletions, or subtle rearrangements near the vidual is already struggling with a developing sense of self
tips of chromosomes, are estimated to be a common cause of and sexuality.
mental retardation, with or without dysmorphic features.
Unbalanced subtelomere rearrangements are reported to Klinefelter Syndrome
occur in 7.4% of individuals with moderate to severe mental Klinefelter syndrome, 47,XXY, affects approximately one in
retardation and can be detected with FISH probes for the 500 males and is a common cause of male infertility. Men
unique subtelomeric regions of most chromosomes (see who are affected with this condition tend to be tall and thin.
Chap. 17) [29]. The identification of such an unbalanced The genitals, particularly the testes, are usually small and
rearrangement in a phenotypically abnormal individual there can be gynecomastia (male breast enlargement). The
allows subtelomeric FISH studies to be offered to the par- development of secondary sex characteristics is incomplete.
ents, and other at-risk family members, to determine if one of As testosterone production is often insufficient, testosterone
them carries a balanced subtelomeric rearrangement. Based replacement therapy is often utilized to minimize the fea-
upon the results of the parental analyses, recurrence risks can tures of this condition related to testosterone insufficiency.
be more accurately quoted. Certain other clinical indications Learning difficulties are common. The IQ is usually average
for subtelomere analysis, such as characterization of known but may be lower than that of siblings. A wide range of IQs
chromosomal abnormalities, have been noted in the literature has been noted, including some well above and well below
[30, 31]. Subtelomeric abnormalities are now more often average. Behavioral differences, such as shyness and insecu-
diagnosed with microarray analysis (see Chap. 18). rity, can be present [8, 21, 32].
There are many chromosomal variants of this condition.
Chromosome Instability Syndromes Some of these variants are associated with a less severe phe-
As discussed in Chap. 14, there are a number of genetic syn- notype, such as some cases of 47,XXY/46,XY mosaicism.
dromes of which a notable feature is an increased incidence Other variants are associated with a more severe phenotype,
of chromosome breaks and instability. The majority of these such as 48,XXYY, which is associated with a greater likeli-
syndromes, including Fanconi anemia, Bloom syndrome, hood of mental retardation [21, 32].
ataxia telangiectasia, and Roberts syndrome, follow an
autosomal recessive pattern of inheritance. Therefore, the Turner Syndrome
presence of one of these conditions in a family can have Turner syndrome, 45,X, is estimated to affect approximately
significant implications for recurrence [18, 21]. one in 2,500 live-born females. The infertility associated with
Turner syndrome results from ovarian degeneration. state, in one member of the couple. Carriers of structural
Affected individuals experience delayed and/or incomplete chromosome rearrangements are often at increased risk to
puberty, and the majority do not menstruate. For this reason, produce unbalanced gametes. When such an unbalanced
estrogen replacement therapy is often utilized to stimulate gamete is fertilized, this imbalance can result in miscarriage.
menstruation and pubertal development. Stature is often It is estimated that in approximately 4% of couples with
short, with an average height of 55 inches. Growth hormone two or more miscarriages, one of the partners carries a bal-
therapy may be used in an effort to increase stature. Kidney anced translocation [19]. Blood chromosome analysis should
and heart defects, along with other anomalies, can be pres- be offered to any individual with a personal or family history
ent. Congenital lymphedema can result in puffiness of the of repeated pregnancy loss.
fingers and toes and a neck that appears webbed. This As previously noted, an unbalanced chromosome rear-
lymphedema, in the form of a cystic hygroma and/or rangement not only has the potential to cause miscarriage but
hydrops, is sometimes identifiable on prenatal ultrasound. can also result in live-born offspring with birth defects and/
Certain health conditions, such as diabetes, high blood pres- or mental retardation (Fig. 21.1). The risk for an abnormal
sure, and thyroid disease, are more common. In addition to live-born child associated with a given balanced chromo-
appearing webbed, the neck tends to be short, the chest is some rearrangement can be difficult to predict precisely. This
often broad, and the nipples widely spaced. Learning risk depends on a number of factors, including the family
difficulties can be present, although most affected individu- history, mode of ascertainment, predicted type of segregation
als have a normal IQ [8, 21, 32]. leading to viable gametes, sex of the carrier parent, and
As with Klinefelter syndrome, there are several chromo- degree of imbalance of the viable gametes [18]. Genetic
somal variants of Turner syndrome. Individuals with mosaic counseling can be vital in helping the individual or couple to
Turner syndrome and those who are missing only part of one understand the reproductive risks associated with a balanced
X chromosome can be less severely affected [8, 21, 32]. Only chromosome rearrangement. Often, the finding of a chromo-
50% of Turner patients present with the classic 45,X karyo- some rearrangement comes as a shock to the couple follow-
type. The remainder have some form of mosaicism and/or ing the frequently frustrating and emotionally distressing
structurally abnormal X chromosome [8]. loss of wanted pregnancies.
For a detailed discussion of chromosome rearrangements,
47,XYY refer to Chap. 9.
Although certain phenotypic features have been associated
with this condition, affected individuals frequently go unde- Advanced Maternal Age
tected, as the features are generally nonspecific. Males with During a genetic counseling session for advanced maternal
47,XYY usually have an IQ that, although in the normal age, the maternal age-related risks for a chromosome abnor-
range, is below that of their unaffected siblings. These indi- mality are discussed. The risks, benefits, and limitations of
viduals tend to be relatively tall, frequently have severe invasive diagnostic testing for chromosome abnormalities
acne, and could experience certain behavior problems in (i.e., CVS and amniocentesis) are also discussed, as are the
childhood, such as hyperactivity and attention deficit disor- benefits and limitations of prenatally available aneuploidy
der. However, it should be noted that violence and psycho- screening tests. Prenatal chromosome analysis is routinely
pathology are not more common in these males. This is performed via CVS or amniocentesis (see Chap. 12). It is
particularly important in light of the fact that some early, stressed to the patient or couple that although greater than
erroneous studies reported that 47,XYY males were over 99% of chromosome abnormalities are detectable by CVS or
represented in prisons and mental hospitals. Fertility is usu- amniocentesis, other genetic, nonchromosomal conditions
ally normal [8, 21, 32]. are not routinely detectable via this testing. If there is an
indication for additional genetic testing, such as a positive
47,XXX family history, such testing can, at times, be performed on
Females with 47,XXX could be of above average height and the sample obtained during one of these procedures.
experience learning disabilities, behavior problems, and The decision to pursue or decline invasive prenatal testing
delayed motor milestones with subsequent poor coordination is a highly personal and, at times, complicated decision. It
and “awkwardness.” Otherwise, there are no remarkable involves weighing the risks and benefits, the individual or
phenotypic features that are associated with this condition couple’s psychosocial circumstances, religious beliefs, per-
[8, 21, 32]. sonal experiences with disability, pregnancy history, and a
multitude of other issues. Genetic counseling can be helpful as
Recurrent Spontaneous Abortion the individual or couple considers these issues, as a major goal
One cause of recurrent spontaneous abortion is a structural of genetic counseling is to enable the individual or couple to
chromosome rearrangement, usually found in a balanced make a thoughtful, well-informed decision. Two common
508 S.H. Clark
reasons that prenatal diagnosis is pursued are if the couple/ that, in the prenatal setting, clinical decision making based
patient would consider pregnancy termination for the condi- upon FISH results should be supported by a confirmatory
tion in question and if they would want knowledge of the chromosome analysis and/or consistent clinical information
diagnosis to prepare for the birth of a child who could have [39]. FISH can also be performed on prenatal specimens for
special needs. the detection of several microdeletion syndromes when the
Chorionic villus sampling is generally performed between ultrasound findings or family history indicates an increased
9 and 12 weeks of pregnancy. During this procedure, a small risk for such a condition. Additionally, FISH or microarray
sample of the placenta is removed either transabdominally or can be performed on prenatal specimens for the detection of
transcervically under ultrasound guidance. The chorionic submicroscopic abnormalities. See also Chap. 12.
villi present in this sample are then placed in culture, and the
chromosomes are analyzed. The risk of a miscarriage associ- Abnormal Prenatal Screen
ated with a CVS was previously quoted as approximately While Down syndrome and trisomy 18 are commonly
one in 100 or 1%, although more recent studies indicate a screened for prenatally, other chromosome abnormalities
lower procedure related risk [14, 16, 33]. One advantage of can, at times, be detected using certain screening methods,
CVS, as compared to amniocentesis, is that it is performed although that is not the goal of such screening. Given that
during the first trimester of pregnancy, allowing for an earlier maternal age alone is a poor screening criterion for fetal
termination of pregnancy if an abnormality is identified. One aneuploidy, prenatal aneuploidy screening should be offered
potential disadvantage of CVS is that approximately 1–2% to all pregnant women [16]. The patient or couple should be
of samples result in a mosaic karyotype. The cause of the fully counseled about the benefits and limitations of screen-
mosaicism can be that the placenta has a different chromo- ing. It is important for the patient to appreciate the distinc-
some constitution than the fetus. This is known as confined tion between screening, which is designed to provide a risk
placental mosaicism. Even when the chromosomally abnor- estimate, and diagnostic tests, which are designed to diag-
mal cells are confined to the placenta, there can still be nose or rule out a chromosome abnormality. When screening
adverse effects on the fetus, as a chromosomally abnormal indicates that there is an increased risk for a chromosome
placenta can cause fetal growth restriction and adverse preg- abnormality in a pregnancy, the pregnant woman or couple
nancy outcome and can raise the possibility of uniparental should be counseled about the implications of this result and
disomy in the fetus. In these situations, follow-up testing, the options for further testing, such as CVS or amniocente-
such as amniocentesis, is often performed in an attempt to sis. An individual or couple may be referred for genetic
clarify the fetal karyotype [8, 14, 16, 18, 34, 35]. counseling prior to pursuing a prenatal screen so that an
Amniocentesis is generally performed at about informed decision can be made about whether or not to pur-
16–18 weeks of pregnancy, although this procedure can be sue the testing.
performed either earlier or later in gestation. During this pro- First trimester screening is, as its name implies, performed
cedure, a small amount of amniotic fluid is removed transab- during the first trimester of pregnancy. This screening
dominally under ultrasound guidance. The fetal cells involves biochemical analysis of the levels of certain preg-
(amniocytes) present in this sample are cultured, and the nancy-related proteins in the maternal circulation, namely,
chromosomes are analyzed. The level of a-fetoprotein (AFP) pregnancy-associated plasma protein A (PAPP-A) and
in the amniotic fluid can also be analyzed to screen for open human chorionic gonadotropin (hCG). To increase the
fetal defects, such as open neural tube defects and abdominal number of affected pregnancies detected by this screening,
wall defects. The risk of a miscarriage associated with an the biochemical analyses can be used in conjunction with
amniocentesis was previously quoted as approximately one a nuchal translucency ultrasound measurement, a measure-
in 200 or 0.5%, although more recent studies indicate a lower ment of the amount of fluid between the skin and soft tis-
procedure related risk [10, 14–16, 33]. sue over the cervical spine of the developing fetus.
When rapid information about the fetal chromosomes is Combined with additional information about the preg-
needed, generally the result of a particularly high risk of nancy and family history, this data is used to generate esti-
aneuploidy or a late gestational age, FISH (see Chap. 17) for mated risks for Down syndrome and trisomy 18 [16,
chromosomes 13, 18, 21, X, and Y can be performed on the 40–43]. In addition to being associated with an increased
direct amniotic fluid or chorionic villi. Chromosomes 13, 18, risk for aneuploidy, an increased nuchal translucency mea-
21, X, and Y are the most common chromosomes involved in surement is also associated with other fetal abnormalities,
a prenatally diagnosed, potentially viable chromosome particularly cardiac malformations, and some genetic syn-
abnormality and are, therefore, the focus of prenatal FISH dromes [16, 40, 44–47].
analysis [36–38]. Although FISH can yield important infor- Second trimester maternal serum screening is generally
mation in a short period of time, it is not a substitute for performed between 15 and 20 weeks of gestation. This
routine cytogenetic analysis. As such, it is recommended screening usually involves analyzing the maternal blood for
the levels of four pregnancy-related proteins, a-fetoprotein Prenatal Identification of a Chromosome

(AFP), human chorionic gonadotropin (hCG), unconjugated Abnormality
estriol (uE3), and dimeric inhibin A (DIA) and is often When a chromosome abnormality is identified prenatally, the
referred to as the quad screen. As with first trimester screen- genetic counselor provides information to the patient or cou-
ing, the levels of these analytes, combined with certain other ple regarding the phenotype associated with the abnormality
information, yield a risk estimate for Down syndrome and in question. Options for continuation or termination of the
trisomy 18. Unlike first trimester screening, second trimester pregnancy and adoption are also discussed, as is the fact that
maternal serum screening also screens for the presence of many chromosomally abnormal pregnancies are at an
open fetal defects, such as a neural tube or abdominal wall increased risk to miscarry, and not only in the first trimester
defects, through the analysis of the level of AFP present in of pregnancy [51]. For example, this risk is particularly high
the maternal serum [8, 16, 40]. in pregnancies affected with Turner syndrome, with at least
Several screening modalities, including integrated and 99% of affected pregnancies aborting spontaneously early in
sequential approaches, have been developed to take advan- pregnancy [8]. The prenatal identification of a chromosome
tage of both first and second trimester aneuploidy screening. abnormality (or any anomaly or genetic condition for that
These screens seek to increase the Down syndrome and tri- matter) can be traumatic and heartbreaking for a couple as
somy 18 detection rates and, in some situations, decrease the they face difficult decisions about an often much wanted
overall chance of a woman receiving a “screen-positive” pregnancy. It is especially important for the genetic coun-
result (i.e., elevated screening risk Down syndrome and/or selor to support the individual, couple, and family during and
trisomy 18) when the fetus is unaffected. It is currently rec- after such a diagnosis. No matter what the final decision
ommended that all women, regardless of age, who present regarding the future of the pregnancy might be, the emotional
for prenatal care prior to 20 weeks gestation be counseled support of the counselor, as well as referrals to appropriate
regarding the option to pursue a fetal aneuploidy screening resources and support groups, can be vital in helping the
test. Counseling should include a thorough discussion of the pregnant woman and/or couple cope with the diagnosis.
benefits and limitations of such screening [16, 40, 48]. The Although the majority of the common chromosome
American College and Obstetricians and Gynecologists abnormalities are associated with a rather well-defined phe-
(ACOG) specifically recommends that integrated or sequen- notype, results associated with unclear or ill-defined pheno-
tial screening be offered to women who seek prenatal care types can understandably be anxiety provoking. This is
during the first trimester [40]. particularly true if the couple/patient is struggling to make a
An ultrasound examination to evaluate a pregnancy for decision regarding termination versus continuation of the
the presence of certain birth defects and sonographic pregnancy.
findings associated with aneuploidy can also be used to The general phenotypes associated with the more com-
screen for Down syndrome and certain other chromosome mon autosomal chromosome aneuploidies, trisomies 13, 18,
abnormalities. Such an ultrasound is generally performed and 21, are described in a previous section and in Chap. 8.
during the second trimester of pregnancy, although some Although these phenotypes are well defined, there is a range
aneuploidy markers are identifiable during the first trimes- of severity particularly associated with Down syndrome or
ter, as is the case with increased nuchal translucency (see with mosaicism where a normal cell line is also present. As
the section “First Trimester Screening”). The percentage of noted previously, the degree of severity of the condition can-
aneuploid pregnancies with a demonstrable abnormality not be predicted from the karyotype. Some individuals find
on ultrasound depends upon the particular chromosome this to be a difficult situation, as they may feel capable of
abnormality and the experience of the sonographer. Some caring for a child with mild disabilities but unable to care for
of the aneuploidy markers that are potentially detect- a child with more severe disabilities.
able with prenatal ultrasound include cardiac malforma- The common sex chromosome abnormalities are gener-
tions, altered fetal growth, duodenal atresia, and cystic ally associated with less severe phenotypes than the afore-
hygroma. In addition to conferring an increased risk for mentioned autosomal trisomies. Although for some this is
aneuploidy, certain congenital anomalies identifiable on encouraging, for others the milder phenotypic features com-
ultrasound could be associated with certain genetic syn- plicate the decision of whether to continue the pregnancy or
dromes. At times, when prenatal chromosome analysis terminate [52].
produces an ambiguous or unclear result, ultrasound is As previously noted, mosaicism can make the prognosis
utilized in an attempt to evaluate the fetal anatomy and to less clear. One example of this is 45,X/46,XY mosaicism.
search for any fetal abnormalities that could be associated The majority of prenatally diagnosed affected individuals,
with the karyotype. As with all other screening, the limita- approximately 85–95%, are phenotypically normal males
tions of ultrasound should be made clear to the patient or externally. However, a range of phenotypes, from a female
couple [8, 22, 49, 50]. See Chap. 12. with Turner syndrome to ambiguous genitalia to externally
510 S.H. Clark
normal males, is possible. In phenotypic males, there can be “laboratory error” but is, rather, an occasionally unavoidable
variation with respect to the size of the phallus, descent of result of growing cells in vitro.
the testes, and scrotal fusion. Hypospadias and other con- • Level III mosaicism is defined as two or more cells with
genital abnormalities have also been noted. There is a risk, the same chromosome abnormality in two or more colo-
estimated to be approximately 27%, for abnormal gonadal nies. This finding is likely to represent true mosaicism
histology, which increases the risk for a gonadal tumor and raises the level of concern that there is an abnormal
(gonadoblastoma). Therefore, close follow-up to monitor cell line in the fetus [18].
for tumor development is warranted. The degree of mosa- When mosaicism is identified prenatally, particularly
icism does not appear to be a reliable predictor of the phe- level III mosaicism, follow-up testing, such as a detailed
notype. Of note, the majority of cases of 45,X/46,XY ultrasound to evaluate the fetal anatomy and/or repeat chro-
mosaicism diagnosed postnatally were associated with an mosome analysis, via amniocentesis or percutaneous umbili-
abnormal phenotype. The reason for this discrepancy is that cal blood sampling (PUBS)—in which fetal blood is obtained
the postnatally diagnosed cases reflect an ascertainment bias from the umbilical cord under ultrasound guidance—can be
[22, 53, 54]. pursued. It is important to realize, however, that such testing
When an apparently balanced chromosome rearrange- is unlikely to completely clarify the fetal karyotype. Again,
ment is identified by CVS or amniocentesis, the first step is the limitations of ultrasound in identifying certain pheno-
to perform chromosome analyses on the parents. If one of the typic abnormalities, such as mental retardation, must be
parents carries the same rearrangement and is phenotypically made clear to the patient or couple. Furthermore, a normal
normal, it is felt that the rearrangement is unlikely to confer repeat chromosome analysis, although encouraging, does not
a significantly increased risk of abnormality. It is important guarantee the absence of an abnormal cell line in the fetus.
to note that there are some mechanisms, such as uniparental Likewise, an abnormal repeat chromosome analysis does not
disomy (see Chap. 20), by which a balanced translocation necessarily mean that the abnormal cell line is present in the
inherited from a phenotypically normal parent can be associ- fetus. Genetic counseling to help the patient/couple interpret
ated with an increased risk for abnormalities. These mecha- this information is particularly important in such complex
nisms seem to be relatively uncommon [18]. If the situations. If the pregnancy is terminated or aborted sponta-
rearrangement is de novo, the risk assessment becomes more neously, chromosome analysis of a variety of fetal tissues
difficult. It has been estimated that the risk for abnormality should be considered. If the pregnancy is carried to term,
associated with a de novo reciprocal translocation is approxi- follow-up analysis of blood and/or skin might also be
mately 6.1%. The estimated risks for abnormality associated indicated.
with a de novo Robertsonian translocation and inversion are Although, as previously stated, mosaic chromosome
3.7 and 9.4%, respectively [55]. However, it can be difficult, abnormalities can be associated with milder phenotypes, the
if not impossible, to predict specific abnormalities. clinical features associated with true mosaicism cannot be
When a structural chromosome rearrangement is unbal- entirely accurately predicted from the karyotype. One reason
anced, whether it is de novo or results from the segregation for this is that it is impossible to know the distribution of
of a balanced rearrangement in a carrier parent, the pheno- normal and abnormal cells in the various tissues of the body.
type is likely to be abnormal. Again, however, it can be In some cases there can, however, be a correlation between
difficult to predict the specific abnormalities. Ultrasound the percentage of abnormal cells and the degree of abnormal-
examination and a literature review might lend some infor- ity. A review of the pertinent literature might provide useful
mation about the clinical picture. information regarding the general phenotype [56–59].
The issue of confined placental mosaicism was introduced It has been estimated that the prevalence of supernumer-
in the previous section regarding CVS, as such mosaicism is ary marker chromosomes at the time of CVS and amnio-
more likely to be found at CVS than at amniocentesis (see centesis is approximately 0.6–1.5 per thousand [60]. The
also Chap. 12). Mosaicism is not, however, always confined discovery of such a marker can be frustrating for the par-
to the placenta. Mosaicism is classified as follows: ents, as there is a lack of substantial information about
• Level I mosaicism is defined as a single abnormal cell. many of these. The limitations of prenatal ultrasound in
This almost always represents a cultural artifact and, in identifying fetal abnormalities can often compound this
the vast majority of cases, is of no clinical significance to frustration. The risk for abnormalities in the light of a
the pregnancy [18]. marker chromosome can depend on the amount of euchro-
• Level II mosaicism is defined as more than one cell with matin present, whether the origin of the marker is an acro-
the same chromosome abnormality in one colony. This centric or nonacrocentric chromosome, whether the marker
type of mosaicism is, in the majority of cases, pseudomo- is familial or de novo, and, if familial, whether the marker
saicism, which is the result of cultural artifact [18]. It is is found in a mosaic state in the carrier parent [60]. One
important to note that cultural artifact does not mean source quotes a 10.9% risk for abnormality associated with
a de novo satellited marker and a 14.7% risk for a de novo larger than 1,000 bases up to several megabases) of uncertain
non-satellited marker [55]. clinical significance is identified [62]. This rate applies to
Certain supernumerary chromosomes are, however, asso- targeted arrays, in which the study is designed to analyze
ciated with well-defined clinical features. For example, an chromosome abnormalities associated with known genetic
isochromosome for the short arm of chromosome 12 [i(12p)] syndromes. Genome-wide arrays are, understandably,
causes Pallister-Killian syndrome, which is associated with expected to identify a higher rate of copy number variants of
profound mental retardation, seizures, characteristic facial unknown significance, but can also identify novel pathogenic
features, and pigmentary abnormalities. Cat-eye syndrome, variants. While the analysis of parental specimens can assist
which is usually caused by a marker that results in tetrasomy in the characterization of these variants, as most inherited
22q11.2, can be highly variable and can cause mental retar- copy number variants are benign, such testing is not always
dation, as well as abnormalities involving the eyes, heart, and informative [62, 63, 66]. Additional limitations of array tech-
urogenital system. Additionally, the “inverted duplicated 15” nology include the inability to detect abnormalities such as
[inv dup(15)] can be associated with varying features, rang- balanced rearrangements, ploidies (i.e., some cases of trip-
ing from mental retardation and clinical features of Prader- loidy), low-level mosaicism, and single gene mutations.
Willi/Angelman syndrome to a normal phenotype [60]. See Uniparental disomy cannot be detected with array CGH, but
Chaps. 8 and 9. can be identified via SNP array. Furthermore, the cost of the
array, which is not always covered by insurance, can be pro-
hibitive for some patients [61–63].
Microarrays The American College of Medical Genetics and Genomics
(ACMG) recommends that microarrays be used as an adjunct
Interest in the postnatal and prenatal utility of microarrays to standard karyotyping and FISH in the evaluation of indi-
(array comparative genomic hybridization [array CGH] or viduals with mental retardation and/or congenital anomalies
single nucleotide polymorphism [SNP] arrays) has increased [61]. The role of arrays in the prenatal cytogenetics setting is,
substantially of late. This technology has many advantages as yet, not well defined. Both the American College of
over conventional karyotyping, including a higher resolution, Obstetricians and Gynecologists (ACOG) and ACMG recom-
a faster turnaround time, and the fact that it does not require mend against the employment of microarrays for routine pre-
dividing cells [61, 62]. Particularly in the postnatal evaluation natal diagnosis. However, both societies state that targeted
of individuals with congenital anomalies and/or unexplained arrays can be offered as an adjunct to conventional karyo-
mental disability, these advantages make microarrays an typing in the evaluation of fetuses with structural abnormali-
important adjunct to conventional karyotyping [61]. The abil- ties and a normal karyotype [61, 62]. Particularly given the
ity to simultaneously analyze multiple loci dismisses the need limitations of the analysis and the potential for ambiguous
for multiple FISH assays [63]. Furthermore, the lack of a need results, genetic counseling is a critical component of array
for dividing cells makes the microarray an important option in analysis. In fact, to this end, ACOG recommends both pretest
the evaluation of congenitally abnormal fetal demises for and posttest genetic counseling [62].
which a conventional karyotype result could not be obtained Microarray technology is covered in detail in Chap. 18.
[62]. Array technology has proven a useful tool in defining or
redefining the causative genetic mechanism in some genetic
syndromes sometimes leading to the identification of new Summary
syndromes. Studies have shown that arrays are capable of
detecting a causative genomic imbalance in up to 10% of indi- Genetic counseling is a complex, fascinating, and continu-
viduals with unexplained mental retardation and a normal ously evolving field. With the current focus of science and
conventional karyotype [64]. Microarrays can also assist in popular culture on genetics, genetic counseling is becoming
the characterization of a chromosome rearrangement or increasingly important in medicine. As stated in the begin-
marker chromosome identified via standard chromosome ning of this chapter, genetic counselors are increasingly
analysis [61, 63]. Based upon a small data set, microarrays found in a wide variety of settings in clinical, research, and
appear to identify a chromosome abnormality in 5–10% of administrative roles. Furthermore, genetic counselors can
fetuses with multiple anatomic abnormalities and a normal contribute significantly, not just in the setting of prenatal
standard karyotype via CVS or amniocentesis [65]. However, genetics, but also in the pediatric and adult arenas.
larger studies are needed to better define the utility of array Counselors not only play a vital role in explaining genetic
technology in the prenatal setting. concepts, recurrence risks, and genetic testing in understand-
Microarrays can yield results that are difficult to interpret. able terms, but also in helping individuals anticipate and
In an estimated 12–15% of prenatal samples, a copy number cope with the psychosocial consequences that can be associ-
variant (i.e., a deletion or duplication of a DNA segment ated with the diagnosis of a genetic condition. Although
512 S.H. Clark
seemingly straightforward, these can be challenging 14. American College of Obstetricians and Gynecologists. Invasive
tasks, particularly when ambiguous test results, cultural dif- prenatal testing for aneuploidy. ACOG practice bulletin number 88.
Obstet Gynecol. 2007;110(6):1459–67.
ferences, and/or mental handicaps are involved. The unique 15. American College of Medical Genetics. Multiple marker screening
training that genetic counselors receive makes them especially in women 35 and older. ACMG Policy Statement; 1994.
well-suited to tackle such challenges. 16. American College of Medical Genetics. Screening for fetal aneu-
Ethics and genetics are closely intertwined, as genetic ploidy and neural tube defects. ACMG practice guideline. Genet
Med. 2009;11(11):818–21.
counselors continuously encounter a variety of situations 17. American College of Medical Genetics. Statement on guidance for
where ethical principles and guidelines must be consulted and genetic counseling in advanced paternal age. ACMG practice
followed. These situations range from the fairly routine to the guideline. Genet Med. 2008;10(6):457–60.
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contribution to human life and disease. The public, govern- uation and counseling for infertile couples. ASRM and SREI
Practice Committee Report; 2002.
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of increasingly complex ethical dilemmas, particularly in the issues for primary care physicians: down syndrome-an update and
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Index
A ACMG. See American College of Medical Genetics and Genomics

Abbreviations, ISCN (ACMG)
common cytogenetic, t29–t30 aCML. See Atypical chronic myeloid leukemia (aCML)
and symbols used in molecular cytogenetics, t43 ACOG. See American College of Obstetrics and Gynecology (ACOG)
ABC. See Aneurysmal bone cyst (ABC) Acrocentric, 13, 14, 17, 23, t25, 26, 36, 49, 61, t61, 62, 129, 141, 146,
Abdominal wall defect, 477, 508, 509 163–165, 168, 198, t311, 416, 446, 510
Abelson (ABL1) oncogene, 424 Acute B-cell lymphoblastic leukemia/lymphoma (B-ALL/LBL)
ABGC. See American Board of Genetic Counseling (ABGC) chromosome abnormalities, 327, t327, 328
ABL1, 44, 317, 328, 334, 371, 423–425, t423, 502 del(9)(p21.3), t327
ABL2, t321 FISH, 327–329, 331–333
ABL1-BCR, t327 hyperdiploidy, 330–331
ABL1-ETV6, t321 hypodiploidy, 329–330
ABL2-ETV6, t321 Philadelphia chromosome, 328
ABMG. See American Board of Medical Genetics (ABMG) rearrangements
ABN. See Advanced beneficiary notification (ABN) BCR-ABL1, 328
Abnormality CDKN2A, 332
acquired, 31, 40, 41, 44, 83, 84, 121, 310, 374 ETV6-RUNX1, 331, 332
autosomal, 5, 28, 177, 218–220, 223, 224, 263 IGH@, 333
clonal, 41, t48, 84, 335 MLL, 328–329, f 329
numerical, t28, 31, 32, 41, 85, 124, 177, 223, 237, 277, t311, 330, MLL-AFF1, 328
331, 335, 341, 355, 421, 422, 434 MLL-MLLT1, 329
order of in nomenclature, 26 9p, 332, f 333
sex chromosome, 28, 41, 177–187, 213, 218, 219, t221, 221–224, PBX1-TCF3, t327
256, 422–423, 506, 509 11q23, 328, f 329
structural, 5, 31, 32, 56, 58, 62, 84, 113, 126, 142, 157, 179, 180, 14q32.3, 333
188–199, 218, t221, 262, 263, 277, 278, 280, 335, 341, recurrent chromosome abnormalities and involved genes, t327
347, 348, 355, 384, 385, 422, 511 RUNX1 amplification, t327
Abnormal prenatal screen, 503, 508–509 t(1;19)(q23.3;p13.3), t327, 329
Abortion t(4;11)(q21.3;q23), 328
chromosomal abnormalities, 178, 231, t233, 277, t277, 284, 285 t(9;11)(p22;q23), 329
incidence, 231, 256 t(9;22)(q34;q11.2), t327, 328, f 328
induced, 230 t(11;19)(q23;p13.3), 328–329
recurrence rate, 396 Acute myeloid leukemia (AML)
recurrent, 275, 279, 284–289, 504, 507 ambiguous lineage, 326–327
spontaneous. See Fetal loss; Pregnancy loss; Spontaneous abortion; basophilic, 323
Spontaneous loss; Spontaneous pregnany loss blastic plasmacytoid dendritic cell neoplasm, 326
ACC. See Adrenal cortical carcinomas (ACC) classification, t323
Accreditation definition, 322
cytogenetics education programs, 92 erythroid, t323
genetic counseling programs, 499 FISH, 327–329, 331, 423, t423
ace. See Acentric fragments (ace) with gene mutations, 326
Acentric chromosomes, 155–158, f 155, 163, 220, t311. CEBPA, t323, 326
See also Acentric fragments (ace) FLT3, t323, 326
Acentric fragment (ace), t48 KIT, t323, 326
Acetic acid, glacial, 58, 97 NPM1, t323, 326
Acetylcholinesterase (AChE), 56, 64, 235 WT1, t323, 326
aCGH. See Array-based comparative genomic hybridization (aCGH) with inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2), 325
AChE. See Acetylcholinesterase (AChE) with inv(16)(p13.1q22.1) or t(16;16)(p13.1;q22.1), 323–324, t323
Achondroplasia, 259, 503 with maturation, 322, t323
S.L. Gersen and M.B. Keagle (eds.), The Principles of Clinical Cytogenetics: Third Edition, 515
DOI 10.1007/978-1-4419-1688-4, © Springer Science+Business Media New York 2013
516 Index
Acute myeloid leukemia (AML) (cont.) Advanced paternal age, 259–260, 503
megakaryoblastic with t(1;22), t323, 325–326 AFH. See Angiomatoid fibrous histiocytoma (AFH)
with minimal differentiation, t323 AFP. See Alpha-fetoprotein (AFP)
monoblastic/monocytic, t323 Age
with myelodysplastic-related changes, t323 maternal, 114–117, t118, 119, 120, 122, 125, 127, 153, 160,
myeloid proliferation related to Down syndrome, t323 177, 182, 184, 186, 230, 231, t232, 233, 234, 237,
myeloid sarcoma, t323 238, 240–242, 244–246, f 245, 251–253, t255, 257,
myelomonocytic, 323, t323, 324 264, 278, 280–283, f 280, 289, 421, 486, 503, t503,
not otherwise specified, t323 507–508
panmyelosis with myelofibrosis, t323 paternal, 184, 259–260, 503
with recurrent gene rearrangements, 322, t323, 326 AGT. See Association of Genetic Technologists (AGT)
with recurrent genetic abnormalities AHO. See Albright hereditary osteodystrophy (AHO)
with CEBPA mutation, t323 AIRE, t216
with FLT3 mutation, t323 AITL. See Angioimmunoblastic T-cell lymphoma (AITL)
with inv(3) or t(3;3), t323 Alagille syndrome, t148
inv(3)(q21.3q26.2), 315, 322, t323 Albright hereditary osteodystrophy (AHO), 476
with inv(16) or t(16;16), t323 ALCL. See Anaplastic large cell lymphoma (ALCL)
inv(16)(p13.1q22.1) t323, 323–324 ALCL-ALK+. See Anaplastic large cell lymphoma, ALK-positive
with KIT mutation, t323 (ALCL-ALK+)
(megakaryoblastic) with t(1;22), t323 ALCL-ALK-. See Anaplastic large cell lymphoma, ALK-negative
with MLL (11q23) translocations, f 325 (ALCL-ALK-)
with NPM1 mutation, t323 ALK-CLTC, t391, 396
t(1;22)(p13;q13), t323 ALK-RANBP2, t391, 396
t(1;22)(p13.3;q13.1), 325–326 ALK-TPM4, t391, 396
t(3;3)(q21.3;q26.2), 315, 322, t323 Alkylating agents, 312, 313, 343
with t(6;9) ), t323 All-trans retinoic acid (ATRA), 324, 371, 423
t(6;9)(p23;q34.1), t323, 325 Alpha-fetoprotein (AFP)
with t(8;21), t323 in amniotic fluid, 56
t(8;21)(q22;q22.3), 322, t323 and Beckwith-Wiedemann syndrome, 386
with t(9;11), t323 and chromosoamla abnormalities, 256
t(9;11)(p22;q23), 322, t323, 324–325 elevated and
with t(9;11)(p22;q23) and oher tanslocations Involving MLL, paternal triploids, 283
324, f 235 trisomy 16, 262
with t(15;17), t323 elevated coexisting, and hyperechogenic fetal bowel, 253
t(15;17)(q24.1;q21.2), 322, t323 and hemihyperplasia, 193
t(16;16)(p13.1;q22.1), t323, 323–324 and hepatoblastoma, 384–386, t385, f 386
with WT1 mutation, t323 high maternal serum, 233, 256
with t(6;9)(p23;q34.1), 325 low levels, and
with t(9;11)(p22;q23) and other translocations involving MLL, 324 maternal triploids, 283
therapy-related myeloid neoplasms, t323 multiple marker screening, 256
treatment-related (t-AML), 324, 326 low levels, associated with
without maturation, t323 trisomy 18, 129
Acute promyelocytic leukemia (APL) with t(15;17)(q24;q21.1), 324 trisomy 21, 253
Acute T-cell lymphoblastic leukemia (T-cell ALL/LBL), maternal serum, 262
333–334, t334 and neural tube defects, 256
Acute undifferentiated leukemia (AUL), 326–327 and quadruple marker screening, 256–257
add. See Additional material of unknown origin (add) and triple marker screening, 256
add(2)(q23), f 386 Alpha-satellite
add(2)(q31), f 386 DNA, 14, 37, 60, 129, 157, 300, 415
add(3)(p21), f 386 FISH probe results, t420
add(6)(q23), f 386 probes, 416, 420, 421, f 420
add(14)(q13), f 386 for amplification of ERBB2 (HER2), 430, f 435
add(16)(p13.3), f 394 for baldder cancer screening, 431, f 435
Additional material of unknown origin (add) for pimed in situ labeling (PRINS), 434
definition, t48 Alpha-thalassemia/mental retardation (ATRX), 201
description, t29 Alternate segregation, 219
long ISCN, t29 Alu repeats, 141
nomenclature, 37 Alu sequences, 14, 139, 140
short ISCN, t29 Alveolar rhabdomyosarcoma (RMS), 397–398, t398, f 399
Adjacent segregant, 161 Alveolar soft part sarcoma (ASPS), t379, t391, 395
Adrenal cortical carcinomas (ACC), t398, 403 Amenorrhea. See also Premature ovarian failure
Adult T-cell leukemia/lymphoma (ATLL), 353–354, f 354 and balanced translocations, 188–191
Advanced beneficiary notification (ABN), 78 and female infertility, 213
Advanced maternal age, 114, 117, 122, 125, 153, 160, 231, 234, 237, primary
238, 242, 244, 245, f 245, 251, 264, 421, 486, 503, and FSHR mutation, t216
t503, 507–508 and 46,XY karyotype, 315
Index 517
secondary MAP3K5, 393

in unbalanced translocations, 190 MDM2, t391, 393
in 45,X and 45,X mosaicism, 213 MUC1, 350
and X chromosome deletions, 215 MYC, t373, 376, 378
and 45,X and 45,X mosaicism, 213 MYCN, 371, t373, 376, 378, t398, 400, 401
and Xp duplications, 191 12p, t379, 383
and (X;Y) translocations, 195 RUNX1, t327, 332
American Board of Genetic Counseling (ABGC), 499 TSPAN31, t391, 392
American Board of Medical Genetics (ABMG), 92 Amplified gene sequences (amp) in nomenclature, t43
American College of Medical Genetics and Genomics Analysis worksheets, 80, 82, 86, 90
(ACMG), 77, 82, 84–86, 91, 164, 259, 260, 264, Anaphase
415, 421, 432, 464, 511 meiosis I, 19, f 19
American College of Obstetrics and Gynecology (ACOG), 240, meiosis II, 20, f 20
445, 509 mitosis, 18, f 18
American Society for Clinical Pathology (ASCP), 92 Anaplastic large cell lymphoma (ALCL)
American Society for Clinical Pathology Board of Certification ALK-negavive (ALCL-ALK-), 356
(ASCP BOC), 92 ALK-positive (ALCL-ALK+), 353, 356
American Society of Clinical Oncology (ASCO), 390 Anaplastic lymphoma kinase gene (ALK), 356
American Society of Human Genetics (ASHG), 421, 499 Anaplastic oligodendrogliomas, 375
(+) Amethopterin, 62 Androgen insensitivity syndrome, 201, f 214, t217
AML. See Acute myeloid leukemia (AML) Androgen receptor gene (AR), 184, 201, t217
Amniocentesis Aneuploidy. See also Autosomal aneuploidy; Monosomy; Tetrasomy;
accuracy of, 233 Trisomy
conventional, 233–235, t236, 238 definition, 113
early, comparison with CVS, 236–238 incidence of
and erythroblastosis fetalis, 229 autosomal trisomies, 117
fetal loss with, 233–238, t236 newborn, 113
genetic, 232–233, t232 oöcyte, 113
history of, 229 sperm, 113
incidence of chromosomal abnormalities spontaneous abortion, 113
autosomal trisomies, 117 interchange, f 160
de novo balanced structural rearrangements, 231, t232, t233 and maternal age, 114–117, f 118, 119, 120, 122, 125, 127
sex chromosome abnormalities, t231 monosomy, 124
structural balanced arrangements, t231 parental origin, 125, 126
structural unbalanced arrangements, t231 sex chromosome, 125, 126
risks of, 233 tertiary, f 160
timing, 229 tetrasomy, 127–129, f 128
Amniocytes, 54, 56, 63, 123, 127, 129, 198, 242, t260, 261, t263, trisomy, t114, 116–123, f 118, t118
286, 454, 508 Aneurysmal bone cyst (ABC), t391, 395, f 396
Amniotic fluid AneuVysion, 421, f 422
collection and handling, 54 Angelman syndrome (AS)
and culture failure, 263 clinical features, 417, t418
flask method of culture, 83 etiology, t477
in situ method of culture, 55 genetic counseling, 505
and maternal cell contamination (MCC), 263 and genomic imprinting, 474, 476–477
volume per week gestation, 254 laboratory diagnostic approach, f 478
amp. See Amplified gene sequences (amp) phenotype, 476
Amplification of recurrence risk, t477
ABL1, 334 Angioimmunoblastic T-cell lymphoma (AITL), t353, 356
ARNT, 350 Angiomatoid fibrous histiocytoma (AFH), 396
BCL9, 350 Angle brackets (< >) in nomenclature, 32
CCND1, 384 Antibiotics, 54–55, 61, 310
CDK4, 393 Anticodon, 11
CDK6, 378 Anti-Müllerian hormone, 217
DDIT3, f 394 Anus, imperforate, 118, 224, 236, 262, 487
EGFR, 374, 384 Aperture diaphragm, 69
ERBB2, 376, t385, 387, 390, 430, f 430 APL. See Acute promyelocytic leukemia (APL) with t(15;17)
ERG, 382 (q24;q21.1)
GLI1, 393 Approximate sign (~) in nomenclature, t28, 39
HER2 (see ERBB2) AR. See Androgen receptor gene (AR)
HGMA2, 387 Archival device, 102
IGF1R, f 398 Argininosuccinate synthetase (ASS1), 424, f 425
JUN, 393 ARNT, 350
KIT, 384 arr. See Microarray or results from microarray testing (arr)
KIT/PDGFRA, 384 Array-based comparative genomic hybridization (aCGH), t43
MAD2L1, t391, 394 Array CGH, 86, 128–130, 264, 310, 383, 404, 511
518 Index
Arrow (®) in nomenclature, t28 tetrasomy 12p, 127–129, f 128

ARSE, 194, 195 tetrasomy 18p, 129
ART. See Assisted reproductive technology (ART) trisomy 8, 121, f 121
ARX, 201 trisomy 9, 121–122
AS. See Angelman syndrome (AS) trisomy 13, 120–121
ASCO. See American Society of Clinical Oncology (ASCO) trisomy 16, 122
ASCP. See American Society for Clinical Pathology (ASCP) trisomy 18
ASCP BOC. See American Society for Clinical Pathology Board of incidence, 119
Certification (ASCP BOC) phenotype, 119–120, f 120
Asherman syndrome, f 214 recurrence, 120
ASHG. See American Society of Human Genetics (ASHG) trisomy 20, 122
ASPS. See Alveolar soft part sarcoma (ASPS) trisomy 21
ASPSCR1, 395 incidence, 117, f 118, t118
ASPSCR1-TFE3, t379, 380, t391, 395 phenotype, 117–119, f 119
ASS1. See Argininosuccinate synthetase (ASS1) recurrence, 119
Assisted reproductive technology (ART), 213, 222, 224, 225, trisomy 22, 122–123
478–480 Autosomal dominant inheritance, 201, t217, 502, 505
and imprinting disorders, 478–479 Autosomal recessive inheritance, 200, t216, t217, 501,
Astrocytomas, 372, t373, 374, f 374, 375 502, 506
ASXL1, 321 Autosomal trisomy
Asynchronous replication, 300, 476 frequency in aborted specimens, t234
Ataxia telangiectasia (A-T), 6, 14, 38, 59–61, 296–299, f 298, outcome of mosaicism diagnosed in amniocytes, t260
343, 506 parental and meiotic/mitotic origin, t114
Ataxia-telangiectasia-mutated (ATM), associated risk of breast trisomy 8, 121, f 121
cancer, 297 trisomy 9, 121–122
ATF1-EWSR1, t391, 396, t398, 403 trisomy 13, 120–121
ATF1-FUS, t391, 396 trisomy 16, 122
ATLL. See Adult T-cell leukemia/lymphoma (ATLL) trisomy 18
ATP6E, 130 incidence, 119
ATP6V1E1, t420 phenotype, 119–120, f 120
ATRA. See All-trans retinoic acid (ATRA) recurrence, 120
AT/RT. See Atypical teratoid/rhabdoid tumor (AT/RT) trisomy 20, 122
ATRX. See Alpha-thalassemia/mental retardation (ATRX) trisomy 21
Atypical chronic myeloid leukemia (aCML), 321, t321, 322, t322 incidence, 117, f 118, t118
Atypical teratoid/rhabdoid tumor (AT/RT), 371, t373, 378 phenotype, 117–119, f 119
Autofluorescence, 75, 76 recurrence, 119
Automated imaging systems trisomy 22, 122–123
application, 102–107, f 102–f 108 AZF. See Azoospermia factor (AZF)
archival device, 102 Azoospermia, in infertile men, 218
CGH, 100, 106–107, f 108 Azoospermia factor (AZF)
components, 101, f 101 AZFa, 177, 197, 220, 222, t222
fluorescent spot counting, 103–105, f 105 AZFb, 177, 197, 220, 222, t222
karyotyping, 101–103, f 102, 105, 106 AZFc, 177, 197, 220, 222, t222
limitations, 101 gene regions and usual phenotypes, t222
M-FISH, 105–106, f 107 Azoospermia, nonobstructive, 218–219
microscope with camera adapter, 102
printer, 102
scanning and metaphase finding, 103, f 103, f 104 B
software, 102 Bacterial artificial chromosomes (BACs), 45, 264, 416, 441,
Autosomal aneuploidy t443, t444
mechanism and etiology Balanced rearrangements, involving three or more chromosomes,
chromosome glue, 116 nomenclature of, 37
limited oöcyte pool model, 115 Balanced translocation(s), 144, f 145, 159–161, 285, t312, t501, 510.
maternal age, 115–117, f 118, 119, 120, 122, 125, 127 See also Reciprocal translocation(s)
meiosis II nondisjunction, f 116 description, t30
meiosis I nondisjunction, f 115 in hematologic neoplasms, 312
oöcytes, 113–117 nomenclature of, 37
parental and meiotic/mitotic origin, t114 B-ALL/LBL. See Acute B-cell lymphoblastic leukemia/lymphoma
production line hypothesis, 115 (B-ALL/LBL)
monosomy 21, 124 Band
monosomy 22, 124 definition, 24
mosaic trisomy, 119–124, f 121, 127 designation
nonmosaic trisomy, 117, 119, 121, 122 breakpoint, 42
supernumerary marker chromosomes, 129–130 centromere, 26, t26, 38
tetrasomy 5p, 127 region, 39
tetrasomy 8p, 127 sub-band, t26, 43
Index 519
Banding osteosarcoma, t391, 395

chromosome, f 6, 24–26, t25, f 26, 58, 95, f 108, 109, 146, 151, synovial sarcomas, t391, 392, f 392
154, 441 Bone marrow
resolution, 25, 26, 33, 58, 64, 65, 82, 146 abnormal proliferation of myelocytes and monocytes of,
technique, 14, 17, 24, 25, 37, 42, 58–61, 129, 151, 310 in JMML, 322
Barr body, 4, 176 aspirate(s)/aspiration 5, 53–54, 84, 90, 97, 309, 417 (see also Bone
Barr, Murray, 4, 176 marrow sample(s); Bone marrow tap(s))
B-cell lymphoma based LPL, 342
diffuse large, 335–338, t335, f 337 cell(s), 126, 127, f 310
nodal marginal zone, 341–342 culture(s), 54–56, 62, 372
splenic marginal zone, 341 failure, in Fanconi anemia, 296, 448
unclassifiable, 316 harvest, robotic processors for, 96
B-cell prolymphocytic leukemia (B-PLL), t335, 340, 345, t346 hypercellular, in myelodysplastic/myeloproliferative
BCL2, 335, 336, 338, f 339, 341, 343, 428 neoplasms, 321
BCL3, 343 plasma cells in, PCM, 346
BCL6, 336–338, 352 preparations
BCL8, 338 X-chromosome aneuploidy in, 187
BCL9, 350 Y-chromosome loss in, 188
BCL10, 341 presence of myeloblasts, in AML, 322
BCL11A, 343 sample(s), 53, 55, 56, 63, 64 (see also Bone marrow aspirate(s);
BCL11B, 334 Bone marrow tap(s))
BCL2 overexpression, 335, 343 sex chromosome loss in, of patients with hematologic
BCR, 317, 328, 334, 371, t423, 424, f 425, f 427, 502 neoplasms, 315
BCR-ABL1, 44, 317, 319–322, 328, 334, 371, 424, 425, f 425 specimen(s), 54, 64, 84, 89, 143 (see also Bone marrow
Beam splitter, 71, 76 sample(s))
Beckwith-Wiedemann syndrome (BWS) tap(s), 54, 63 (see also Bone marrow aspirate(s))
association with assisted reproductive technology, 479 transplant(s)/transplantation (see also Stem cell transplant(s)/
clinical features, 478 transplantation) nomenclature of, 39, 45
and imprinting, 478–479 opposite sex, t429
mechanisms, 477 reporting mosaicism, chimerism, and chimerism
Bednar tumor (BT), 388 secondary to, t28
Benchtop drying chamber, f 97 sex-mismatched, 428–429
Beta satellite probes, 416 for treatment of B-CLL, 328
Biochemistry, ultrasound, nuchal translucency (BUN) for treatment of Fanconi anemia, 297
study, 257 B-PLL. See B-cell prolymphocytic leukemia (B-PLL)
Biotin, 415, 417 Brackets
BIRC3, 340, 341 Angle (< >) in nomenclature, t28, 32
BIRC3-MALT1, 340, 341 square ([ ]) in nomenclature, t28, 30, 31
Bivalents, 3, 19, 114, 116, 144, 160, 166, 167, 219, 220, 261 BRAF, tandem duplication, t373
BL. See Burkitt lymphoma (BL) BRAF V600E, 346
Bladder cancer, UroVysion, 431, f 431. See also Urothelial cancer Brain tumor. See also Central nervous system (CNS) tumors
Blastic plasmacytoid dendritic cell neoplasm, t323, 326 FISH, 431
BLM, 302 1p/19q loss in, 431
Bloom syndrome (BS) BRCA1, 296, 390
BLM, 302 BRCA2, 296, 390. See also FANCD1
clinical features, 302 BrdU. See Bromodeoxyuridine (BrdU)
definition, 302 Break-apart FISH probes, nomenclature, 44–45
incidence, 302 Breakpoint, definition, t30
sister chromatid exchange, 302, f 302 Breast cancer/carcinoma, 389–390
b-MCL. See Mantle cell lymphoma, blastoid type (b-MCL) in ataxia telangiectasia, 298
BOC. See American Society for Clinical Pathology Board and ERBB2, 430, t430
of Certification (ASCP BOC) FISH for ERBB2 analysis, 99
Bone and soft tissue tumors Invasive, 390
alveolar soft part sarcoma, t391, 395 intraductal, 390
aneurysmal bone cyst, t391, 395, f 396 metastatic, 390
angiomatoid fibrous histiocytoma, t391, 396, f 397 secretory (SBC), t379, 390
congenital fibrosarcoma/infantile fibrosarcoma, t379, susceptibility and BRCA2, 296
390–392, t391 Brightfield microscopy
extraskeletal myxoid chondrosarcoma, t391, 395 beam splitter, 71
inflammatory myofibroblastic tumor, t391, 396–397 coordinate location, 71–72
leiomyoma, t391, 394 coverglass, 71
leiomyosarcoma, t391, 394–395 coverslips, 72
lipoblastoma, t391, 393 differential interference contrast, 73–74, f 74
lipoma, t391, 393 eyepieces, 71
liposarcoma, t391, 393–394, f 394 microscope slides, 72
malignant rhabdoid tumors, t391, 397 microscope stage, 71
520 Index
Brightfield microscopy (cont.) CDK4, t391, 393, f 394

mounting media, 72 CDK6, t373, 378
objective lens CDKN2A, t327, 332, 334, 373–375, t373, t379, 382, 384, t385, 389,
correction collars, 71 390, t398
coverglass, 71 CDK2NA, t334
immersion objectives, 70–71 CDKN1C, 478–480
immersion oil, 70–71 Cd staining. See Centromeric dot/kinetochore staining (Cd staining)
objective types, 70 Cell cycle
optical and conjugate focal planes errors of, 16
aperture diaphragm, 69 meiosis, 18–21
condenser, 69 mitosis, 17–18
field, 69 Cell division, 9–21, 53, 55, 62, 116, 139, 155, 156, 158–160, f 158,
Köhler illumination, 67–68, f 68 165, 283, 309
transmitted light source, 68–69 Cell harvest, t53, 57
phase contrast, 72–73, f 73 Cell preservation, 63
resolution vs. magnification, 70 Cell synchronization, 62
Bromodeoxyuridine (BrdU), t25, 62, 176, 294, t295 CEL, NOS. See Chronic eosinophilic leukemia, not otherwise
BS. See Bloom syndrome (BS) specified (CEL, NOS)
BT. See Bednar tumor (BT) cen. See Centromeres (cen)
Buffy coat, 56 Central nervous system (CNS) tumors
BUN study. See Biochemistry, ultrasound, nuchal translucency astrocytomas, 372–375
(BUN) study choroid plexus, t373, 376, t377
Burkitt lymphoma (BL), 335, t335, 336, t336, 338, f 338, 345, embryonal, t373, 376–378
428, 502 ependymoma, 372, 373, 375–376
BWS. See Beckwith-Wiedemann syndrome (BWS) gliomas, 372
meningioma, t373, 378
oligoastrocytomas, 375
C oligodendroglioma, 372–375, t373
c. See Constitutional anomaly (c) Centromere (cen)
Ca-ex-PA. See Carcinoma ex pleomorphic adenoma (Ca-ex-PA) ancient sequences, 157
Camera adapter, 101, 102 definition, 12, t48
Canadian Society of Medical Laboratory Science (CSMLS), 92 designation, 26
Cancer karyotypes fusion, 13
clone, 40 idieogram, 26
composite karyotype, 41 inactive, 36
describing, 40 instability, 299
mainline (ml), 40–41 location, 142
sidelines (sdl), 40–41 misdivision, f 158
stemline (sl), 40–41 neocentromeres, 37, t49, 156–158
unrelated clones, 41 nomenclature, 13, t48
CAP. See College of American Pathologists (CAP) position, 5, f 24, 58, 151
Carcinoma ex pleomorphic adenoma (Ca-ex-PA), 385, 387 premature division/separation of, 115, 116, 187, 300
Cardiovascular abnormalities probes, 45, 181, f 427, f 430, f 435
in children with, t249 splitting, 300
in William syndrome, 157 Centromeric dot/kinetochore staining (Cd staining), 61
Cardiovascular defects in William syndrome, 157 CFS. See Congenital fibrosarcoma (CFS)
Carrier testing, indications for, 465 CGG repeat, 455–465, f 457, f 459
Caspersson, Torbjörn, 6 cgh. See Comparative genomic hybridization (cgh)
Cat-cry syndrome, 5, 146. See also Cri du chat syndrome CGH. See Comparative genomic hybridization (CGH)
Cat-eye syndrome, 130, t150, t420, 511 Charcot-Marie-Tooth type 1A (CMT1A), 147, 148, t150, 151, 418
C-bands/banding (constitutive heterochromatin banding), 60 Chargaff, Erwin, 9
CB-banding, t25 Chargaff’s rule, 9, 10
CBFB. See Core-binding factor b (beta) (CBFB) CHD11-COL1A1, t391
CBG-bands/banding, t25, 60 chi. See Chimera (chi)
CBL, 319, 321 Chiasma/chiasmata (xma), 19, 115–117
CCAAT, 326, 333 definition, t48
CCAAT/enhancer-binding protein-a (CEBPA), 321, 323, 326 nomenclature, t48
CCND1, t336, 338, f 340, 350, 384, 423, 428 CHIC2, 320, 428
CCND2, t334, t336, f 340 Children’s Oncology Group (COG), 327, 425
CCND3, t336, f 340, 350 Chimera (chi)
CCND1-IGH@, 350 definition, t48
CCND2-TRA@, t334 nomenclature, t48, 282
CCR. See Complex chromosome rearrangements (CCR) in ovotesticular disorders of sex development, 200–201
CCSK. See Clear cell sarcoma of the kidney (CCSK) Chimeric
CDH13, 403 fusion, 395, 396
CDH11-USP6, 395 gene, 402
Index 521
gene fusion, 395 Chromosomal abnormalities, 6, 44, 45, 84, 86, f 105, 219, 251, t276,
gene protein, 388 t277, 276–278, 280, 284, 288, 289, 313, 372, 381, 417,
transcript, 316–317 421, 423, 426, 441, 443, 506
transcritption factor, 392 additional material of unknown origin, t29, 37, 39, t48, 416
Chimerism and advanced maternal age, 153
and bone marrow transplants, t28 approximate sign (~) in nomenclature, t28, 39
constitutional, 39 balanced rearrangements, 37
vs. mosaicism, t28, 31, 39 chromosome breakage, 23, 38
reporting, t28, 39 clinical suspicion of, 504
reporting secondary to bone marrow stem cell deletions, 33
transplantation, t28, 39 derivative, t29, 32, 34–37, 39, 40, t43, t48, t49
spontaneous abortion, 282–283 dicentric, t25, t29, 36, 37
CHL. See Classic Hodgkin lymphoma (CHL) duplications, 126
Chloroma, 326 effect on preimplantation embryo development, 223–224
Chondrodysplasia, 191, 194, 195 family history of, 500–502
CHOP, 429. See also DDIT3 fragile sites, 38, t48
Choriocarcinoma, 283, 383 frequency in stillbirths and neonatal deaths, t234
Chorionic gonadotropin, human (hCG), 122, 256, 262, 275, 282–284, incidence of, 230–231, 246, 288
508, 509 incomplete karyotypes, 38, t48
Chorionic villus sampling (CVS) and infertility, 184
analysis protocols, 82 insertions, 34
benefits, 238 inversions, 33
cultured preparations, 243 isochromosomes, 35
diagnosis, 238 isoderivative chromosomes, 36
direct preparations, 243 isodicentric, 36, 129, 193–194
vs. early amniocentesis, 235 in liveborn babies, 224
fetal loss, 240–241 marker chromosomes, 37–38
genetic counseling, 240, 257 maternal age-specific rates for, t232
heterochromatin decondensation, 240 mosaicism and chimerism, 39
history, 238–239 multiple copies of rearranged chromosomes, 38
limb reduction, 238–239 nomenclature of, 23–49, f 24, t25, f 26–f 27, t28–t30, f 31, t43,
limitations, 239 t48–t49
maternal age, 240 prenatal identification of, 509–511
maternal cell contamination, 243 previous child with, 257–258
microarrays, use of, 264 previous pregnancy with, 257–258
mosaicism, 241–242, 260–262 risk for, 231, 245, 253, 508
pseudomosaicism, t260, 260–262 of sex chromosomes, 177
risks associated with, 238–240 spontaneous abortion, 278
specimen requirements, 243 Chromosomal syndromes, 4, 5, 443
transabdominal, 237, t241, 241 Chromosome (chr), t48
transabdominal vs. transcervical, t241, 241 Chromosome breakage, 5, 13, 23, 38, 86, 139, 142, 293, 296–298,
transcervical, 230, 239 f 297, 303
turnaround time, 238–240 Chromosome instability
Choroid plexus atypical papilloma, t373 ataxia telangiectasia (A-T)
Choroid plexus carcinoma (CPC), t373, 376, f 377 ATM, 297–299
Choroid plexus cysts (CPC), 252–253, t255, 256 cancer risk, 298
Choroid plexus papilloma (CPP), t373, 376, f 377 clinical findings, 294, 296
chr. See Chromosome (chr) immunoblotting, 298
Chromatid (cht) incidence, 297
definition, t48 spontaneous chromosome breakage, 298
nomenclature, t48 syndromes, 293, 296–304, f 297–f 303
Chromatid gap (chtg) Chromosome paint, t48, 416, 433, 435
definition, t48 Chromosome rearrangements/abnormalities
nomenclature, t48 acentric chromosomes, 156–157
Chromatin acentric fragment, t311
compaction, 294, 474, 476 definition, t311
constitutive heterochromatin, 15, t25, t48, t49, 60, deletion
61, 299 distal short arm, f 146, 159, 165
euchromatin, 14, 129, 130, t420, 510 high-resolution banding, 26, 33, 146
facultative heterochromatin, 15 interstitial, 146
heterochromatin, 14, 15, t25, 39, t48, t49, 60, f 61, 129, f 151, LCR sequences, 147, 149
152, 156, 157, 167, 168, 176, 199, 240, f 299, long arm, 39
300, 485 pathological significance, 146
structure of, 141 syndromes, 140, t148
X, 4, 230 (see also Barr body; Sex chromatin body) terminal, 146
Chromophobe carcinoma, renal (RCC) and oncocytoma, 381 Williams syndrome, 147
522 Index
Chromosome rearrangements/abnormalities (cont.) IGH@ abnormalities, 343–345, f 344

dicentric chromosomes, 155, f 156, 156, 164 non-IGH@ reciprocal translocations, 345
duplication prognostic markers, 345
direct, 149 recurrent chromosome abnormalities, 342
distal long arm, f 149, 162, 165, 167 trisomy 12, 343
inverted, 149 Chronic myelogenous leukemia (CML)
microdeletion-microduplication syndromes, 151 accelerated phase, 317
microduplication syndromes, 140, 147–149, t148–t149, 151 acute phase, 317
phenotypes associated, 147, 149, 151 BCR/ABL1, f 425
proximal long arm, 141, 147, 149, f 151, 152, 162, 165, 167 blast crisis, 315, 317, f 318, 325, 328
insertion chronic phase, 317, f 318
complex chromosome, 167 clonal evolution, 317
direct, 165 FISH, 317
incidence, 165 Philadelphia rearrangement, 328
interchromosomal, f 166, 166–167 recurrent chromosome abnormalities, 317
intrachromosomal, 156, 165, f 166, 166 t(9;22), 317
inverted, f 165, 165 Chronic myelomonocytic leukemia (CMML)
variant chromosomes, 167–168 FGFR1, 321
inversion PDGFRA, 321
mechanism, 151–152 PDGFRB, 321
paracentric, 154–155 PDGFRB-ETV6, 321
pericentric, 152 recurrent chromosome abnormalities, 321
isochromosome, 157, f 158 Chronic neutrophilic leukemia (CNL), 316, t317, 320
jumping translocation, 165 chromosome abnormalities, 322
mechanism of formation cht. See Chromatid (cht)
Alu/satellite DNA, 139, 140 chtg. See Chromatid gap (chtg)
balanced and unbalanced rearrangements, 142 CKS1B, f 427
de novo rearrangements, 143 Classical Hodgkin lymphoma (CHL), 338, 352
direct LCRs, 139, 140, 155 Classical satellite probe, 421
duplications and deletions, 155, 166–168 Clear cell carcinoma, renal (clear cell RCC)
familial rearrangements, 142–144 PBRM1, 380
NHEJ, 141, 147, 161 PTH1R, t379, 380
risk factors, 247 VHL, t379, 380
sporadic rearrangements, 139 Clear cell sarcoma of the kidney (CCSK), t379, 381, 383
prenatal cytogenetics, 230–231, t231 Client-centered approach, 499, 500
reciprocal translocations Clinical cytogenetics, history of
de novo mutations, 142, 161, 168 autosomal abnormalities, 5
long arm of chromosomes, f 161 chromosomal abnormality, 5
short arm of chromosomes, 159, f 160 “CLIA ‘88” (see Clinical Laboratory Improvement Amendments
(4;8) translocation, 140, 154, 161, 162 of 1988 (“CLIA ‘88”))
(8;22) translocation, 142, 161–163 human chromosome, 6
(11;22) translocation, 141, f 161, 161–163 hypotonic shock procedure, 3
reproductive risks for carriers, 164–165 phenotypic anomalies, 5
ring chromosome plant chromosomes, 6
carriers of, 159 sex chromosome configuration, 3
centromere lack, 158 Clinical findings amenable to ultrasound detection consistent with
instability, 158 22q11.2 deletion, t249
molecular studies, 158 Clinical Laboratory Improvement Amendments of 1988 (“CLIA ‘88”),
phenotype, 159 78, 91
Robertsonian translocations CLL/SLL. See Chronic lymphocytic leukemia/small lymphocytic
homologous, 142, 164 lymphoma (CLL/SLL)
nonhomologous, 163–164 Clonal aberrations
syndromes, 146–149, t148–t149 additional, 40
translocation, 165 related, 40
uniparental disomy, 144, 147, 164–165 stemline, 40
Chromosome variants, 168 Clonal evolution, 30, 40, 41, 310, 317, 343, 443, 447
Chronic eosinophilic leukemia, not otherwise specified (CEL, NOS) definition, t311
FIP1L1-PDGFRA, 320 Clonal rearrangements, 299
PDGFRA, 320 definition, 40
PDGFRB, 320 size, t28, 39–42
Chronic lymphocytic leukemia/small lymphocytic lymphoma subclone, 30, 40, 41, t48, 84
(CLL/SLL) unrelated, 40, 41, 84
del(6q)/MYB, 343 CML. See Chronic myelogenous leukemia (CML)
del(11q)/ATM, 343 CMML. See Chronic myelomonocytic leukemia (CMML)
del(13q), and BCL2 overexpression, 343 CMN. See Congenital mesoblastic nephroma (CMN)
del(17q)/TP53, 343 CMT1A. See Charcot-Marie-Tooth type 1A; Charcot-Marie-Tooth
FISH, 343 type 1A (CMT1A)
Index 523
CNBP-USP6, t391 Constitutive heterochromatin, 15, t48–t49, 61, 299

CNL. See Chronic neutrophilic leukemia (CNL) bands/banding (C-bands/banding), t25, 60
CNVs. See Copy number variants (CNVs) Contamination
COG. See Children’s oncology group (COG) bacterial, 55, 80
COL1A1-PDGFB, t385, 388, f 389 fungal, 55
Colcemid, 57, 62, 89, 372 microbial, 54, 56, 63, 93
Colchicine, 3, 4, 57 mycoplasma, 55
College of American Pathologists (CAP), 64, 91, 390 viral, 55
Colon, double (::) in nomenclature, t28 Contiguous gene syndromes, 146, 147, 190, 417
Colon, single (:) in nomenclature, t28 Contracting, 500
Colony of origin, 55 Coordinate location, 71, 72
Combined screening, 257 COPG2IT1, 484
Comma (,) in nomenclature, t28 Copy number variants (CNVs), 86, 168, 192, 264, 511
Comparative genomic hybridization (cgh), t43. See also Comparative Core-binding factor genes, 326
genomic hybridization (CGH); Array CGH Core-binding factor b (beta) (CBFB), 324, t423, 424
Comparative genomic hybridization (CGH). See also Array CGH Cornelia de Lange syndrome, 149
definition, t48 Cot-1 DNA, 416
epifluorescence microscopy, 74 Coverglass, 71
instrumentation, 106–107 Coverslips, 55, 56, 58, 71, 72, f 97, 97, 179, 372
laboratory challenges with, 447 cp. See Composite karyotype (cp)
on metaphase, 432 CPA4, 484
in nomenclature, t43, 45, t48 CpG island, 176, 456, 462, 475
profiles, 106–107, 421, 429, 432 CpG-oligdeoxynucleotides (CpG-ODNs), 335, 345
on solid tumors, 383 CPM. See Confined placental mosaicism (CPM)
Complementation groups CPP. See Choroid plexus papilloma (CPP)
in Fanconi anemia, 296 CREB1, 396
in xeroderma pigmentosum, 303 CREB1-EWSR1, f 391
Complex chromosome rearrangements (CCR), de novo incidence Crick, Frances, 9
in prenatal diagnosis, 233 Cri du chat (cat-cry) syndrome, 5, f 146, t148, 252
Composite karyotype (cp) CRLF2, 333
definition, t48 Cross(ing) over, 15, 19, 20, t30, 35, t49, 147, 166, 175, 417, 418
nomenclature, 40, 41, t48 within paracentric inversion loop, 155, f 155, 156
Computerized imaging systems, 87, 95 within pericentric inversion loop, 152, f 152, 153
con. See Connected signal (con) U-type exchange, f 155, 157, 158
Condensers Crosslinking agents
Abbe, 69 diepoxybutane, 297
achromatic, 69 mitomycin C, 297
aplanatic, 69 CRTC1-MAML2, 387, 389
aplanatic/achromatic, 69 Cryopreservation, 63
brightfield microscopy, 68 Cryptic
Confined placental mosaicism (CPM) deletions, 145, 196, 345
definition, 241 mosaicism, 179, 180
diagnosis, 243 rearrangements, 142, 144, 310, 317, 329
errors in mitosis, 278 subtelomeric rearrangements, 419
interphase FISH, 243 t(2;14)(p24;q32.3), 340
in recurrent spontaneous abortion, 288 t(12;14)(p13;q32;3), 340
uniparental disomy in, 242 t(12;21)(p13.2;q22.3), 327, t327, 331–332, f 332
Congenital fibrosarcoma (CFS)/infantile fibrosarcoma (IFS), translocation, 333, 417, f 422
390–392, t391 CSMLS. See Canadian Society of Medical Laboratory Science
Congenital mesoblastic nephroma (CMN), t379, 381, f 382, 390 (CSMLS)
Connected signal (con) CTNNB1-PLAG1, 385, 387
definition, t43, 44 Culture
in nomenclature, 44 amniotic fluid, 54, 83, 262
Consanguinity, 117, 300, 301, 303, 502 bone marrow, 54–56, 62, 372
Constitutional failure, 62–63, 79, 89, 233, 235, 237, 263, 448
anomaly (c) harvest, 80–82
definition, t48 initiation, 53–56, 63
arrays, 46 maintenance, 53, 56–57
chimerism, nomenclature, 39 peripheral blood, 53, 55, 56, 62, 243
chromosome aberration, 41 primary, 55, 56
chromosome abnormality, 64, 90, 217, 218 protocols, 80
comstitutional cytogenetic studies of infertile men, 218 solid tissues, 54–57
FISH studies, 417 systems
+21, in megakaryoblastic AML with t(1;22), t323 closed, 56
mosaicism, 39 open, 56
nomenclature, t48 vessels, 54–56
trisomy 8, 121 worksheets, 80, 90
524 Index
Cutaneous melanoma, t385 DDIT3, 393, f 394, 429. See also CHOP
CVS. See Chorionic villus sampling (CVS) DDIT3-EWSR1, t391, 393
CYP17A1, t216 DDX3Y, 197, 222
CYP19A1, t216 DEB. See Diepoxybutane (DEB)
Cystic fibrosis (CF) Decimal point (.) in nomenclature, 26, t28
and hyperechoic bowel, 253, t254, f 254 Degeneracy of DNA code, 11
and uniparental disomy, 481, 484 DEK, 325, t398, 403
Cytogenetic abnormalities, 16, 47, 84, 113, 121, 146, 182, 213, 217, del. See Deletion (del)
222, 223, 231, 238, 271, 275–278, t276, t277, 280, del(1p)
f 281, 282, 287–289, 300, t311, 312, 315, 316, 326, in follicular lymphoma (FL), 335–336, t336
327, 333, 334, 336, 353, 378, 443–445, 477, 502 in Hodgkin lymphoma (HL), t336
Cytogenetics, definition, 3 in neuroblastoma, t398
Cytogenetics laboratory procedures in SS, t353
cell harvest in T-cell lymphoma, 352, t353
fixative, 58 +del(1)(p13p36), in MDS, f 314
hypotonic solution, 57–58 del(1)(q21.3q26.2)
mitotic inhibitor (mitogen), 57 in heptaoblastoma, mixed embryonal and fetal, f 386
slide preparation, 58 in MDS with other chromosome abnormalities, 315
chromosome analysis, 53, f 57, 64–65 del(3p), in primary MDS and t-MDS, t313
chromosome elongation, 62 del(3)(p21.3), in MDS, f 314
chromosome staining and banding techniques del(3)(q21.3q26.2), in MDS with other chromosome
centromeric dot (Cd)/kinetochore staining, 61 abnormalities, 315
constitutive heterochromatin (C) banding, 60, f 61 del(4q)
4’,6-Diamino-2-Phenole-Indole/Distamycin A (DAPI-DA) in blastic plasmacytoid dendritic cell neoplasms, t323, 326
staining, 61–62 in PCM, t336
fluorescence in situ hybridization (FISH), 62 in T-cell lymphoma, 352, t353
Giemsa at pH 11 (G-11) banding, 61 del(5p), 252
Giemsa (G-) banding, 59, f 59 del(5)(p15.3), 33, f 146
quinacrine (Q-) banding, 59, f 59 del(5q)
reverse (R-) banding, 59–60, f 60 in AML (megakaryoblastic), adults, t323
silver staining for nucleolar organizer regions (NOR) staining, in AML (megakaryoblastic) with t(1;22), t323
61, f 61 in AML myelodysplastic-related changes, t323
telomere (T-) banding, 60–61 in AML (panmyelosis with myelofibrosis), t323
culture failures, 62–63 in AML with inv(3) or t(3;3), t323
culture initiation in AML with inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2), t323
antibiotics, 54–55 in AML with minimal differentiation, t323
culture vessels, 55 in blastic plasmacytoid dendritic cell neoplasms, t323, 326
direct, 55 in chronic myelogenous leukemia (CML), t316, 319, 325
flask method, 55 in chronic myelomonocytic leukemia (CMML), 321, t322
growth factors, 55 in erythroleukemia (erythroid/ myeloid), t323
growth media, 54 in essential thrombocythemia (ET), t317, 319
in situ method, 55 in juvenile myelomonocytic leukemia (JMML), 315, 322, t322
L-glutamine, 54 in mature T-cell lymphoma, t353
mitotic stimulants (mitogens), 55 in MDS, 313, t313, 314, 315, t316, t317, 319, 322, 325, 326
serum, fetal bovine, 54 in MDS/MPN, t322
specimen labeling, 78–79 in MDS/MPN,U, t322
specimen preparations, 78 in MDS with deletion of 7q or monosomy 7, 315
culture maintenance, growth interval, 56–57 MDS with isolated del(5p), t317
instrumentation (see Instrumentation, cytogenetics laboratory) in MDS with trisomy 8, 315
preservation of cells, 63 in polycythemia vera (PV), t317, 319
specimen collection and handling in primary MDS and t-MDS, t313
amniotic fluid, 54 in primary myelofibrosis (PMF), t317, 319
bone marrows, 53–54 in pure erythroid leukemia, t323
peripheral bloods, 53 in therapy-related myeloid neoplasms, t353
solid tissues, 54 del(5)(q23q34), in MDS, f 314
Cytokinesis, 18, f 18, 20 del(6p)
in AML (megakaryoblastic), t323
in AML myelodysplastic-related changes, t323
D in AML (panmyelosis with myelofibrosis), t323
DA-DAPI, t25 in erythroleukemia (erythroid/ myeloid), t323
DA staining. See Distamycin A (DA) satining in MDS, t313
Database, patient, 79 in MDS with deletion of 7q or monosomy 7, 315
DAX1, 191, 199, 201 in primary MDS and t-MDS, t313
DAZ, 197, 199, 222 in pure erythroid leukemia, t323
DBY, 222 in therapy-related myeloid neoplasms, t353
DCDF. See Dual color, dual fusion probe set (DCDF) del(6)(p21.3p24), in MDS, f 314
Index 525
del(6q) del(9)(p21.3), in B-ALL, t327

in B-ALL, t327, 331 del(9)(p21.3) (homozygous/hemizygous), in T-ALL, t334
in blastic plasmacytoid dendritic cell neoplasms, t323, 326 del(9q)
in CLL, 343, 426 in acute promyelocytic leukemia with t(15;17)
in CLL/SLL, t336 (q24.1;q21.2), 324
in diffuse large B-cell lymphoma (DLBCL), 335, t336, 337 in AML myelodysplastic-related changes, t323
in follicular lymphoma (FL), 335, t336 in AML without maturation, t323
in hairy cell lymphoma (HCL), t336 in AML with t(8;21), t323
in Hodgkin lymphoma (HL), t336 in AML with t(8;21)(q22;q22.3), 322
in lymphoplasmacytic lymphoma (LPL), t336 in AML with t(15;17), t323, 324
in mycosis fungoides (MF), t353 in blastic plasmacytoid dendritic cell neoplasms, t323, 326
in NK-cell leukemia (aggressive type), (NKCL), t353 in MDS with deletion of 7q or monosomy 7, 315
in peripheral T-cell lymphoma, not otherwise specified, in primary MDS and t-MDS, t313
(PTCL-NOS), t336 in therapy-related myeloid neoplasms, t323
in plasma cell myeloma, (PCM), t336 del(9)(q22q34), in MDS, f 314
in prolymphocytic leukemia (PLL), t336 del(10q)
in Sézary syndrome (SS), t353 in angioimmunoblastic T-cell lymphoma (AITL), t353
in T-ALL, t334 in diffuse large B-cell lymphoma (DLBCL), 335, t336, 337
in T-cell lymphoma, 352, t353 in follicular lymphoma (FL), 335–336, t336, t353
in T-cell prolymphocytic leukemia (T-PLL), t353 in peripheral T-cell lymphoma, not otherwise specified,
del(6)(q21-q23), in CLL, t426 (PTCL-NOS), t353
del(6)(q23.3)/MYB, in CLL/SLL, 342 in Sézary syndrome (SS), t353
del(7q) in T-cell lymphoma, 352, t353
in acute promyelocytic leukemia with t(15;17) del(11q)
(q24.1;q21.2), 324 in AML with t(9;11)(p22;q23) and other translocations Involving
in AML (megakaryoblastic), in children, t323 MLL, 324–325, f 325
in AML (megakaryoblastic) with t(1;22), t323 and association with trisomy 12 in CLL, 343
in AML myelodysplastic-related changes, t323, 326 and ATM mutation in CLL, 343
in AML with inv(16) or t(16;16), t323 in B-ALL, t327
in AML with inv(16)(p13.1q22.1) or t(16;16) with t(12;21)(p13.2;q22.3), 331
(p13.1;q22.1), 324 in chronic neutrophilic leukemia (CNL), t317, 320
in AML with minimal differentiation, t323 in CLL/SLL, t336, 343
in AML (panmyelosis with myelofibrosis), t323 in diffuse large B-cell lymphoma (DLBCL), t336, 337
in AML with t(8;21), 322–323, t323 in essential thrombocythemia (ET), t317
in AML with t(15;17), t323 in MDS, t313, f 314, t317
in chronic myelogenous leukemia (CML), t317 in MDS with deletion of 7q or monosomy 7, 315
in chronic myelomonocytic leukemia (CMML), in NK-cell leukemia (aggressive type) (NKCL), t353
321–322, t322 in primary MDS and t-MDS, t313
in erythroleukemia (erythroid/ myeloid), t323 in prolymphocytic leukemia, (PLL), t336, t353
in essential thrombocythemia (ET), t317, 319 in systemic mastocytosis (SM), t317, 320
in hairy cell leukemia (HCL), t336 in T-cell lymphoma, 352, t353
in Hodgkin lymphoma (HL), t336 in T-cell prolymphocytic leukemia (T-PLL), t353
in juvenile myelomonocytic leukemia (JMML), 322, t322 del(11)(q14q24), in MDS, f 314
in MDS, 315, t316, t317, 319 del(11)(q22.1)/ATM, in CLL/SLL, 342
in MDS/MPN, t322 del(11)(q22.3-q23.1), in CLL, t426
in megakaryoblastic AML with t(1;22)(p13.3;q13.1), 326 del(11)(q23), FISH for hematologic malignancies, ATM, t423
in polycythemia (PV), t317, 319 del(12p), in
in primary MDS and t-MDS, t313 AML (megakaryoblastic), t323
in primary myelofibrosis (PMF), 317, t317, 319 AML myelodysplastic-related changes, t323
in pure erythroid leukemia, t323 AML (panmyelosis with myelofibrosis), t323
in splenic MZBCL, t336 AML with minimal differentiation, t323
in systemic mastocytosis (SM), 320 B-ALL, t327
in therapy-related myeloid neoplasms, t323 blastic plasmacytoid dendritic cell neoplasms, t323, 326
del(7)(q22q36), in MDS, f 314 chronic neutrophilic leukemia, (CNL), t317, 320
del(9p) erythroleukemia (erythroid/ myeloid), t323
in B-ALL, 328 mature T-cell lymphoma, t353
in B-ALL, in adults, 332 MDS, 315, t317
in blastic plasmacytoid dendritic cell neoplasms, t323, 326 MDS with deletion of 7q or monosomy 7, 315
in Ewing sarcoma/peripheral primitive neuroectodermal tumor pure erythroid leukemia, t323
(EWS/pPNET), t398 T-cell lymphoma, 352, t353
in mycosis fungoides (MF), t353 T-cell prolymphocytic leukemia (T-PLL), t353
in peripheral T-cell lymphoma, not otherwise specified, therapy-related myeloid neoplasms, t323
(PTCL-NOS), t353 del(12)(p11.2p13), in MDS,f314
in Sézary syndrome (SS), t353 del(12q), in angioimmunoblastic T-cell lymphoma
in T-cell lymphoma, 352, t353 (AITL), t353
del(9)(p21.3), t327 del(13), 242
526 Index
del(13q), in MDS, 319

angioimmunoblastic T-cell lymphoma (AITL), t353 MDS with deletion of 7q or monosomy 7, 315
blastic plasmacytoid dendritic cell neoplasms, t323, 326 MDS with other chromosome anbnormalities, 315
CLL/SLL, t336, 342 MZBCL (MALT type), t336
and prognosis, 342 NK-cell leukemia (aggressive type),(NKCL), t353
and trisomy 12, 343 nodal MZBCL, t336
diffuse large B-cell lymphoma (DLBCL), t336, 337 plasmablastic lymphoma (PBL), t336
esential thrombocythemia (ET), 316, 319 plasma cell myeloma (PCM)
Hodgkin lymphoma (HL), t336 high risk, t336
myeloproliferative neoplasm (MPN), 319 and IGH@ rearrangements, 350
and prognosis, 316 polycythemis vera (PV), t317, 319
peripheral T-cell lymphoma, not otherwise specified primary MDS and t-MDS, t313
(TCL-NOS), t353 primary myelofibrosis (PMF), t317, 319
plasmablastic lymphoma (PBL), t336 prolymphocytic leukemia (PLL), t336, 353
plasma cell myeloma (PCM), t336, 349 pure erythroid leukemia, t323
and IGH@ rearrangements, 351 Sézary syndrome (SS), t353
primary MDS and t-MDS, t313 splenic MZBCL, t336
primary myelofibrosis (PMF), 316, 319 T-cell lymphoma, 352, t353
prolymphocytic leukemia (PLL), t336 T-cell prolymphocytic leukemia (T-PLL), t353
T-cell lymphoma, 352, t353 therapy-related myeloid neoplasms, t323
del(13)(q12q21), in MDS, f 314 unclassifiable-DLBCL/Hodgkin, t336
del(13)(q14) del(17)(p11.2p11.2), in Smith-Magenis syndrome, 505–506
in CLL, t423, t426 del(17)(p12p13), in MDS, f 314
FISH probe for MIR16-1, MIR15A, t423, t426 del(17)(p13), in CLL, t426
del(13)(q14.2) in retinoblastoma, t398 del(17)(p13.1), FISH for hematologic malignancies, TP53, t423
del(13)(q14.3), in CLL/SLL, 342 del(17)(p13.1)/TP53, in CLL/SLL, 342
del(13)(q21.3q33), f 146 del(17)(p13.3p13.3), in Miller-Dieker syndrome, 506
del(14p), t221 del(20q), in
del(14q) AML (megakaryoblastic), in adults, t323
in CLL/SLL, t336 AML myelodysplastic-related changes, t323
and trisomy 12, 343 AML (panmyelosis with myelofibrosis), t323
del(14)(q24), in CLL, f 344 atypical chronic myeloid leukemia (aCML), t322
del(14)(q24.1), in CLL with IGH@ abnormalities, 345 blastic plasmacytoid dendritic cell neoplasms, t323
del(14)(q24.1q31.1), in clear cell sarcoma (CCSK), t379, 381 chronic neutrophilic leukemia (CNL), t317, 320
del(15)p, t221 erythroleukemia (erythroid/ myeloid), t323
del(15q), in essential thrombocythemia (ET), t317, 319
blastic plasmacytoid dendritic cell neoplasms, t323, 326 isolated, in MDS, t316
primary MDS and t-MDS, t313 MDS, t317
del(15)(q11.2q13), in Prader-Willi syndrome, 505 MDS/MPN, t322
del(15)(q13q15), in MDS, f 314 MDS with deletion of 7q or monosomy 7, 315
del(16q) in, MPN, 316, t317
B-ALL, t327 myeloid sarcoma or granulocytic sarcoma, 326
and t(12;21)(p13.2;q22.3), 331, t327 plasma cell myeloma (PCM), t336
Hodgkin lymphoma (HL), t336 polycythemis vera (PV), t317, f 319
myeloid sarcoma (MS), 326 primary MDS and t-MDS, t313
plasma cell myeloma (PCM), t336 primary myelofibrosis (PMF), t317, 319
primary MDS and t-MDS, t313 pure erythroid leukemia, t323
del(16)(q22), in MDS, f 314 systemic mastocytosis (SM), t317, 320
del(17p), in therapy-related myeloid neoplasms, t323
adult T-cell leukemia/lymphoma (ATLL), t353 del(22q), in
AML, t323 atypical teratoid/rhabdoid tumor, t373
AML (megakaryoblastic), t323 follicular lymphoma (FL), 335, t336
AML myelodysplastic-related changes, t323 meningioma, t373
AML (panmyelosis with myelofibrosis), t323 rhabdoid tumor (RTK), t379
AML with minimal differentiation, t323 22q11.2 deletion
CLL/SLL, t336 cardiovascular abnormalities in children with, t249
and additional abnormalities, 343 clinical findings amenable to ultrasound detection, t249
and loss of TP53, 343 percentage among children with, t249
and trisomy 12, 343 del(X)(p21), 215
without loss of TP53, 343 del(X)(p22.33p22.33)/del(Y)(p11.32p11.32), in B-ALL, 333
diffuse large B-cell lymphoma, (DLBCL), t336 del(Yq), t218
erythroleukemia (erythroid/myeloid), t323 del(Y)(q11q22), t218
hairy cell leukemia (HCL), t336 del(Y)(q12), t218
Hodgkin lymphoma (HL), t336 Deletion (del)
lymphoplasma-cytic lymphoma (LPL), t336, 342 description, t29
mature B-cll lymphoid neoplasms, t336 long ISCN, interstitial, t29
Index 527
long ISCN, terminal, t29 der(1;16)(q10;p10), in

short ISCN, interstitial, t29 EWS/pPNET, t398
short ISCN, terminal, t29 PCM with chromosome 1 abnormalities, 349–350, f 350
Deletion(s), 145–149 Wilms tumor, t379, 381
definition, t48, t311 der(1;17)(q10;q10), in neuroblastoma, f 401
distal short arm, f 146, 194, 400 der(1;19), 350, 371
high-resolution banding, 146 in anaplastic oligoastrocytoma, t373
interstitial, t29, 33, 42, 146, 191, 333, 348, 378, f 442, 476, 505, 506 in oligoastrocytoma, t373
LCR sequences, 147 der(1;19), oligodendrogliomaf375
long arm, 39, 191, 215, 313 der(1;19)(q10;p10), in
mechanisms for acquiring or retaining a telomere, 146 anaplastic oligoastrocytoma (OA), t373
nomenclature, t29, 33, 34, 38, 39, 42–46, t43, t48 anaplastic oligodendrogliom (OD), t375
pathological significance, 146 embryonal rhabdomyosarcoma, f 400
syndromes oligoastrocytoma, t373
Alagille syndrome, t148 oligodendroglioma (OD), t373, 374
Angelman syndrome, 129, t148, 149, t150, f 151, 417, plasma cell myeloma (PCM) with chromosome 1 abnormalities,
476–477, t477, f 478, 505 349–350, f 350
Cri du chat syndrome, f 146, t148 +der(1;19)(q10;p10)
DiGeorge syndrome, 43, t150, t418, 419, 505 in embryonal rhabdomyosarcoma, f 400
DiGeorge/velocardiofacial syndromes, t149 in oligodendroglioma, f 375
Ichthyosis (X-linked)c syndrome, t149, 191 der(2)(qter ® q24.2::p16 ® q11.2), in neuroblastoma, f 401
Jacobsen syndrome, t148, 295 der(4)t(1;4), in hepatoblastoma, 384, t385
Kallmann syndrome, t149, 191, 195 der(9), 44, f 160
Langer-Giedion syndrome, t148 in CML, 424, f 425
Miller-Dieker syndrome, t148, t150, t418, 418, 506 der(9;18) (p10;q10), in polycythemia vera (PV), 319
Monosomy 1p36 syndrome, t148 +der(13)t(2;13), f 399
Phelan-Mcdermid syndrome, t149 der(13)t(2;13), in alveolar rhabdomyosarcoma, f 399
Potocki-Shaffer syndrome, 141, t148 der(13;13)(q10;q10), in upd(13)pat, 486
Prader-Willi syndrome, t148, t150, f 151, t418, 476–477, der(13;14)(p11.2;p11.2), f 163
t477, 505 der(13;14)(q10;q10), f 163, f 481
17q21.3 microdeletion syndrome, t148, 154 der(13;21)(q10;q10), t259
22q11.2 deletion/microdeletions syndromes, 258 der(14;21)(q10;q10), f 163, t259
22q11.2 deletion/microdeletions syndromes: cardiovascular in pilocytic astrocytoma, f 374
anomalies in children with, t249 der(15;21)(q10;q10), t259
22q11.2 deletion/microdeletions syndromes: clinical findings der(17)t(X;17)(p11.23;q25.3), in alveolar soft part sarcoma (ASPS),
amenable to ultrasound detection consistent with, t249 t391, 395
recurring, 147, t148, t150, 154 +der(18), in follicular lymphoma (FL), 335, t336, f 337
Rubinstein-Taybi syndrome, t148 +der(18)t(14;18, in diffuse large B-cell lymphoma
Smith-Magenis syndrome, t148, t150, 418, t418, 505–506 (DLBCL), 337
terminal15q13.3 microdeletion syndrome, t148 der(19)t(1;19)(q23.3;p13.3), in pre-B-All and B-ALL, 329
velocardiofacial, t418 der(22), 35, 44
Williams syndrome, 142, 147, t148, 154, 419 in CML, 424
Wolf-Hirschhorn syndrome, 148 der(22)r(17;22) in dermatofibrosarcoma protuberans (DFSP) and
X chromosome variants, t385, 388
and premature ovarian failure, 215 +der(22)t(9;22)
and primary amenorrhea, 215 in B-ALL, t327
Xp, 190–191, 195, f 195, 215 in CML, t317
Xq, 191, 215 +der(22)t(17;22)(q21.3;q13.1) in dermatofibrosarcoma protuberans
Y chromosome, 177, 194, 199 (DFSP), f 389
Dendritic spines, 457, 458 der(D;D), t231
De novo rearrangements, 142, 143, t232, 285, 477 der(D;D)(q10;q10), t231
Denys-Drash syndrome, 201, 381 der(D;G), t231
Deoxyribonucleic acid (DNA) der (D;G)(q10;q10), t231
biochemical analyses, 9 der(X), 215
classification, 464 der(X)t(X;Y)(p22.3;q11.2), f 195
molecular structure, 9, 294 der(X)t(X;Y)(Xqter®Xp22::Yq11®Yqter), 194, 195
organization, 11–12, f 16 der(X)t(X;Y)(Xqter®Xp22.3::Yp11®Ypter), 195
phosphate and sugar backbone strands, f 10 Derivative chromosome (der)
protein synthesis, 10–11, f 15 definition, t48
synthesis, 9–10, f 11, 13, 17, 18, 62, 303, 456 description, t29
der. See Derivative chromosome (der) nomnclature, t29, 32, 34–37, 39, 40, t43, t48, t49
der(1), f 160 Dermal tumors, t385, 388–389
der(1)t(1;13), in alveolar rhabdomyosarcoma, f 399 dermatofibrosarcoma protuberans (DFSP), t385, 388
der(1;7)(q10;p10), in primary MDS and t-MDS, t313 hidradenoma, t385, 389
der(1;15)(q10;q10), in PCM with chromosome 1 abnormalities, malignant melanoma, 389, t398, 403
349–350 variants of DFSP, 388
528 Index
Dermatofibrosarcoma protuberans (DFSP), t385, 388, t389 functional, 182, 190, 192, 193
Designation, band, 26, 33, 39, 42, 44, 46 inability to detect by array CGH, 86, 511
Desmoplastic small round cell tumor (DSRCT), t398, 402 maternal uniparental, in Prader-Willi syndrome, 505
DFSP. See Dermatofibrosarcoma protuberans (DFSP) in sperm, 280
DFSP and variants, t385 X-chromosome, 189
DFSP, with fibrosarcoma (FS)-like changes (DFSP-FS), 388 XY, 219
DHCR7, 201 Disorders of gonadal (testicular) development, 177, 201
DH11-USP6, 395 Disorders of sex development (DSD)
DIA. See Dimeric inhibin A (DIA) X chromosomes, 175–195, f 176, f 181, f 189, f 195,
Diakinesis, 19 199–201
4’,6-Diamino-2-phenole-indole (DAPI) staining, t25, 61–62, f 418, Y chromosomes, 175–177, f 176, 180–182, 185–188, 191,
f 422, f 436 194–200, f 195, f 197
DIC. See Differential interference contrast (DIC) Dispersed repetitive DNA, 14
dic(5;17)(q11.2;p11.2), in MDS, 314 Distamycin A (DA) staining, t25, 61–62
dic(9;12) (p13.2;p12.2), 322 DLBCL. See Diffuse large B-cell lymphoma (DLBCL)
dic(9;20)(p13.2;q11.2), 322, f 333 DLK1, 480, 486
dic(17;20)(p11.2;q11.2), in primary MDS and t-MDS, t313 DM. See Myotonic dystrophy (DM)
dic(X;Y)(Xqter®Xp22::Yp11®Yqter), 195 DMDs. See Differentially methylated domains (DMDs)
Dicentric dmin. See Double minutes (dmin)
chromosomes, 156, f 420 DMRT1, 201
C-banding for determing presence of, t25, 60 DMSO. See Dimethyl sulfoxide (DMSO)
produced by carrier parent with paracebtric inversions, 258 DNA. See also Deoxyribonucleic acid
fragments in Fanconi anemia, 297 alpha-satellite (a-satellite), 14, 25, 37, 49, 60, 129, 157, 300, 415,
inverted duplication of short arm of the X, 192 420, f 420, 421
isochromosome X, 181 helicase, 10, 296, 301
marker chromosome, 129 highly repetitive sequence, 13
problems in cell division, 156 methylation, genomic imprinting, 474, 476, 479
recombinant, 155, f 155, 156 microsatellite, 14, 186
rings, 159 middle repetitive sequence, 13, 14
Robertsonian chromosomes, 163–164 minisatellite, 14
Dicentric (dic) organization, 11–12, f 16
definition, t48, t311 polymerase I, 10
description, t29 primase, 10
long, t29 repetitive, 13, 14, 23, 25, 60, 141, 163–165, 168, 415, 416
short, t29 replication, 9, 10, 13, 141, 176, 293, 294, 300, 301, 303, 455, 456,
nomnclature, 36–37 475, 476
Dichroic mirror, 75, f 75, 76 satellite, 14, 25, 139–141, 157, 158, 168, 299
Dictyotene, 20 single copy, 13, 420, 421
Diepoxybutane (DEB), 297 structure, 9, 10, 15, 48, 139, 141, 456, 475
Differential interference contrast (DIC), 72–74, f 74 synthesis, 9–10, 13, f 15, 17, 18, 62, 303, 456
Differentially methylated domains (DMDs), 475 unique sequence, 13, 416
Diffuse grade II/anaplastic astrocytoma grade III, t373 DNA based methods, FXS, 464
Diffuse large B-cell lymphoma (DLBCL), 335–338, t335, t336, f 337 DNMT3B, 299, 300
DiGeorge syndrome, 43, 124, 142, t150, 151, t418, 419, 505. See also Dosage compenstaiton, 175, 176, 473
Shprintzen syndrome, velocardiofacial syndrome Double helix, 9, f 10, 12
Dimeric inhibin A (DIA) Double minute (dmin)
and quadruple marker screening, 256–257 in astrocytomas ith EGFR amplification, 374
and trisomy, 256, 509 definition, t48, t311
Dimethyl sulfoxide (DMSO) in hematologic neoplasms, 312
Diploid nomnclature, 35, 42
cell line Doubling
in mosaicism, 282 contiquous, in tandem duplications, 149
in nomenclature, 39 of hypodiploid clone, 330, 347, 348, f 348
chromosome number, 3–4, 18 of near-haploid clone, f 330
clone, in nomenclature, 41 Down syndrome (DS), 4, f 4, 5, f 6, 37, 41, 114, 117–119, f 118, f 119,
definition, t311 f 163, 184, 214, 230, 245, 247, 256, 278, f 279, 280,
zygote in uniparentql disomy, 480 t323, 325, 326, 333, 503–505, 508, 509. See also
Diplotene, 19, 20, 48, 115 Trisomy 21
Direct duplication, t29, 33, 149 Drosophila melanogaster, 457
Disomy. See aslo Uniparental disomy (UPD) Drying chambers
as a cause fetal growth restriction and adverse pregnancy acid fixative, 97
outcome, 508 benchtop device, 97
in comfined placental mosaicism, 242 fixative, aspiration of, 97
in complete hydaditiform mole, f 285 in situ culture and harvesting, 97
definition, t49 D15S10, t418
detection by SNP array, 385, 511 D22S75, t418, 419
Index 529
DSD. See Disorders of sex development (DSD) Duplication/triplication syndromes

DSP30, neoplasms stimulated with and associated cytogenetic Beckwith-Wiedemann syndrome, t150
abnormality rates, 335 Cat eye syndrome, t150
DSRCT. See Desmoplastic small round cell tumor (DSRCT) Charcot-Marie-Tooth 1A (CMT1A) syndrome, t150
D trisomy, 5. See also Trisomy 13 Duplication 3q syndrome, t150
Dual color, dual fusion probe set (DCDF), 423, t423, 424, f 425, Pallister-Killian syndrome, t150
426, 428 Potocki-Lupski syndrome, t150
For BIRC3-MALT1, 341 Proximal 22q11.2 microduplication syndrome Duplication/
use in AML, 243 triplication syndromes, t150
use in CML, 424, f 425 Pseudodicentric 15 (“inverted duplicated 15”), t150
Dual-fusion probes recurring, t150
nomenclature, 44 7q11.23 microduplication syndrome, t150
Duchenne muscular dystrophy, 143, 191, 216, 259, 488 17p13.3 duplication syndrome, t150
dup. See Duplication (dup) DXZ1 (X centromere), t420, 429
dup(1q), in B-ALL, t327 DYZ3, 181
dup(1)(q23q43) in embryonal rhabdomyosarcoma, f 400
dup(15)(q11.2q13.1), f 151
dup(15)(q24q26.3), f 149 E
dup(17)(p11.2p11.2), f 418 Early amniocentesis
Duplication (dup) comparison with chorionic villus sampling, 237–238
description, t29 outcome, 236
detailed ISCN, t29 specimen requirements, 238
direct, t29, 33 EB. See Ethidium bromide (EB)
inverted, t29, 33 EBV. See Epstein-Barr virus (EBV)
long ISCN, direct, t29 Echogenic bowel, 250, 253
long ISCN, inverted, t29 Echogenic foci, intracardiac, 248–250, f 249
nomenclature, 33, 35, 43, 48 Edwards syndrome, 5, 278. See also Trisomy 18
short ISCN, direct, t29 E2F3, t398, 403
short ISCN, inverted, t29 EGFR, t373, 374, 378, 384, t385, 390
Duplication(s), 349 EGR1, 313
definition, t48, t311 Elastin (ELN) gene, 147, t418, 419, 505
direct, 33, 149 ELL, 325
distal long arm, 149 ELN. See Elastin (ELN) gene
inverted, t29, 33, 129, 140, 142, 149, t150, 154, Embryonal CNS tumors, 376–378
192, 511 Embryonal rhabdomyosarcoma (RMS), t385, 398,
and marker chromosomes, 129–130, 139, 417, 419–421, f 419, 400, f 400
t420f420, 433, 434, 441 EMC. See Extraskeletal myxoid chondrosarcoma (EMC)
mechanism of formation, 129, 139–142, f 140 EML4-ALK, 385, 390, 431
microdeletion-microduplication syndromes, 151 end. See Endoreduplication (end)
microduplication syndromes, 140, 149, t149 Endocrine disorders, and pregnancy loss, 276
phenotypes associated with, 149 Endometrial stromal tumors (EST), t379, 382–383
proximal long arm, 149, f 151, 197, f 197 Endometriosis and female infertility, 213, 216
of SNRPN probe, t418 Endoreduplication (end)
syndromes definition, 32, t48
Beckwith-Wiedemann, 149, t150, f 419, 477–478 in hypodiploidy, 329
cat eye, t150 in omenclature, 32
Charcot-Marie-Tooth, t150, 151, 418 origin og paternally derived triploid, 283
Cornelia de Lange, 149, t150 England finders, 72
duplication 3q, t150 Ependymoma
duplication 7q, t150 intracranial, t373, 375
duplication 17p, t150 posterior fossa, 373, 375, f 376
duplication 17q, t150 spinal, t373, 375
microduplication 22q, t150 Epifluorescence microscopy, 74–76. See also Fluorescence
Potocki-Lupski, t150 microscopy
proximal 15q, t150 Epithelial cancer
types of rearrangements involving breast cancer, 389–390
derivatives, 149 lung cancer, 390
dicentrics, 142, 149 Epstein-Barr virus (EBV), 55, 63, 352, 353, 356
isochromosomes, 139, 142, 149 Equal sign (=) in nomenclature, t28
markers, 149 Equipment maintenance, 80–81
recombinants, 149, 153 ERBB2, 45, 99, 371, t373, 376, t385, 387, 390,
rings, 142, 149 430, f 430
of X chromosomes, 188, 200 ERG
of Xp, 191–192 in Ewing sarcoma, 401
of Xq, 190, 192–193 in prostate cancer, 382
Duplication-deficiency chromosomes, f 153, 201 overrepression in, t379
530 Index
Errors FANCB, 296

clerical, 77, 87 FANCD1, 296
in fertilization, 283–284 FANCD1/BRCA2, 296
in gametogenisis, 278 FANCD2, 296
harvesting, 89 FANCJ, 296
human, 89, 90 FANCL, 296
imprinting, 225 spontaneous chromosome breakage, 297
labeling, 78–80, 88, 90 telomere shortening, 297
leading to chromosomally abnormal conceptus, 278–286, f 279, treatment, 297
t279, t280, f 281, f 284, f 285 FAP. See Familial adenomatous polyposis (FAP)
in meiosis, 114, 278–280, 282, 283 FBS. See Fetal bovine serum (FBS)
in mitosis, 278, 282–283 FDA. See US Food and Drug Administration (FDA)
in recombination, 350 Female infertility
in transcription, 79 endometriosis, 216
typographical, 5 hypothalamic and pituitary, 216–217
Erythroblastosis fetalis, 229 ovarian dysfunction, 213–217
Erythroleukemia (erythroid/myeloid), t323 45,X and 45,X mosaicism, 213–214
ESCO2, 300 X-autosome translocation, 215–216
Essential thrombocythemia (ET), 316, t317, 319 X-chromosome deletions, 215
EST. See Endometrial stromal tumors (EST) Femur, short, 253, t255
ET. See Essential thrombocythemia (ET) Fertilization
Ethics policies, 93–94 errors in, 278, 283–284
Ethidium bromide (EB), for chromosome elongation, 62 in vitro, 113, 114, 213–215, 224, 225, 289, 422, 478
E trisomy, 5. See also Trisomy 18 in vivo, 224
ETS, 381 Fetal bovine serum (FBS), 54, t233
ETV6, 320, 321, 331, 332, t334, 381, 390, t423, 425 Fetal chromosome anomalies, microarrays, 445
ETV1-EWSR1, t398 Fetal diagnosis, noninvasive, 265
ETV4-EWSR1, t398 Fetal DNA in maternal circulation, 265
ETV6-FLT3, t321 Fetal loss, 214, 231, 233, 234, 236, t236, 237, 238. See also Stillbirths
ETV6-NTRK3, 321, t379, t385, 390, t391 and neonatal deaths; Pregnancy loss; Spontaneous
ETV6-RUNX1, t327, 328, 331, 332, 425 abortion; Spontaneous loss; Spontaneous pregnany loss
Euchromatin, 14, 129, 130, t420, 510 following chorionic villus sampling (CVS), 233, t233, 236, 237,
Ewing sarcoma (EWS), 162, t398, 401–402, 429 240–241
family of tumors, 401 following conventional amniocentesis, 233–236, 238
EWSR1, 393, 396, 402, 403, 429 following early amniocentesis (EA), 235–238
EWSR1-ATF1 protein, 403 following percutaneous umbilical blood sasmpling (PUBS), 244
EWSR1-WT1, 402 in pregnancies with combined placental mosaicism (CPM), 242
Exchange, 15, 34, 56, 139, 147, 149, 155, 159, 164, 194, 302, FEV-EWSR1, t398
t312, 419 FGFR1, 320–321, 428
Extragonadal germ cell tumors, 383 FGFR3, 259, 350, 351, f 427
Extraskeletal myxoid chondrosarcoma (EMC), t391, 395 FGFR3-MMSET, 350
Eyepieces, microscope, 71, 73 Fiber FISH, 434
Fibrosarcoma, 379, 390, 392
Fibrosarcoma-like changes (DFSP-FS), 388
F Filters
FA. See Fanconi anemia (FA) bandpass, 75, 76, 104, 433
Facultative heterochromatin, 15 barrier, 75, 81
Failure, culture, 62–63, 79, 89, 233, 237, 263, 448 emission, 75, f 108
Familial adenomatous polyposis (FAP), 385–386 excitation, 75
FAM22-YWHAE, t379, 381, 383 fluorescence, 75–76
FANCA, 296 infrared, 69, 75
FANCA-null patients, 296 interference, 69, 75, 81
FANCB, 296 multiband, 76
FANCD1, 296. See also BRCA2 neutral density, 68, 75
FANCD1/BRCA2, 296 wheel, 104, 106, f 108
FANCD2, 296 Final reports, 88
FANCJ, 296 FIP1L1, 321, 428, f 428
FANCL, 296 FIP1L1-PDGFRA, 320, 428
Fanconi anemia (FA), 5, 38, 296–297, 506 First trimester screening, 508, 509. See also Combined screening;
breast/ovarian cancer risk, 296 Integrated screening
carrier frequency, 296 fis. See Fission (fis)
chromosome breakage, 296 FISH. See Fluorescence in situ hybridization (FISH)
clinical features, 297 Fission (fis), definition, t48
complementation groups, 296 Fixative, 58, 89, 90, 95–97
FANCA, 296 FL. See Follicular lymphoma (FL)
FANCA-null patients, 296 Flask culture method, 56
Index 531
FLI1, t418, 429 Fluorescence microscopy, 5, 74–75. See also Epifluorescence

FLI1-EWSR1, t398, 402 microscopy
Floor model drying chamber, f 98 Fluorescent spot counting, 103–105, f 105
FLT3, f 320, 321t, 323t, 326 5-Fluorodeoxyuridine (FUdR), 62, f 294
Fluorescence filter cube, 75 FMC7, 345
Fluorescence in situ hybridization (FISH) FMR1
acute lymphoblastic leukemia, 425–426 associated disorders, 459–460
acute myeloid leukemia, 423–424 epigenetic changes, 456, 458
applications for studies of acquired chromosomal aberrations, methylation of CGG repeat, 458
423–431, t423, f 424, f 425, t426, f 427–f 431 molecular rules of ninheritance, 463
automated imaging systems, 100–107, f 101–f 107 prenatal diagnosis, 460
B cell neoplasms, 426–428, f 427 trinucleotide repeats, 463
bladder cancer, 431, 431f FMR2, 465
break-apart probes, 340, 341, 423, 424, 426, 429 FMRP. See Fragile X mental retardation protein (FMRP)
nomenclature, 43–45 Folate-sensitive fragile sites, characteristics of, t465
chimerism, 45 FRAXA, t465
chronic disorders with eosinophilia, 428 FRAXE, t465
chronic myelogenous leukemia, 424–425, 425f FRAXF, t465
clinical applications, 417–431, f 418–f 420, f 422, f 424, f 425, FRA16A, t465
f 427, f 429–f 431, f 428, t418, t420, t421, 423, t426 FRA11B, t465
comparative genomic hybridization, 429, 432, 433f Folic acid
copy number aberrations, microarray analysis, 421 deficient medium, 294
cryptic subtelomeric rearrangements, 419 pathway, 62
definition, 415 supplimentation, 258
dual-fusion probes, 423, 424 Follicle stimulating hormone (FSH), 115, 213–215, f 214,
duplications and marker chromosomes, 419–421, 420f, 420t, 433 219, 223
enrichment, 428 Follicular lymphoma (FL), t335, t336, 335, f 337, 341, 428
ERBB2 and breast cancer, 430 Food and Drug Administration, US (FDA), 78, 85, 86, 91, 258, 382,
fiber, 434 390, 430–432
gene amplification, 429, 430 Fork stalling and template switching (FoSTeS), 141, 147
for hematologic malignancies, 423, f 424, 428, 445 FOXO4-PAX3, 398, t398
hybridization, 415–436, f 416, t418, f 418, f 434, f 435 FOXP1, 341
instrumentation for, 99 fra. See Fragile sites (fra)
interphase, 78, 85, 100–102, 104–106, f 105, 243, f 394, 417, 418, FRA16A, t465
426 (see also Nuclear FISH) FRA11B, 295, 296, t465
nomenclatre, 42–45 Fragile sites (fra)
labeling and detection, 417 aphidicolin inducible, t295
mBAND analysis, 433, f 435 5-azacytidine inducible, t295
metaphase, 85, 417, 426, 429, 432, f 434, 436 BrdU requiring, t295
nomenclature, 42–43, 45 5-bromodeoxyuridine (BrdU) inducible, t295
methodology, 415–417, f 416, 431 classification, 293–294, f 294
microdeletions and microduplications, 417–419, f 418, t418 clinical significance, 294–296
multicolor, 422, f 434 common, 293–296
multiplex, 432–434 common and rare, t295
nomenclature, 415, 432 definition, t48
non-small cell lung cancer (NSCLC), 431 definition and classification, 293–294
nuclear, 85 (see also Interphase FISH) description, 293
nomenclature, 44–45 distamycin A inducible, t295
oncology FISH probe panels, 45 folate sensitive, 62, t295, t465
panels, 425–427, f 427 folate/thymidylate depletion, 294
prenatal chromosomal abnormalities, 421 in humans, 293–296, f 294, t295
pretreatment, 99, f 99 classification, 293–294, f 294
PRINS, 432, 434, 435, f 435 clinical significance, 294–296
probe types, 415–417, 423, f424 definition, 293–294, f 294
procedure, 415, 416, 436 folate/thymidylate depletion, 294, f 294
reverse, 434–436, f 436 nomenclature of, 38, t48
schematic representations, 416 rare, 293–295
sex-mismatched bone marrow, 428–429, f 429 X chromosome, on the, t454
single-copy, 417, f 418, 421, 434 Fragile sites on the X chromosome
soft tissue tumors, 429, f 430 FRAXA, t454
solid tumors, 417, 423, 433 FRAXB, t454
diagnosis, 429 FRAXC, t454
special quality considerations, 432 FRAXD, t454
sub-telomere (subtel), 506 FRAXE, t454
nomenclature, 43–44 FRAXF, t454
Fluorescence light sources, 75 Fragile X gene, 456–458, f 457
532 Index
Fragile X mental retardation protein (FMRP), 456–461, f 457, f 459, FRAXF, t454
464, 465 cytogenetic expression, 453
Fragile X premature ovarian insufficiency (FXPOI), 461, 462 characteristics of, t465
Fragile X primary ovarian insufficiency (FXPOI), 461, 462 Free b-hCG, 246, 251, 257
Fragile X-related (FXR) genes, 456 Friedreich Ataxia, t455
Fragile X syndrome (FXS) FSH. See Follicle stimulating hormone (FSH)
aging, 460–461 FSHB, t217
behavioral phenotype, 460 FSHR, t216
carrier testing, 465 FUdR. See 5-fluorodeoxyuridine (FUdR)
clinical aspects, 456, 460–462 Fungicides, 54
cognitive phenotype, 460 FUS, 393, 396, 429
cytogenetic testing, f 454, 463, 464 FXPOI. See Fragile X premature ovarian insufficiency
diagnostic laboratory testing, 463–465 (FXPOI)
epidemiology, 453, 462–463 FXR1, 456
inheritance, 453 FXR2, 456
molecular aspects FXR genes. See Fragile X-related (FXR) genes
analysis using linked polymorphisms, 454 FXS. See Fragile X syndrome (FXS)
classification, trinucleotide repeats, 455 FXTAS. See Fragile X tremor ataxia syndrome (FXTAS)
instability of repeats, 455–456
molecular testing, 464
DNA-based methods, 464 G
PCR-based methods, 464 GACI, t398
phenotype, 38, 456–458, 460, 461 GALT, t216
premutation carrier phenotypes Gametogeneis, 141, 142, 165, 181, 186, 278, 280, 474–476
fragile X premature ovarian insufficiency (FXPOI), Gap 1 (G1), 16, 17
461, 462 Gap 2 (G2), 16, 17
fragile X tremor ataxia syndrome (FXTAS), 461, 462 Gap zero (G0), 17
premutation expansion, timing of, 462 Gastrointestinal tumors
protein/mRNA-based diagnosis, 464–465 gastrointestinal stromal tumors (GIST), 371, 384, t385
Fragile X tremor ataxia syndrome (FXTAS), 461, 462 hepatic mesenchymal hamartoma, t385, 386
Fragments, t28, 60, t232, 258, 264, 285, 297, 480. See also hepatoblastoma, 384–386, f 386
Acentric fragments (ace) liver tumors, 384–386, t385, f 386
fetal deaths subsequent to amniocentesis, t232 GATA1, 325, 326
Franklin, Rosalind, 9 G-11 banding, 61
fraX G-bands/banding. See Giemsa (G)-bands/banding
cytogenetic expression of, 453 GBY, 177, 198
cytogeneticn testing, 463 GCF. See Giant cell fibroblastoma (GCF)
inheritance of, 453 Gene amplification, t29, 42, 48, 85, t311, 371, 384, 387, 390, 398,
molecular analysis, 455, 464 429, 430
prenatal diagnosis, 454 detected by FISH, nomenclature, 45
FRAXA, t454 GenePix microarray scanner, f 108
appearance of, f 45 Genetic counseling
CCG repeat in, 294 abnormal prenatal screen, 503, 508–509
characteristics of, t465 advanced maternal age, 503, 507–508
clinical significance of, 295 advanced maternal and paternal age, 259–260, 503
cytogenetic expression of, 453 autosomal trisomies, 504–505, 509
vs. FRAXE phenotype, 465 cancer, 502
trinucleotide repeat size, 455 chromosome abnormality, 500–504, 506–511
full mutation, 455 prenatal, 509–511
gray zone, 455 chromosome instability syndrome, 506
indeterminate, 455 client-centered approach, 499, 500
normal, 455 consanguinity, 502
permutation, 455 counselor, role of, 499, 500
FRAXB, 293, t454 cytogenetic indications, 504–511
FRAXC, t454 definition, 499
FRAXD, 453, t454 genetic syndrome or chromosome abnormality, 500–502
FRAXE indications for referral, 500–504, f 501, t503
analysis using linked polymorphisms, 454–455 infertility, 503–504, 506
CGG repeat instability, 455 microarrays, 504–506, 508, 511
clinical significance of, 295 microdeletion syndromes, 504, 505, 508
cytogenetic expression, 453 principle of nondirectiveness, 499
in affected males and carrier females, 543–454 recurrent spontaneous abortion, 504, 507
expansion, 465 session components, 500
genetic counseling, 465–466 sex chromosome abnormalities, 506–507, 509
phenotype, 465 subtle rearrangements, 506
syndrome, t455, 465 unbalanced chromosome rearrangements, 505, 507
Index 533
Genetic syndrome and isodicentric Xq, 194

clinical suspicion of, 500–502, f 501 in 46,XX, 200
family history of, 500–502, f 501 in 48,XXXX, 184
Genitourinary tumors Gonadoblastoma, 177, 180–182, 194, 196–198, t216, 281, 421,
bladder cancer, f 105, 382, t379, 431 423, 510
chromophobe renal cell carcinoma, 378, t379, 381 Granulocytic sarcoma, 326
clear cell renal cell carcinoma (ccRCC), 378, t379, 380, 381 Gray-zone alleles, of FMR1, 457, 459, 462
clear cell sarcoma of the kidney (CCSK), t379, 381, 383 GRB10, 484
congenital mesoblastic nephroma (CMN), t379, 381, f 382, Growth factors, 55, 310
390, 392 Growth intervals, 56–57
endometrial stromal tumor (EST), t379, 382–383 Growth media
extragonadal germ cell tumors (GCTs), 383 complete, 54
nonseminomatous germ cell tumors (NSGCT), t379, 383 incomplete, 54
oncocytoma, 378, t379, 381 GTG-bands/banding, 14, 25, 59, 435
ovarian dysgerminoma, t379, 383 GTL2, 480, 486. See also MEG3
papillary renal cell carcinoma (RCC), 378, t379, f 380 GTL-bands, t25
postpubertal germ cell tumors (GCTs), t379 GTW-bands, t25
prepubertal germ cell tumors (GCTs), t379
prostate cancer, t379, 381–382
rhabdoid tumor of kidney (RTK), t379, 381 H
seminomatous germ cell tumors (GCTs), t379, 383 H19, 474–476, 478–480, 484
testicular seminoma, t379, 383 Hairy cell leukemia (HCL), 335, 336, 346, f 347
TFE3 and TFEB translocation RCC, 380 Hanabi metaphase spreader, f 98
t(6;11) renal cell carcinoma (RCC), t379 Hanabi-PIV automated slide maker, f 98
Wilms tumor, t379, 381, 479–480 Handling, specimen collection and of
Genomic imprinting amniotic fluid specimens, 54
Angelman syndrome, 474, 476–478, t477, f 478, 486, 487 bone marrow aspirates, 53–54
assisted reproductive technology, 478–479 peripheral blood specimens, 53
Beckwith-Wiedemann syndrome, 477–479, f 478 solid tissue biopsies, 54
cancer, 474, 476, 479, 480 Haploid
description, 473 bands per, set of chromosomes, 59
and human diseases, 476, 486 chromosome number, 32, 473
mechanism, 474–482, f 475, t477, f 478, f 481, f 482, 485, 487 in meiosis, 18, 278
paraganglioma, 479 in oogenesis., 20
Prader-Willi syndromes, 474, 476–478, t477, 486 in telophase 19
rhabdomyosarcoma, 477, 479–480 definition, t311
Wilms tumor, 477, 479–480, 485 egg, 283
X-chromosome inactivation, 473, 474 gains relative to, background, 376
Gentamicin, 54 karyotype, f 27
Germ cell tumors (GCT) pronucleus, maternal, 283
ovarian dysgerminoma, t379, 383 set of chromosomes,
postpubertal germ cell tumors, t379, 383 origin of in triploidy, 125
prepubertal germ cell tumors, t379, 383 in unique sequence DNA, 13
Gestational age, 63, 235, 239, 240, 244, 246–248, f 247, 251, 252, sperm, 283
256, 276–278, 419, 508 Harvest(ing)
Gestation, multiple, 224 cell, 53, f 57
Giant cell fibroblastoma (GCF), t385, 388 errors, 89
Giemsa (G)-bands/banding, 17, 24–27, 48, 59, 60, 84–86, 180, 230, flask method, 55–56
419, 434, 436 in situ method, 55–56, 95
idiograms of, 26, f 27 protocols, 62, 81
pattern, 25 slide preparation, 58, 77, 80–86
characteristic size and centromere position, 24 suspension, 95, 96, 371
idiograms/diagrammatic representations, 26 Harvester, robotic, 89, 95, f 96
GIST. See Gastrointestinal tumors (GIST) HB. See Hepatoblastoma (HB)
Gleevec®, 320, 334, 428 hCG. See Human chorionic gonadotropin (hCG)
Glioblastoma (GB), t373, 374 HCL. See Hairy cell leukemia (HCL)
Gliomas, 297, 372, 374 HD. See Huntington’s disease (HD)
Glycerol, 10%, 63 Health Insurance Portability and Accountability Act of 1996 (HIPAA),
GNAS, 476 93–94, 101
GNRHR, t217 HEB. See Hyperechoic bowel (HEB), second trimester finding
Gonadal (testicular) development, disorders of, 177, 201 Helicobacter pylori (H. pylori), 340
Gonadal dysgenesis Hematologic neoplasms
and deletions of Xq, 191 atypical chronic myeloid leukemia, 321, t322
in disorders of gonadal (ovarian) development, 200–201 characterization, 346
in disorders of gonadal (testicular) development, 201 chromosome abnormalities, 309–313, t313, 315–317, 319–324,
and isodicentric Xp, 193–194 326–328, 333–336, 341, 342, 346, 347, 351–353, 356
534 Index
Hematologic neoplasms (cont.) hsr. See Homogeneously staining regions (hsr), intrachromosomal
chronic eosinophilic leukemia (CEL), 316, 317, t317, homogeneously staining regions
320, 321 HSTL. See Hepatosplenic T-cell lymphoma (HSTL)
chronic myelogenous leukemia (CML), 316–317 htz. See Heterozygous (htz) in nomenclature
chronic myelomonocytic leukemia (CMML), 321, t322 Human chorionic gonadotropin (hCG), 122, 256, 262, 275, 282–284,
chronic neutrophilic leukemia (CNL), 316, t317, 320, t322 508, 509
classification of, 320 Human chromosomes
description, 309 balanced paracentric inversion, 30, 156
essential thrombocythemia (ET), t317, 319 balanced translocation, 30, t30, 37, 144, f 145, 159–161, 165
juvenile myelomonocytic leukemia (JMML), 315, 321, t322 banding and identification, 24, t25, 26
myelodysplastic/myeloproliferative neoplasms (MDS/MPNs), cancer karyotypes, 31, 32, 36, 38, 40, 41
321, t321 centromere, 12–15, 23, 24, f 24, t25, 26, t29, 31, 33–38, 42, 45, 46,
myelodysplastic syndromes, 312, t313, t321, t322, 326 (see also 142, 151–153, f 151, 155–159, f 158, 161, 163–165
Myelodysplastic syndromes (MDS) characteristic size and centromere position, 24, f 24
myeloid and lymphoid neoplasms, 320–321 chromosome aberrations, 26, t28, 30, 32, 41, 46, 147
myeloproliferative neoplasms (MPNs), 312, 316, t317, 321 chromosome abnormalities, 504
polycythemia vera (PV), 316–318, t317, f 319 clones, 30–32, 35, 38–40
primary myelofibrosis (PMF), 316, 317, 319 comparative genomic hybridization, 42, t43, 45, 48
systemic mastocytosis (SM), 316, t317, 320 constitutional and acquired, 31
with trisomy 8, 315 definition, 3, 58
types of, 316–356 FISH, 34, 42–45, t43
unclassifiable, 321 functional and structural components, f 17
Heparin, sodium, 53, 54, 244 Giemsa/G-bands/banding, 25, t25
Hepatic mesenchymal hamartoma (HMH), t385, 386 idiograms/diagrammatic representations, 26
Hepatoblastoma (HB), 384–386, t385, f 386, 477 karyotype descriptions, 26, t28, 30–32, t30
Hepatosplenic T-cell lymphoma (HSTL), t353, 355 microarray analysis, 45–46
HER2, 45, 390, 430, f 430. See also ERBB2 molecular cytogenetic techniques
HESX1, t217 mosaicism and chimerism, 39
Heterochromatin nucleolar organizer regions, 12, 13, 163
centromeric, 156, 157, 485 numerical aberration, 28, 31, 32
constitutive, 15, 25, t25, t48, 60, 61, 299 numerical abnormalities and ploidy, 32
decondensation, 240 Q-bands/band, t25, 59
facultative, 15 R-bands/banding, 24, 25, t25
paracentromeric, 299 staining techniques, t25
pericentromeric, 299, f 299 telomeres, 13, 24–26, t25, 46, 48
Heterochromatin repulsion (HR), 300 Human error
Heterodisomy, 159, 442, 480, 486, 488 culture failure, 89
Heterozygous (htz), 147, 162, 164, 201, 296, 298, 303, 454, 476, 482, post-culturing errors, 89–90
483, 485, 487 Humerus, short, 253, t255
in nomenclature, t48 Huntington’s disease (HD), 64, t455
Hidradenoma, t385, 387, f 388, 389 Hybrid gene, definition, t311, 317
HIPAA. See Health Insurance Portability and Accountability Act of Hybridization, in situ, 6, 34, 42, 44, 48, 59, 62, 67, 74, 77, 85, 95, 99,
1996 (HIPAA) 113, 124, 142, f 151, 154, 157, 165, 168, 179, 180, 215,
HIRA, 43, 47, t418, 419 233, 243, 263, 280, 286, 310, 317, 372, 374, 415–436,
Histone proteins (histones), 176 f 416, f 418, t435, f 436
HIV-related lymphoma, 351 HYMAI, 484
HL. See Hodgkin lymphoma (HL) Hyperdiploid, 35, 329
HLF, 329 in atypical choroid plexus tpapilloma (CPP), t373, 376
HMGA2, t385, 387, t391, 393 in B-ALL with t(1;19)(q23.3;p13.3), 329
hmz. See Homozygous (hmz) in nomenclature clones in plasma cell myeloma (PCM), 426
Hodgkin lymphoma (HL), 335, t335, t336, 338, 352 with MYC rearrangements, 351
Holoprosencephaly, 120, f 120, 122, 124 definition, t311
Homogeneously staining regions (hsr), intrachromosomal in Hodgkin lymphoma (HL), 352
homogeneously staining regions, t29, 42, 48, 312 karyogram in B-ALL, f 331
definition, t311 karyotype(s), 348, 349
description, t29 true, 330
long ISCN, t29 Hyperdiploidy
short ISCN, t29 in acute lymphoblastic leukemia (ALL), 425
Homozygous (hmz), 117, 296, 332, t373, t379, 381, 384, 431, 464, in B-ALL, 327, t327, 330–331
481–483, 485, 487 difficulty identifying with microarray testing, 447
definition, t48 high, 331
in nomenclature, t43 in Hodgkin lymphoma (HL), t336
HOXA10-TRB@, t334 in plasmablastic lymphoma (PBL), t336
HOXA13, t217 in plasma cell myeloma (PCM), 347–348, f 349, 428
H. pylori. See Helicobacter pylori (H. pylori) standard risk, t336
HR. See Heterochromatin repulsion (HR) for the X chromosome, 187
Index 535
Hyperechoic bowel (HEB), second trimester finding, 253, f 254, t254 i(17)(q10) in
Hypereosinophilia, 320, f 428 atypical chronic myeloid leukemia (aCML), 322
Hypodiploid chronic myelogenous leukemia (CML), 317, 328
in chromophobe RCC, 381 chronic myelomonocytic leukemia (CMML), 322
chromosome complement in B-ALL, 329–330 chronic lymphoid leukemia (CLL), 343
definition, t311 in primary MDS and t-MDS, t313
karyogram in high risk in plasma cell myeloma, f 348 i(18p), 129
multiplex or multicolor FISH (M-FISH), f 434 i(18)(p10), 129
in plasma cell myeloma (PCM), 347, 349, 351 i(21q), 258
of the X chromosome, 187 i(21)(q10), in primary MDS and t-MDS, t313
Hypodiploidy i(Xp), 193, 194
in B-ALL, 327, t327, 329, f 434 i(X)(p10), t28
in chromophobe RCC, 381 i(Xq), 181, 186, 213
difficulty identifying with microarray testing, 447 i(X)(q10), 181, f 181, 186, 194
formation in chromosome instability, 292 i(Yp), 198
in plasma cell myeloma (PCM), 347 IC. See Imprinting center (IC)
high risk, t336 ICF syndrome, 296, 299–300, t299
with SNP-based arrays, 443 clinical features, 299
Hypopigmentation in cytogenetic findings, 299
Angelman syndrome, t418, 477 DNMT3B, 299–300
Prader-Willi syndrome, 476 Ichthyosis, X-linked, 191
tetrasomy 12p (Pallister-Killian syndrome), 128, t150 ICSI. See Intracytoplasmic sperm injection (ICSI)
trisomy 6, 123 ID4, 333
trisomy 16, 122 idem
upd(15)mat, 486–487 definition, 40, t48
Hypothalamic in nomenclature, 40
causes female infertility, 216, t217 ider. See Isoderivative chromosome (ider)
gene mutations affecting function, t217 ider(20)(q10)del(20q), in primary MDS and t-MDS, t313
Hypotonic shock procedure, 3 IDH1, t373, 374, 375
Hypotonic solution, 3, 57, 58, 89, 95, 310, 441 IDH2, t373, 374
IDH mutations, t373, 374, 375
idic. See Isodicentric chromosome (idic)
I idic(1)(p22), in malignant mixed germ cell tumor, f 384
i. See Isochromosome (i) idic(15), t420
i(1)(p10), 482 idic(22), t420
i(1)(q10), 482 idic(X)(p11.2), 194
i(2)(p10), 483 idic(X)(q13), in primary MDS and t-MDS, t313
i(2)(q10, 483 idic(Yp), 197, 198
i(4)(p10), 483 Idiograms
i(4)(q10), 483 definition, 48, 311
+i(5)(p10), 127 of G-band patterns, f 27
i(7)(q10), in hepatosplenic T-cell lymphoma (HSTL), t353, 355 of X and Y chromosomes, f 176
i(8)(p10), 127 IFS. See Infantile fibrosarcoma (IFS)
i(8q), in malignant melanoma, 389 IGF2, 474, 475, 478–480
i(8)(q10), in T-cell prolymphocytic leukemia (T-PLL), t353, 353 IGF2/H19, 477, 478
i(9p), 127 IGF1R, t398, 400
i(9)(p10), 485 IGF2R, 474–476
in B-ALL, 127 IGH@, 333, 335, 336, f 338, f 340, 341, 343–345, 347, 348, 350–351,
i(9q), in B-ALL, t327 t423, 426–428
i(9)(q10), 127 IGHV@, 343, 345
in B-ALL, 485 IGK@, 335, 336, t338, t340, 341
i(12p) in IGL@, 335, 336, f 338, f 340
germ cell tumors, t379, 383 IL2. See Interlikin-2 (IL2)
malignant mixed germ cell tumor from the mediastinum, f 384 IL3-IGH@, 333
ovarian dysgerminoma; testicular, seminoma, nonseminoma; Imaging systems
extragonadal, t379 automated, 100–109, f 101–f108
tetrasomy 12p, 127 (see also Pallister-Killian syndrome) components, 101, f 101
i(12)(p10), in tetrasomy, 12p, 127, 384. See also Pallister-Killian computerized, 87, 95
syndrome and karyogram production, 86–87
+i(12)(p10), in malignant mixed germ cell tumor, f 384 Imatinib mesylate. See Gleevec®
i(17q) in Immersion oil, 71, 76
B-ALL, t327 Immunoglobulin genes, 335, 336, 338, 340, 345, 350, 351, 428
chronic myelogenous leukemia (CML), 317, t317, f 318 Imprinting. See also Genomic imprinting; Uniparental disomy (UPD)
chronic myelomonocytic leukemia (CMML), 318, t322 disorders and assisted reproductive technology, 478–479
CLL, 343 IVF and ICSI, 224
pediatric medulloblastoma, t373 Imprinting center (IC), 477, 478
536 Index
IMT. See Inflammatory myofibroblastic tumor (IMT) Instability syndromes. See Chromosome instability syndromes
Inactivated X chromosome, 4 Instrumentation, cytogenetics laboratory
Inc. See Incomplete karyotypes (inc) acid fixative, 97
Incomplete karyotypes (inc), 38 application, 100–103
Induced abortion, 230 archival device, 102
Inducible T-cell kinase (ITK) gene, 356 automated imaging systems, 102–106, f 102–f 106
Infantile fibrosarcoma (IFS), 390, 392 benchtop device, 97
Infertility camera, 102
birth defects, 224–225 CGH, 106–107
chromosomal mosaicism, 224 components, 101, f 101
delayed puberty, 213, f 214 computer, 102
endometriosis, 216 drying chambers, 96–99, f 97, f 98
female, causes of, 213 enrichment, 100
hypothalamic and pituitary, 216–217, t217 for FISH, 99, 107
imprinting and IVF, 225 fixative, aspiration of, 97
inversions, 220 fluorescent spot counting, 103–105, f 104
male, causes of, 217 hybridization, 99–100, f 100
microdeletions of Y chromosome, 220, 222 in situ culture and harvesting, 97
micromanipulation, 222 in situ cultures, 96, 97
mitochondria of spermatozoa, 225 karyotyping, 101–103, f 102, 106
ovarian dysfunction, 213–217, f 214, f 215, t216, t217 limitations, 101
ovum and spermatozoan, f 223 M-FISH, 105–106, f 106
pregnancy rates, 214, 224–225 microscope with camera adapter, 102
preimplantation embryo development, 223–224 multipurpose instruments, 107, f 108, 109
robertsonian translocations, t218, 219, 220 pretreatment, 99, f 99
semen analysis, 217–218 printer, 102
semen specimens, 217–218 robotic harvesters, 95–96, f 96
sex chromosome abnormalities, 219 scanning and metaphase finding, 103
SRY gene and genetic sex, 217 slide-making process, f 98, 99
45,X and 45,X mosaicism, 213–214 software, 102
X-autosome translocation, 215, 216 suspension cultures, 95
X-chromosome deletions, 215 Integrated screening, 256, 257. See also Combined screening
Inflammatory myofibroblastic tumor (IMT), t391, 396, f 397 Interchange aneuploidy, 160
Infratentorial primitive neuroectodermal tumor (iPNET), 376 Interchromosomal aberrations, M-FISH, 105. See also Jumping
Inhibin A. See Dimeric inhibin A (DIA) translocations
Inhibitor Interchromosomal insertions, 166–167, f 166
microbial, 53, 54 Interlikin-2 (IL2), 55, t335, 338
mitotic, 57, 310 International MDS Risk Analysis Workshop (IMRAW), 316, t317
tyrosine kinase, 319–321, 371, 384, 385, 388, 390, 428 International Prognostic Scoring System (IPSS), MDS, 314–316
ins. See Insertions (ins) International System for Human Cytogenetic Nomenclature (ISCN),
ins(5;11)(q13.1;p15.3p13), f 165 23, 26, 31, 32, 35, 39, 41, 42, 44, 46, 47
ins(12;9)(p13.2;q34q34), t321 cytogenetic aberrations, short and derailed, t29–t30
Insertion (ins) definition, t311
definition, t49 description in final report, 88
description, t29 glossary of cytogenetic terminology used in hematologic
detailed ISCN, t29 neoplasms, t311
nomenclature of, 31, 34 punctuation and significance, t28
long ISCN, paracentric, t29 Interphase FISH, 44–45, 85, 86, 100–102, 104, f 105, 106, 243,
long ISCN, pericentric, t29 f 394, 418, 426. See also Nuclear (interphase) in situ
short ISCN, paracentric, t29 hybridization (nuc ish)
short ISCN, pericentric, t29 in confined placental mosaicism, 243
Insertion(s) Intersex conditions. See Disorders of sex development
complex chromosome, 165 Interstitial deletions, t29, 33, 42, f 146, 191, 333, 348, 378, 442, 476,
definition, t311 505, 506
incidence of, 165 Intrachromosomal
interchromosomal, 166–167, f 166 amplification, 295
intrachromosomal, 166, f 166 insertions, 156, 166, f 166
model for meiotic pairing of, f 166 mBAND, 433, f 435
Philadelphia chromosome, 6, 35, 317, 328 Intracytoplasmic sperm injection (ICSI), 222–223, f 223
of unknown material, f 433 and birth defects, 224–225
variant chromosomes, 167–168 chromosomal mosaicism, 224
In situ culture method, 260 imprinting and IVF, 225
In situ hybridization (ish), 43–44, 62, 263. See also Metaphase FISH micromanipulation, 222
nomenclature, 43 mitochondria of spermatozoa, 225
Inspections, laboratory, 93 ovum and spermatozoan, f 223
Instability of CGG repeat, 455–456 pregnancy rates, 214, 224–225
Index 537
preimplantation embryo development, 223–224 inv(17)(p13.2q21.33), in aneurysmal bone cyst, t391, 395, f 396
semen specimens, 217–218 inv(19)(p13.3q13.4), in children with B-ALL, 329
Intrauterine growth restriction (IUGR), 122, 124–126, 184, 242, 253, inv(X)(p11;q22), 193
t254, t260, 261, 262, 288, 473, 483, 484, 486–488 inv(X)(p11.23q13.1)/TFE3-NONO, in papillary RCC (pRCC), 380
Intrauterine mortality, t276 inv(X)(q26q28), f 193
inv. See Inversion (inv) inv(Y), in men with infertility, t218
inv(1), in men with infertility, t218 inv(Y)(p11q11), f 197
inv(2) in men with infertility, t218
common pericentric inversion, 258 inv(Y)(p11.2q11.2), 199
in mature T-cell lymphomas, t353 “inv dup(15)”, 129, 511. See also Inverted duplication of
in men with infertility, t218 chromosome 15
inv(2)(p11q13) Inversion (inv)
in men with infertility, t218 definition, t49
pericentric inversions, 152 description, t29
recurrent pericentric, 151, f 151 long ISCN, t29
inv(2)(p21p23.2), in non-small cell lung cancer (NSCLC), 323, nomenclature, 33
t385, 390 short ISCN, t29
inv(3) in Inversion(s), 151–156
AML, t323 de novo balanced structural rearrangements in amniocentesis, t232
AML (megakaryoblastic), t323 de novo incidence in prenatal diagnosis, 233
erythroleukemia (erythroid/ myeloid), t323 fetal deaths subsequent to amniocentesis, t232
men with infertility, t218 incidence in liveborns, 231
inv(3)(p13q25), in men with infertility, t218 in infertile men, t221
inv(3)(p25q21), recurrent inversion, 151 loop, 147, f 152, 153, f 154, 155, f 155, 156
inv(3)(q21.3q26.2) in, male infertility, t218
AML with inv(3) or t(3;3), t323, 325 mechanism, 139, 152, 155
AML with other chromosome abnormalities, 315 meiotic behavior of, 152–153, f 154, 155–156
AML with recurrent genetic abnormalities, 322, t323 multicolor banding of pericentric, f 435
inv(5), in men with infertility, t218 nomenclature of, 30
inv(5)(p13q13) paracentric, t29, 154–156, f 155, 220, t311
in men with infertility, t218 in a carrier parent, 258
recurring inversion, 151 definition, t311
inv(7)(p14q35), in ataxia telangiectasia, f 298 meiotic behavior, 155–156
inv(7)(p15.2q34), in T-ALL, t334 with multicolor banding, f 435
inv(8)(p23q22), recurring inversion, 151 nomenclature, 33
inv(9), population variant, 258 of the X chromosome, 193, f 193
inv(9)(p11q12), in men with infertility, t218 of the Y chromosome, 199
inv(9)(p11q13), f 151 pericentric, t29, f 151, f 152, f 154, 151–154, t311
inv(9)(p23q33), in men with infertility, t218 chromosomal variants, 167
inv(10) (p11.2q21.2), recurring inversion, 151 definition, t311
inv(11)(q21q23), recurring inversion, 151, 154 meiotic behavior, 152–154, f 154
inv(14) in in a member of a couple, 258
mature T-cell lymphomas, t353 and men with infertility, t218
T-PLL, t353, f 353 nomenclature, 33
inv(14)(q11q32), f 298 polymorphic variants, 139
inv(14)(q11.2q32.1) in prenatal results for, t259
T-ALL, 334, t334 risks for carriers, 152–154, f 154
T-PLL, 353, t353, f 353 sperm karyotyping on men with, 220
inv(14)(q11.2q32.3), in B-ALL with rearrangements of 14q32.3 of the X chromosome, 193
(IGH@), 333 of the Y chromosome, f 197, 199
inv(16), in AML, 324 polymorphism
inv(16)(p13q22) of chromosome 9, 218
in AML, 424 8p, 154
FISH for hematologic malignancies, t423 8p23, 154, 162
inv(16)(p13.1q22.1) in, Yp, 155
AML with inv(16) or t(16;16), t323, 323–324 recurring, 141, 151–153, f 151
AML with recurrent genetic abnormalities, 322, t323 risks for carriers, 152–154, f 154
CML, 317 unbalanced, 193
inv(16)(p13.1q22.1)/t(16;16)(p13.1;q22.1), in AML with gene of X chromosomes, 193
mutations, 326 of Y chromosomes, 154
inv(16)/t(16;16) in Inverted duplication, 129, 149, 192
AML with inv(16) or t(16;16), 323–324 of chromosome 15 (“inv dup(15)”), 129
AML with KIT mutation, t323 description, t29
myeloid sarcoma, 326 nomenclature, 33
systemic mastocytosis (SM), 320 8p, 30
inv(17)(p13.2q11), in aneurysmal bone cyst, 395 Inverted Y chromosome, 199
538 Index
In vitro fertilization (IVF), 113, 114, 213, 224, 225, 289, 422, 478 K
and 45,X and 45,X mosaicism, 214 KAL, t217
Ionizing radiation (IR), 69, 75, 76, 299 Kallman syndrome, 195
hypersensitivity to, in Nijmegen syndrome, 299 Kanamycin, 54
and transmitted light source, 69 Karyogram(s)
iPNET. See Infratentorial primitive neuroectodermal tumor (iPNET) in analysis protocols, 82
IR. See Ionizing radiation (IR) complex, f 339, f 341, f 350, f 354
IR emission, 76 definition, t49
IR excitation, 76 with gain of chromosome 15 and loss of Y chromosome
ISCN. See International System for Human Cytogenetic in MDS, f 316
Nomenclature (ISCN) hyperdiploid, of patient with B-ALL, f 331
ish. See Metaphase FISH (ish) M-FISH, f 107
Isochromosome (i) of a normal bone marrow cell, f 310
definition, t49, t311 partial
description, t29 illustrating common MLL (11q23) translocations observed in
long ISCN, t29 AML, f 325
nomenclature, 35 illustrating recurrent abnormalities observed in CLL, f 342
short ISCN, t29 recurrent abnormalities in MDS, f 314
Isochromosome(s), 157 showing recurrent (or specific) rearrangements in AML, f 324
and homologous Robertsonian translocations, 164 the most common translocations involving PDGFRA,
and Klinefelter syndrome, 186 PDGFRB, and FGFR, f 320
in MDS with other chromosome abnormalities, 315 of patient with
mechanisims for formation of, f 158 aggressive “double-hit” lymphoma, f 339
in plasma cell myeloms (PCM) with chromosome 1 adnormalities, atypical CLL, f 344
349, 350 B-ALL, f 332, f 333
of 12p, 127–128, t150, 157, f 384, 511. (see also Pallister-Killian B-cell PLL, f 346
syndrome; Tetrasomy 12p) B-CLL, f 328
of 7q, in hepatosplenic lymphoma, f 355 CLL, f 344
of 8q, in T-cell prolymphocytic leukemia (T-PLL), f 354 CML in blast crisis, f 318
supernumerary, 127, 129 CML in chronic phase, f 318
in upd(2)mat, 483 follicular lymphoma, f 337
in upd(9)mat, 485 Hairy cell leukemia, f 347
in upd(13)mat, 485 Hepatosplenic lymphoma, f 355
in upd(13)pat, 485–486 high-risk plasma cell myeloma, f 348
of X chromosome, 181, f 181 mantle cell lymphoma at diagnosis, f 339
Isoderivative chromosome (ider), 36 plasmablastic lymphoma, f 352
definition, t49 plasma cell myeloms (PCM), f 349, f 351
Isodicentric (idic) chromosome polycythemia vera (PV), f 319
definition, t49 T-cell prolymphocytic leukemia, f 354
Isodicentric chromosome(s) preparation using automated imaging, 102–103
chromosome X, with multicolor FISH, f 435 production, 77, 86–87, 100
formation of, 157, f 158 requirements for
nomenclature, 36 amniotic fluid, flask method, 83
supernermerary, 129 amniotic fluid, in situ method, 83
Xp chromosomes, 186, 193 bone marrow and unstimulated peripheral blood, 84
Xq chromosomes, 194 chorionic villus samples (CVS), 83
Y chromosomes, 180, 197–198, f 197 percutaneous umbilical blood sampling (PUB), 83
Isodicentric Y chromosome, 197, 198 PHA-stimulated blood (non-neoplastic disorders), 83
Isodisomy, 159, 164, 182, 184, 442, f 145, 480, 481, 483–488 solid tissue (non-neoplastic studies), 83
Isolated bright hyperechoic bowel, clinical outcome of solid tumors, 84
second-trimester finding of, t254 of tetrasomy 12p, t128
ITK-SYK, 356 of triploid fetus, f 125
IUGR. See Intrauterine growth restriction (IUGR) of trisomy 21, f 118
IVF. See In vitro fertilization (IVF) Karyotype(s)
abnormal, 36, 87, 215, 220, 223, 246, 275, 276, 278, 280, 287,
288, t288, 313, 319, 320, 326, 387
J balanced, 145, t373, 375
Jacobsen syndrome, 295, t465 cancer, t28, 31, 32, 36, 38, 40, 41
JAK2, 319, 322, 332, 333 complex, 84, 313–315, 325, 326, 337, 343, 346, 351, t353, 378,
JAK2-BCR, t321 t398, 403, 429, 443, 447
JAK2-ETV6, t334 composite (cp)
JAK2 V617F, 319 definition, t48
JAZF1-SUZ12, t379, 383 nomenclature, 40, 41
JMML. See Juvenile myelomonocytic leukemia (JMML) constitutional, 36, 156, 165, 167, 223, f 377
Jumping translocations, 165. See also Interchromosomal aberrations definition, 3, t49, 81
Juvenile myelomonocytic leukemia (JMML), 315, 321, 322, t322 describing cancer, 40–42
Index 539
descriptions for, 26–32, f 27, t28–t30, f 31 Leri-Weill dyschondrosteosis, 175, 196

detailed form, 44 and SHOX deletion, 195
detailed system, 31, 32, 35 Letter “c” in nomenclature, t28
diagrammatic representations, f 27 Letters/triplets, for chromosome aberrations, 28
fetal, 243, 263, 282, 286, 289, 508, 510 Leydig cells, 184, 200, 217
incomplete, 38, 248 L-Glutamine, 54
of normal G-banded male, f 24 LH. See Luteinizing hormone (LH)
short form, 44 LHB, t217
short system, 33 LHCGR, t216
string, 30, 32, 36 Light-emitting diodes (LEDs), 68, 69, 75
unbalanced, 34, 143, 157, 160, 162, 165–167, 277, 287 Light microscopy
Karyotyping. See also Cancer karyotypes beam splitter, 71
automated imaging systems, 101 brightfield microscopy, 67, 68, 72, 74, 76, 432
balanced paracentric inversion, 30 considerations, 76
balanced translocation, 30, 37 coordinate location, 71
chromosome aberrations, 30, 41 DIC, 72
clones, 30, 41 epifluorescence microscopy, 74
constitutional and acquired, 31 eyepieces, 71
definition, 143 filters, 75–76
mosaicism and chimerism, 39 fluorescence microscope, 74–75
numerical aberration, 31 light sources, 75
KCl. See Potassium chloride (KCl) microscope slides, coverslips, and mounting media, 72
KCNQ1, 477, 478 microscope stage, 71
KCNQ1OT1, 477–479 objective lens, 69–70, 74, 82
Kennedy’s disease, t455 optical and conjugate focal planes, 67
Köhler, August, 67 phase contrast, 72, 73
Köhler illumination, 67–69, 72, 74 Light path, 68, 69, 72, f 73, 74, f 75, f 108
KIAA1549/BRAF, t373, 374 Lipoblastoma, t391, 393
Kidney tumors. See Renal tumors Lipoma, t391, 393
Kinetochore Lipopolysaccharide from E. coli (LPS), t391, 393
apparatus, 13 Liposarcoma (LPS), t391, 393, f 394, 429
staining, 61 List of
KIT, t317, 320, 323, t323, 324, 326, 384, t385, f 428 common symbols and abbreviations used in molecular
KIT-PDGFRA, 384 cytogenetics, t43
Klinefelter syndrome (KS) commonly used banding and staining techniques in human
definition, 506 cytogenetics, t25
development, 185 glossary of commonly used ISCN terms, t48–t49
origin, 184 glossary of cytogenetics terminology used in hematologic
phenotype, 184, 186 neoplasms, t311–t312
and possible paternal age association, 503 ISCN abbreviations, t48–t49
KRAS, 326, 390 ISCN punctuation and significance, t28
KS. See Klinefelter syndrome (KS) short and detailed ISCN for common cytogenetic aberrations,
t29–t30
LLGL1, t418
L LMO1-TRA@, t334
Labeling LM02-TRA@, t334
and detection of probes, 42 LNX, f 428
errors, 78–80, 88, 90 Locus
fluorescent, 86, 104 definition, t311
primed in situ, 113, 434 designations, 42, 43
specimen, 78–79, 90 specific probe, 419, 431
Laboratory procedures, cytogenetics. See Cytogenetics laboratory Logbook, 79
procedures LOH. See Loss of heterozygosity (LOH)
Laboratory reports, 87–88, 240 LOI. See Loss of imprinting (LOI)
LAMB1, 42 Long interspersed elements (LINEs), 14
LAMB3, 482 Looped domains, 12
Landmark, 24,–25, f 26, 58 Loss of heterozygosity (LOH), 46, 254, 282, 287, 386, t398,
definition, 49 400, 442, 479
Langer-Giedion syndrome, t148 Loss of imprinting (LOI), 475, 478, 480
LCR. See Low-copy repeats (LCR) Loss of X, age-associated, 187–188
Leiomyoma, t391, 394 Loss of Y chromosome, age-associated, 188, 280
Leiomyosarcoma, t391, 394–395 Low-copy repeats (LCRs), 147, 149, 154, 157
Lejeune, Jérôme, 4, f 4, t391, 394–395 LPD. See Lymphoproliferative disorder (LPD)
LEP, t217 LPL. See Lymphoplasmacytic lymphoma (LPL)
LEPR, t217 LPS. See Lipopolysaccharide from E. coli (LPS);
Leptotene, 19 Liposarcoma (LPS)
540 Index
Lung cancer sex chromosome abnormalities, 219

non-small cell lung carcinoma, 390, 431 SRY gene and genetic sex, 217
squamous cell, 295 47,XXY, 222
Luteinizing hormone (LH) Male-specific region of the Y (MSY), 177
and chromosomally normal pregnancy loss, 286 Malignant peripheral nerve sheath tumors, 392
and female infertility, 213 Malignant rhabdoid tumor (MRT), 381, t391, 397
Lymph nodes, 54, 64, 353, 428 Malsegregation, 114, 144, 160, 164, 282, 481, f 481
Lymphoblastoid cell lines, 63, 479 MALT1, 340, 341
Lymphoma MALT lymphoma. See Lymphoma
acute B-cell lymphoblastic leukemia/lymphoma, 327–333 MAML2-CRTC1, t385, f 388
adult T-cell leukemia/lymphoma, 353, t353, f 354 Mantle cell lymphoma (MCL), 335, 336, 338, f 339, f 340, f 341,
anaplastic large cell, 353, 356 350, 428
anaplastic large cell l, ALK-positive (ALCL-ALK+), 353, t353 Mantle cell lymphoma, blastoid type (b-MCL), 335
angioimmunoblastic T-cell (AITL), t353, 356 mar. See Marker chromosomes (mar)
B-cell, 334–336, t336, f 337, 338, 340, 346, f 346, 351, 428 Marginal zone B-cell lymphoma (MZBCL)
Nijmegen breakage syndrome (NBS), 299 MALT type, t336
Burkitt (BL), 335, 336, t336, 338, f 338, 345, 428, 502 nodal, t336
Chronic lymphocytic leukemia (CLL)/small lymphocytic splenic, t336
lymphoma (SLL), 342 Marker chromosome (mar)
cutaneous T-cell lymphomas definition, 37, t49, t311
mycosis fungoides (MF), t353, 355 nomenclature, 33, 35, 37–38
Sézary syndrome (SS), t353, 355 Marker chromosome(s)
diffuse large B-cell lymphoma (DLBCL), 336, t336, f 337, 338 and advanced maternal age, 244
follicular lymphoma (FL), 335, t336, f 337, 341, 428, 445 assessment, t420
hepatosplenic T-cell lymphoma (HSTL), t353, 355 derived from chromosome 22, 130
Hodgkin lymphoma (HL), t353, t336, 338, 342, 352 derived from X chromosome, 180–181
human immunodeficiency virus (HIV)-related, 351 and duplications, 419–421, 433
lymphoplasmacytic lymphoma (LPL), t335, 336, 342 in either member of a couple, 258–259
mantle cell (MCL), t335, t336, 338, f 339, f 340, f 341, 350, 428 fetal deaths subsequent to amniocentesis, t232
marginal zone B-cell lymphoma (MZBCLa), t335, t336, 340–342 formation secondary to LCR-mediated nonallelic homologous
mature B-cell, ot lymphoid neoplasms, 334, t336 recombination, 139
mature T-cell, 352, t353, 355 found on CVS, 263
mucosa-associated lymphoid tissue (MALT) lymphoma, identication of origin with FISH, 85, 180–181, 233, 417, t420,
t335, 340–341 433, 434
nodal marginal zone B-cell (MZBCL), 341–342 identication of origin with microarray, 441, 444, 447
non-Hodgkin, 335, t335, 341, 342, 356, t423, 428 in infertile males, t221
peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), and interference with meiosis, 258
t335, 355–356 risk of de novo non-satellite marker, 511
plasmablastic (PBL), t336, 351–352, f 352 risk of de novo satellite marker, 510–511
precursor B-cell lymphoblastic leukemia/lymphoma (pre-B-ALL/ stains for identification of, 60–62
LBL), 327 supernumerary, 113, 129–130, 441, 489, 510
precursor T-cell lymphoblastic leukemia/lymphoma (pre-T-ALL/ in Turner syndrome, 182, 251
LBL), 327 of the X chromosome, 180, 420
small lymphocytic (SLL), t336, 342 of the Y chromosome, 180, 194
splenic marginal zone B-cell lymphoma (sMZBCL), t335, 341 Markers
Lymphoproliferative disorder (LPD), 55, t335, 426 biochemical, 250, 251, 257
Lyon hypothesis, 5, 175 cell surface, in acute leukemia of ambiguous lineage, 326–327
Lyon, Mary, 5, 175 definition, 419
giant, in soft tumors, t391
immunohistochemical, in clear cell sarcoma, 403
M Laboratory Challenges with Microarray Testing of, 447
MAD2L1, t391, 394 maternal serum markers, 247, 254, 256
MAF, 350, 351 microsatellite, 186, 330
MAFB, 350 polymorphic, 186
MAGEL2, 476 prognostic, in acute lymphoid neoplasms, 327–328
Magnification, vs. resolution, 70 prognostic, in B-ALL, 331
Mainline (ml), 40, t49 prognostic, in CLL, 345
Male infertility prognostic, in plasma cell myeloma, 349, f 350
causes of, 217 serum markers, 198
cytogenetic findings, t218 serum screening, 252, 254
and intracytoplasmic sperm injection, 222 sex chromosome, 420–421
inversions, 218, t218 ultrasound, of fetal aneuploidy, 253, 254, t255, 509
Klinefelter syndrome, 184 Mastocytosis, systemic (SM), 316, 317, 320
microdeletions of Y chromosome, 222 Maternal
Robertsonian translocations, 219 carrier for pericentric inversions, t259
semen analysis, 217–218 carrier for Robertsonian translocations, 259, t259
Index 541
circulation, fetal DNA in, 265 peripheral T-cell lymphoma, not otherwise specified
habitus, limitations imposed by, 254, 256 (PTCL-NOS), t353
nondisjunction, 116, 182, 184 Sézary syndrome (SS), 353, 355
triploid, 284, f 284 T-cell prolymphocytic leukemia (T-PLL), t353, f 354
uniparental disomy, 482 MB. See Medulloblastoma (MB)
47,XXY (Klinefelter syndrome) mBAND, analysis, 433, f 435. See also m-banding;
47,XXY (Klinefelter syndrome): of autosomal Multicolor banding
trisomies, t114 m-banding, 432. See also mBAND analysis; Multicolor banding
Maternal (mat) MCC. See Maternal cell contamination (MCC)
origin MCL. See Mantle cell lymphoma (MCL)
in nomenclature, t49 MDM2, t373, t391
Maternal age MDM4, t398
advanced MDS. See Myelodysplastic syndromes (MDS)
and aneuploidy detection with FISH, 421 MDS/MPN, U. See Myelodysplastic/myeloproliferative neoplasm,
and chromosome abnormality, 503 unclassifiable (MPN/MPD, U)
definition, 234, 244 MEC. See Mucoepidermoid carcinoma (MEC)
genetic counseling for, 507 MECOM, 315, 325
and incidence of trisomies, 231 Medicare, 78
and increased aneuploidy frequency, 114 Medulloblastoma (MB)
and nondisjunction, 117 adult, t373
and risk associated with inversion, 153 pediatric, t373
and risks for chromosome abnormalities, 244 MEG3, 480, 486. See also GTL2
and triploidy, 125 Meiosis, 8, 16, 18–20. See also Meiosis I; Meiosis II
and UPDs that result from “trisomy rescue”, 486 abnormal segregation during, 285
effect of, 421 crossing-over in pseudoautosomal regions during, 175
and incidence of chromosome abnormalities, 230, t232, 246 errors in, 114, 142, 278–280, 282
and monosomy X, 282 fertilization, 20
and nondisjunction, 114 formation of double-loop structures during, 166
and risks for chromosome abnormalities, 232, t503 isochromosome formation during, f 158
and risks for trisomy, f 21, t118 malsegregation, 160
and trisomy 9, 122 nondisjunction, 113, 114, 126, 282, 486
and trisomy 18, 119–120, f 120 oögenesis, 20, f 21
and 47,XXY (Klinefelter syndrome), 186 pairing in pseudoautosomal regions during, 200
Maternal cell contamination (MCC) spermatogenesis, 20, f 21
in amniotic fluid, 263 toleration of chromosome rearrangements in, 167
in chorionc villus (CVS) samples, 243 Meiosis I
in CVS for prenatal fragile X testing, 465 errors in, 114, 283
in early amniocentesis (EA), 238 and 47,XXY, 184
low, 243 nondisjunction, 19, 114, f 115
in samples from recurrent pregnancy loss, 286 and 47,XXX conceptions, 182
and trisomy 18, 243 and 49,XXXXY, 185
and trisomy 21, 263 substages of
Maternal serum alpha-fetoprotein (MSAFP), 121 anaphase I, 19, f 19
elevated metaphase I, 19, f 19
in mosaic trisomy 16, 262 prophase I, 19, f 19
in trisomy 8, 121 prophase I: diakinesis, 19
high, 233, 256 prophase I: diplotene, 19
Maternal serum screening prophase I: leptotene, 19
first trimester, 257 prophase I: pachytene, 19
second trimester, 247 prophase I: zygotene, 19
Mature B-cell disorders, stimulation of B-lymphocytes in, 55 telophase I, 19, f 19
Mature B-cell lymphoid neoplasms, 335 Meiosis II, 18, 20, f 20, f 21, 114, 126, 184, 186, 279, 283
associated recurrent chromosome abnormalities, t336 errors in, 114, 283
Mature B-cell neoplasm(s) and 47,XXY, 184
associated recurrent chromosome abnormalities, t336 failure, in origin of triploid, 126
culturing of, 334 nondisjunction, 114, f 116
Mature T-cell lymphomas and 47,XYY, 186
adult T-cell leukemia/lymphoma (ATLL), 353, t353, f 354 and 49,XXXXY, 185
anaplastic large cell lymphoma, ALK-positive Meiotic
(ALCL-ALK+), t353 behavior, for carriers of parscentric inversions, 155
angioimmunoblastic T-cell lymphoma (AITL), t353, 356 behavior, for carriers of pericentric inversions, 152
biologic and genetic characteristics of, t353 cell division in isochromosome formation, f 158
culturing method for, 334 configurations in Y;autosome translocation, 196
hepatosplenic T-cell lymphoma (HSTL), t353 crossing-over, 35
mycosis fungoides (MF), t353, 355 division, failure of in triploidy, 125
natural killer cell leukemia (NKCL), 353, t353 error in tetrasomy 12p, 127
542 Index
Meiotic (cont.) single nucleotide polymorphism, 442–443

errors in men with gonadal failure and low FSH undergoing types, 441
ICSI, 223 Microbial inhibitor, t53, 54
events resulting in unbalanced conceptions, in familial Microdeletion(s)
rearrangements 143 findings in men with infertility, t218, 220
malsegregation and predisposition to UPD, 481 genetic counseling, 258, 465–466, 505
nondisjunction, 114 male infertility, 196, 217
and UPDs that result from “trisomy rescue,” 486 and microduplications, 145, 147, 417–419
in 48,XXXY, 186 recurring, t148
in 48,XXYY, 185 syndromes, 149–151, 154, 258, t418, 418–419, 504, 505, 508
in 49,XXXXX, 184 of the Y chromosome, 220–222, t221, t222
pairing Microdeletion syndromes
abnormalities in isodicentric X chromosomes, 194 Angelman syndrome, 149–151, t150, 476–477, 505
abnormalities in X chromosome rearrangements, 190 DiGeorge syndrome, 43, 151, 418, 419, 505
in complex chromosome rearrqngements, 167 Kallman syndrome, 195
configuration for (1;9) translocation, f 160 Miller-Dieker syndrome, 150, 418–419, 506
of insertion chromosome, 166, f 166 Phelan-Mcdermid syndrome, 149
in insertions, 166, f 166 Prader-Willi syndrome, 151, 418, 477, 505
products, f 115, f 116 22q11.2 deletion syndrome, 505
segregation, 163 Smith-Magenis syndrome, 150, 418, 505–506
Melanoma velocardiofacial, 149
cutaneous, t385 Williams syndrome, 147, 154, 505
malignant, 302, 389, t398, 403 Microduplications, 140, 147–149, t148, 151
uveal, t385 and microdeletions, 417–419
Meningioma, 300, t373, 378, 446 recurrent, 192
atypical/anaplastic, t373 syndromes, 147, t148
Mesoblastic nephroma, congenital. See Congenital mesoblastic Microsatellite DNA, 14, 186
nephroma (CMN) Microscope slides, 42, 57, 71, 72, 80–82, 90, 96, 98, 107
Messenger RNA (mRNA), 10, 13 Microscopy. See Fluorescence microscopy; Light microscopy;
MEST, 484 Phase contrast microscopy
MET, t379 Miller–Dieker syndrome, t150, 418, 506
Metacentric, 13, 23, t150, 163, t311, 420, t420 Minisatellite DNA, 14
Metaphase(s) Minus sign (−) in nomenclature, t28, 43, t43
chromosome harvester, Hanabi-PII, 96 MIR16-1, t423, t426
definition, t311 MIR15A, t423, t426
finding, 100, 103, 106 Misdiagnosis, 90–91, 422
finding system, 103, f 104 Mitochondria
first pictograph of, f6 paternal, 225
labeling of, prints, 79 of the spermatozoa, 225
meiosis, 19 Mitogens
mitosis, 17, f 18 B-cell, 55, 334, 335, 338
Q-banded, f 5 CpG-oligodeoxynucleotides, 335, 345
scanning, automated, 101 DSP-30, 55, 335, 338, 426
selection of, 64 Epstein-Barr virus, 352
spreader, Hanabi, 98 interleukin-2, 55, 335
Metaphase FISH, 42–43, 45, 85 lipopolysaccharide from E. coli, 55
Methotrexate (MTX), 62 phytohemagglutinin, 55, 352
MF. See Mycosis fungoides (MF) pokeweed, 55
M-FISH. See Multicolor FISH (M-FISH) protein A, 55
mGluR theory, 458, 459 T-cell, 55
Microarray analysis, 45–47, t49 12-0-tet-radecanoylphorbol-13-acetate, 55
Microarray-based comparative genomic hybridization (aCGH), Mitomycin C (MMC), 297
441–442 Mitosis, 17, f 17, t18, f 21
Microarray or results from microarray testing, nomenclature (arr), t43 anaphase, 18, f 18
Microarrays. See also Comparative genomic hybridization (CGH); definition, 16
Array-based comparative genomic hybridization (CGH); errors in, 282
Array CGH; aCGH errors of, 18
advantages of, 447–448 metaphase, 17, f 18
challenges with, 447 nondisjunction, 18, 117
clinical applications, 443–447 prometaphase, 17
cytogenetic abnormalities, in children, 443–445 prophase, 17, f 18
fetal chromosome anomalies, 445 telophase, 18, f 18
genetic counseling, 447 Mitotic inhibitor, 57, 310
interpretation, 446–447 Mitotic stimulants, 53, t53, 55, 63. See also Mitogens
oncology, 445–446 MKRN3, 475, 476
prenatal cytogenetics, 445 ml. See Mainline (ml)
Index 543
MLL, 44, 45, 313, 323–329, t327, 343, 417, 423–425, t446 Mosaic (mos)
MLL (11q23) translocations, f 325 definition, t49
MLL2, 182 Mosaic/mosaixcism
MLL-AFF1, 328 in amniotic fluid, 83
MLL-MLLT1, 329 autosomal structural abnormality, 263
MLLT1, 325 autosomal trisomy, 262
MLLT3, 324, 329, 332 in amniocytes, 242
MLLT4, 325 vs. chimerism, t28, 39
MLLT10, 325 in chorionic villus sampling (CVS), 83, 237, 242–244, 260, 262,
MMC. See Mitomycin C (MMC) 263, 282
MMSET, 350 confined placental, 237, 241–243, 487
mn. See Modal number (mn) constitutional, 55
Modal number (mn) detected by aCGH and SNP arrays, 441
definition, 40, t49 detected by array CGH, 264
in hyperdiplopidy, 330 and dicentric chromosomes, 156
in nomenclature, 39–40 discrepancy in CVS, t242
Molar due to errors in mitosis, 278, 282
phenotype, 125 dynamic, 159
pregnancies, 284, 287 gonadal (germ cell), 119, 129, 142, 201, 257, 258, 378, 464
complete, 283, 284 gonadoblastoma in females with 45,X/46,XY, 177, 423
and errors in fertilization, 283 in situ vs. flask method, 55
Molecular analysis for FXS, 62 interphase FISH in, 85, 243
Molecular cytogenetics. See also Array CGH; Fluorescence in situ involving
hybridization (FISH)causes of male infertility, 217 isochromosome of Yp or Yq and a 45,X cell line, 197, 198
common symbols and abbreviations, t43 isodicentric chromosome Yp, 198
Moles isodicentric X chromosomes, 194
hydatidiform, 284, 287 ring Y chromosome, 198
partial, 287 tetrasomy and pentasomy X, 184
Monosomy uniparental disomy (UPD) in, 164, 481, 483, 485–489
autosomal, 188–189, 262, t277 45,X, in female infertility due to ovarian dysfunction, 213
definition, 124, t311 45,X, in women using in vitro fertilization, 214
mosaic, 124, 262 Y;autosome translocations, 196
partial, in upd(14)mat, 486 Yq deletions and a 45,X cell line, 197
rescue, 159, 481 and jumping translocations, 165
Monosomy 1, f 160 low-level, 55, 86, 123, 264
partial, f 160 in nomenclature, 39
1p36 syndrome, t148, t385 nondisjunction in mitosis, 117
Monosomy 3, in uveal melanoma, t385 in PHA-stimulated blood, 83
Monosomy 7 in pregnancies from couples with infertility, 224
in juvenile myelomonocytic leukemia (JMML), 315 prenatal cytogenetics, 241, 242
MDS with deletion of 7q or, 315 vs. pseudomosaicism in tissue culture, 244
probes for, in myeloid disorders, 416 PUBS to clarify possible, 244
Monosomy 9, f 160 rare trisomy, in in amniocytes, 263, t263
partial, f 160 reporting, secondary to bone marrow stem cell transplantation, t28
Monosomy 17, 262 residual disomy/trisomy, 164
in MDS with other chromosome abnormalities, 315 resolve possible, 88
Monosomy 18, in sperm of men Klinefelter syndrome, 222 r(Y), 198
Monosomy 21, 262 sex chromosome and age, 188
complete, 124 sex chromosome, with ICSI, 184
mosaic/mosaicism, 124, 262 solid tumors, 83
nonmosaic, 124 and supernumerary chromosomes, 157
partial, 124 tissue specific (limited), 127, 128, 142, 179
Monosomy 22, 262 trisomy 22, 122–123, 262
in meningeal tumors, 378 true, 55, 237
mosaic/mosaicism, 124, 262 true vs. pseudomosaicism, 260–261
Monosomy X, 123, 278, 280–282, f 281, 288. See also 45,X in Turner syndrome variants, 179
in age-related sex chromosome aneuploidy, 188 undetected, 178
and blastocyst rate in PGD, 224 with X chromosomes, 179, 180
in CPM with interphase FISH, 243 X/46, 179
inactivated X in, 187 45,X/46,XY, 177, 180, 181, 509, 510
mosaic, 260 45,X/47XXX, 39, 182, 214
nonviable, f 281 XXX, 182
and pregnancy loss, 277 XXY/46, 223, 506
and prenatal loss, t277 XY, 177,
in spontaneous abortions and live births, t277 with Y chromosomes, 177, 180–181, 281
Monosomy Y, mosaic, 260 Mosaics, and prenatal loss, 277
544 Index
Mosaic tetrasomy 12p, t150 pediatric medulloblastoma, t373

Mosaic trisomy 2, 123 soft tissue tumors, 429
Mosaic trisomy 3, 123 supratentorial primitive neuroectodermal tumor, 376
Mosaic trisomy 5, 123 cryptic translocation in blastoid mantle cell
Mosaic trisomy 6, 123 lymphoma, t335
Mosaic trisomy 7, 123, 243 Mycosis fungoides (MF), t353, 355
Mosaic trisomy 8, 121, f 241, 127, 243, 282 MYC-TRA@, t334
Mosaic trisomy 9, 122, 252, 260 Myelodysplastic/myeloproliferative neoplasms (MDS/MPN)
Mosaic trisomy 10, 123, 243 atypical chronic myeloid leukemia (aCML), 321, t321
Mosaic trisomy 12, 261 chromosome abnormalities in, 315
Mosaic trisomy 13, 121, 260, 261 chronic myelomonocytic leukemia (CMML), 321, t322
Mosaic trisomy 14, 123, 258 classification of, 321
Mosaic trisomy 16, 122, 242, 261, 262, 487 del(5q), t322
Mosaic trisomy 17, 124, 487 del(7q), 315
Mosaic trisomy 18, 120, 243, 260, 261 del(17p), 319
Mosaic trisomy 19, 124 description, 321
Mosaic trisomy 20, 122, 261 juvenile myelomonocytic leukemia (JMML), 321
Mosaic trisomy 21, 119, 252, 261 prognostic significance, 316
Mosaic trisomy 22, 122, 262, 488 with trisomy 8, 315
Mounting media, 72 types of, 312
MPL, 319 Myelodysplastic/myeloproliferative neoplasm, unclassifiable
MPN. See Myeloproliferative neoplasms (MPN) (MPN/MPD, U), 321
MPN/MPD, U. See Myelodysplastic/myeloproliferative neoplasm, Myelodysplastic syndromes (MDS)
unclassifiable childhood, t313
MRT. See Malignant rhabdoid tumor (MRT) with deletion 7q or monosomy 7, t315
MSAFP. See Maternal serum alpha-fetoprotein (MSAFP) International Prognostic Scoring System of common abnormalities
MSY. See Male-specific region of the Y (MSY) in, t316
MSY genes, 177 with isolated del(5q), t313
MTX. See Methotrexate (MTX) myelodysplastic syndrome–unclassifiable, t313
MUC, 350 with other chromosomal abnormalities, 315
Mucoepidermoid cancer, t385 percent of chromosomal abnormalities in subdivisions of, t313
Mucoepidermoid carcinoma (MEC), 387–389, f 388 prognostic significance of chromosome abnormalities,
Multicolor banding, 433, f 435. See also mBAND analysis; m-banding 315–316, t316
Multicolor (multiplex) FISH (M-FISH), 84, 105–106, f 107, 419–421, recurrent chromosomal abnormalities in, t313, 314
429, 432–433, f 434, 436 refractory anemia with excess blasts-1 (RAEB-1), t313
Multiple myeloma, 433, 435. See also Plasma cell refractory anemia with excess blasts-2 (RAEB-2), t313
myeloma (PCM) refractory anemia with ring sideroblasts (RARS), t313
Multiplex (multicolor) FISH (M-FISH), f 434 refractory cytopenia with multilineage dysplasia
Multiplication sign (x) in nomenclature, t28, t43 (RCMD), t313
Multiprep robotic harvester, 96 refractory cytopenia with unilineage dysplasia
Multiradial configuration(s) (RCUD), t313
in Fanconi anemia, 297, f 297 revised prognostic scoring system of chromosome abnormalities
in ICF syndrome, 299, f 299 in, t317
MYB, 45, 342, 343, 346, 426, f 427 therapy-related, 312, 313, t313, 314, 315
MYC with trisomy 8, 315
amplification in Myeloid leukemia associated with Down syndrome, t323
gastrointestinal stromal tumors, 384 Myeloid proliferation related to Down syndrome, t323
osteosarcoma, 395 Myeloid sarcoma, t323, 326
in medulloblastoma, t373, 376 Myeloproliferative neoplasms (MPNs), 312, 321, t321
as part of ALL FISH probe panel, f 427 characterization, 316
in pediatric medulaoblastoma, t373 chronic eosinophilic leukemia, not otherwise specified
in pleomorphic adenoma, 387, t387 (CEL, NOS), 320
in prostate cancer, 381 chronic myelogenous leukemia (CML), 316–317, t317
rearrangements in plasma cell myeloma, 351 chronic neutrophilic leukemia (CNL), 320
in supratentorial primitive neuroectodermal tumor, 376–378 classification of, 316–317, t317
translocations in cytogenetic abnormalities, 316
Burkitt lymphoma, 336 essential thrombocythemia (ET), 316, t317, 319
plasmablastic lymphoma, t336, f 352 myeloid and lymphoid neoplasms, 321
plasma cell myeloma, t336 polycythemia vera (PCV), 316, t317
prolymphocytic leukemia, t336 primary myelofibrosis (PMF), 316, t317, 319
MYC-IGH@, 428 systemic mastocytosis (SM), 316, t317, 320
MYC-MYCN amplification, 378 unclassified (MPN,U), 316, t317, 321
MYCN MYEOV, 350
amplification in MYH11, 324, 424
medulloblastoma, t373 Myotonic dystrophy (DM), 455, t455
neuroblastoma, t398, f 401 MZBCL. See Nodal marginal zone B-cell lymphoma (MZBCL)
Index 545
N and 48,XXXY, 186

NAACLS. See National Accrediting Agency for Clinical Laboratory and 48,XXYY, 185, 186
Personnel (NAACLS) and 49,XXXXX, 184
Nasal bone, absent or hypoplastic, and trisomy 21, 250–251, t255 and 49,XYYYY, 187
National Accrediting Agency for Clinical Laboratory Personnel Nonhistone proteins, 11–13
(NAACLS), 92 Non-Hodgkin lymphoma (NHL), 335, t335, 342, t423, 428
National Society of Genetic Counselors (NSGC), 499 B-cell lymphoma(s), 299, t335, 340
Natural killer cell leukemia (aggressive type) (NKCL), 353, t353 unclassifiable, with features intermediate between diffuse large
NB. See Neuroblastoma (NB) B-cell lymphoma and Burkitt lymphoma, 338
NBS. See Nijmegen breakage syndrome (NBS) unclassifiable, with features intermediate between diffuse large
NCOA1-PAX3, t398 B-cell lymphoma and Hodgkin lymphoma, 338
ND filter. See Neutral density filter Burkitt lymphoma (BL), 355, t335, 336, t336, f 338
NDN, 476 diffuse large B-cell lymphoma (DLBCL), 335, t335, 336,
Near-diploid, 32, 393, 400 t336, 338
Near-haploid, 32, 329, 330 FISH, 428
Near-tetraploid, 32, 41, 347, 393 follicular lymphoma (FL), 335–336, t335, t336, f 337, 428, 445
Near-triploid, 32, 400 lymphoplasmacytic lymphoma (LPL), t335, 342
Neocentromere (neo) mantlecelllymphoma(MCL), 335, t335,t336, 338, f 339, 340, f 339,
definition, t49 f 340, f 341, 350
nomenclature, 37 marginal zone B-cell lymphoma, t336, 340–341
Neocentromere(s), 156–158, 199 mucosa-associated lymphoid tissue (MALT), 335, t335, t336,
Neonatal deaths. See Fetal loss; Stillbirths and neonatal deaths 340–341
NER-deficiency syndromes. See Nucleotide excision and nodal marginal zone B-cell lymphoma (MZBCL), t336, 341–342
repair-deficiency syndromes splenic marginal zone B-cell lymphoma (sMZBCL), t335, 341
Neural tube defects (NTD) Nonhomologous end jointing (NHEJ), 141, 147, 161
evaluation of pregnancy loss, 287 Nonmosaic trisomy, 242
and high maternal serum alpha-fetoprotein, 256 Nonmosaic trisomy 13, 121
multifactorial conditions and, 502 Nonmosaic trisopmy 16, 261
previous child with, 258 Nonmosaic trisomy 18, 243
screening of amniotic fluid for, 508 Nonmosaic trisomy 20, 122, 261
Neuroblastoma (NB), 371, 400–401, t401, 480 Nonmosaic trisomy, 21, 117, 119
chromosomal aberrations in, t398 Nonmosaic trisomy 22, 488
histology, 400 Non-obstructive azoospermia, 218–220
with MYCN amplification, t398 Nonseminomatous NSCTs (gonadal germ cell tumors; NSGCT), 383
without MYCN amplification, t398 Non-small cell lung cancer (carcinoma) (NSCLC), 390, 431
Neutral density (ND) filter, 68, 75 NOR banding (staining), t25, 26, f 61, 87
NF. See Nuchal fold (NF) Normal karyogram, of bone marrow cell, f 310
NF1, 384, t391, 394 NORs. See Nucleolar organizing regions (NORs)
NF2, t373, 375, 378 NOTCH1, t334
NHL. See Non-Hodgkin lymphoma (NHL) NPAP1, t476
Nijmegen breakage syndrome (NBS), 299 NPM1, 321, 324, 326, 356, f 356, 476
clinical features, 299 NR4A3, t391, 395
NMN, 299 NR5A1, t216
Nitrogenous bases, 9, 10, 15 NR4A3-EWSR1, t391
NMN, 299 NRAS, 326
Nodal marginal zone B-cell lymphoma (MZBCL), t336, 341–342 NR4A3-TAF15, t391, 395
Nomenclature, 23–47. See also International System for Human NR4A3-TCF12, t391, 395
Cytogenetic Nomenclature (ISCN) NR0B1, 199, t217
describing cancer karyotypes, 31, 32, 36, 38, 40, 41 NSCLC. See Non-small cell lung cancer (NSCLC)
glossary of terms used inv, t48–t49 NSGC. See National Society of Genetic Counselors (NSGC)
karyotype descriptions, 26 NTD. See Neural tube defects (NTD)
string, 26, t29, 32, 37–42, 44, t48, t49 NTRK3, t321, t379, 381, f 382, t385, 390–392, t391
Nondirectiveness, principle of, 499 nuc ish. See Nuclear (interphase) in situ hybridization (nuc ish)
Nondisjunction Nuchal fold (NF), 245–247, f 246, t255. See also Nuchal thickening;
age-associated (age association), 280, 421 Nuchal thickenss, Nuchal translucency
chromosome susceptibility to, 280 ultrasound marker in fetal aneuploidy, t255
genetic predisposition to, 117 Nuchal thickening, 245–247. See also Nuchal fold; Nuchal thickenss;
maternal, 116, 182, 184 Nuchal translucency
meiosis, 114 ultrasound marker in fetal aneuploidy, t255
meiosis I (MI), 114, 115, f 115, 116, f 482 Nuchal thickness, 245–247, 250, 251. See also Nuchal fold; Nuchal
meiosis II (MII), 114, 116, f 116 thickening; Nuchal translucency
mitotic, 113, 119, 126, 281, 282, f 482 Nuchal translucency, 245–247, 250, 251, t255, 257, 508, 509. See also
and origin of isochromosmes, 157 Nuchal fold, nuchal thickening; Nuchal thickness
paternal, 186, 280 Nuclear (interphase) in situ hybridization (nuc ish), t43. See also
postzygotic, 126, 186, 281, 282 Interphase FISH
recurrent, 257, 279, 282 nomenclature, t43, 44–45
546 Index
Nucleolar organizer/organizing regions (NORs), 12, 13, t49, f 61, Ovarian function, 182, 189, 190, 215, t216
163, 199 gene mutations affecting, t217
Nucleosome, 12 Ovarian teratoma, 473
Nucleotide excision and repair (NER)-deficiency syndromes, 303 Ovotesticular disorders of sex development, 200–201
Numerical aperture (NA), 69, 70, 72, 81 Ovulation disorders, 213
Numerical chromosome abnormalities, and ploidy, 124 Ovum, 20, t21, 125, 126, 186, 214, 222, f 223, 225
blighted, 283
O
OA. See Oligoastrocytoma (OA) P
Obesity, 126, 129, t148, 192, t217, t418, 460, 476, 505 17p13, 381, 426, 487
in Prader-Willi syndrome, f 151, 164 PA. See Pilocytic astrocytoma (PA)
Objective lens Pachytene, 19, 115, 188, 196, 219, 220
achromat, 70 PAFAH1B1, 418, t418
apochromat, 70 Painter, T.S., 3
correction collar, 71 Paint(s)
fluorite, 70 chromosome, t48, 416, 433
immersion, 70–71, 76 partial chromosome, 433, f 435
non-coverglass correction, 71 Pallister-Killian syndrome, 123, 127, 128, t150, 263, 511. See also
oil, 71 Tetrasomy 12p
phase contrast, 72–73, 76 Panel(s), FISH, f 427
plan, 70 for ALL, 425
resolution vs. magnification, 69, 70 for B cell neoplasm (disorders), 426, t427
OCA2, 486 for CLL, 426
Octamer, 11, 12 oncology, nomenclature, 45
OD. See Oligodendroglioma (OD) for plasma cell myeloma, 426, t427
Okazaki fragments, 10, f 11, 456 Panel, triple marker screen, 256
Oligoastrocytoma (OA), anaplastic, t373, 375 Papillary renal cell carcinoma (pRCC), t379, 380, f 380
Oligodendroglioma (OD), 372, t373, 374, 375, f 375, 431 PAPP-A. See Pregnancy-associated plasma protein A (PAPP-A)
anaplastic, t373, 374, 375 PAR1, 175–177, f 176, 196, 199. See also Pseudoautosomal
Oligospermia regions
in infertile men, 218, t221 PAR2, 175–177, f 176, 199. See also Pseudoautosomal regions
and the Y chromosome, 177, 196, 218, 220, t221 Paracentric inversion, 30, 33, 147, 151, 154–156, f 155, 162, 166, 193,
Oligozoospermia. See Oligospermia 199, 220, 258, f 435
OMD, 395 definition, t311
OMD-USP6, t391 description, t29
Oncocytoma, 378, t379 mechanism of formation, 129, 139–143, 162
renal, 381 meiotic behavior and risk for carriers of, 152–156
Oncogene, 295, t311, 312, 335, 338, 351, 378, 381, 390, 393, 395, nomenclature of, 30, 33
403, 429 recurrent, 154
Oncology of the X chromosome, 193
FISH probe panels, 45 Paraganglioma (PGL), 395, 479
microarrays, 46–47 Parental inheritance, 38
Oöcyte, 20, f 21, 113–117, 178, 225, 280, 422, 462 Parental origin, 473
aneuploidy, 113–115, 117, 280, 422 of autosomal trisomies, 114, t114
primary, 20, 283 of monosomy X, 281
secondary, 20 in nomenclature, 38, 46
Oögenesis, 20, f 21, 141, 144, 163, 278, 462, 475 of ring chromosomes, 181–182
errors in, 278 in triploidy, 125–126
and spermatogenesis, 20, 21 of uniparental disomy in confined placental
Oögonia, 20, f 21, 115, 141 mosaicism, 242
Oromandibular-limb hypogenesis syndrome, and CVS, 238 Parentheses ( ) in nomenclature, t28, 30, 32, 42, 44
OS. See Osteosarcoma (OS) Partial gonadal dysgenesis, 201
Osteosarcoma (OS), t373, 378, t379, 382, t391, 395, 400, 480 pat. See Paternal (pat)
Ova, 20, 275, t276, 289 Patau syndrome, 5, 278. See also Trisomy 13
Ovarian failure, 178, 187, 214, 231 Paternal
and balanced X;autosome transloctions, 188 age, advanced, 259–260, 503
complete, 191 allele, 475–476
and deletions of Xq, 191 carrier
and FMR1 muttion, t216 of chromosomal rearrangement, 258
and GALT muttion, t216 for pericentric inversions, t259
premature, 188–191, 215, f 215 for Robertsonian translocations, t259
primary, 184, 188, 191, f 215 deletion in Prder-Willi syndrome, t148, t477
secondary, 188, 189, 191, 214 de novo mutation, in retinoblastoma and osteosarcoma, 480
and X chromosome;autosome translaoctions, 216 diploids, 287
and X chromosome deletions, 215 disomy, f 285
Index 547
duplication Pericentric inversion, 139, 151–155, f 151, f 152, f 154, 167, f 197,
in Beckwith-Wiedemann syndrome, t150, 477 t231, 258, t259
in paraganglioma, 480 definition, t311
gametogenesis, 186, 280 deletion and duplication, 193
genome, 473 description, t29
gonald mosaisism, 119 inversion heterozygote, f 152, 153
meiotic errors, 117 mechanism of formation, 139–142
non-disjunction, 113, 117, 280, 282, 486 meiotic behavior and risks for carriers, 152–156
origin, t49, 283, 285 meiotic pairing, f 152
of autosomal trisomies, t114, 280 in men with infertility, t218, 200
de novo Xp duplications, 191 nomenclature, 33
of extra chromosomes in polysomy X syndromes, 186 prenatal results, t259
of isochromosome X, 181 recurring, f 151
in nomenclature, 38, t49 of the X chromosome, 193
of ring X chromosome, 181 of the Y chromosome, 199
of structural chromosome rearrangements, 142 Pericentromeric
in triploidy, 125–126 heterochromatin, 299
of the X chromosome in Turner syndrome, 178 polymorphic regions, 60
in 47, XXY (Klinefelter syndrome) 164 regions, 59, 61, 157, 167, 197, 216, 443, 446, 487
in 47,XYY, 186 Period (.) in nomenclature, t28, t43
triploids, 283, f 284 Peripheral primitive neuroectodermal tumor (pPNET), t398, 401–402
uniparental disomy (UPD), 164, 242, 385, 386, 477, t477, Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS),
480–489 t353, 355–356
X chromosome, inactivation of, 176 Personnel requirements, 82, 91, 92
Paternal (pat) PEX10, 482
definition, t49 PGD. See Preimplantation genetic diagnosis (PGD)
inheritance, in nomenclature, 38 PGL. See Paraganglioma (PGL)
Patient database, 79 PHA. See Phytohemagglutinin (PHA)
PAX, t398 Phase contrast
PAX3, 398, 429 microscope, 73, 97
PAX5, 332, 333 microscopy, 72, 96
PAX7, 398, 429 objectives, 73
PAX3-FOXO1, 398, t398, f 399 system, f 73
PAX7-FOXO1, 398, t398, f 399 Phase ring, 72, 73, f 73
PAX fusion negative ARMS, 398, t398 Phelan-Mcdermid, t149
PBL. See Plasmablastic lymphoma (PBL) Phenol red, 54, 57
PBRM1, 380, t391 Phenylketonuria (PKU), 503
PBX1, 329, 425 PHF1-EPC1, t379
PBX1-TCF3, t327, 425 PHF1-JAZF1, t379
pcc. See Premature chromosome condensation (pcc) PHI. See Protected health information (PHI)
pcd. See Premature centromere division (pcd) Philadelphia (Ph) chromosome, 5, 6, f 6, 7, 35, 36, 64, 309, 310,
PCM. See Plasma cell myeloma (PCM) 316–317, f 318, 328, 424, f 425, 502
PCM1-JAK2, t321, 322 in B-ALL, 328
PCOS. See Polycystic ovary syndrome (PCOS) in chronic myelogenous leukemia (CML), 316–317, f 317, f 318,
PCR. See Polymerase chain reaction (PCR) 328, 424, 502
PCS. See Premature centromere separation (PCS) discovery of, 7, 309, 317
PDGFRA, 320–321, 384, 385, 428 pH indicator, 54, 57
PDGFRA-FIP1L1, 320, 328, 428 Photograph, of first Q-banded cell, f 5
PDGFRB, 320–321, f 320, 321, 384, t385, 428, f 428 Phytohemagglutinin (PHA), 5, 55, 83, 128, 187, 352
PDGFRB-ETV6, 321 Pilocytic astrocytoma (PA), t373, 374, f 374, t385, 387
Pedigree, f 144, 284, 500, f 501 Pituitary
PEG1, 484 causes of female infertility, 213, 216–217
PEG10, 484 function in females, gene mutations affecting, t217
Penetrance, 145 PKU. See Phenylketonuria (PKU)
of FXS, 453 Placental mosaicism, confined. See Confined placental
incomplete, t148, 168, 477 mosaicism (CPM)
reduced, 453, 463, 502 PLAG1, 387, t391, 393
Penicillin/streptomycin, 54 Plasmablastic lymphoma (PBL), t336, 351–352, f 352
Pentasomy X (XXXXX), 184 Plasma cell myeloma (PCM), t336, 340, 346–352, f 348–f 352, t423,
Percutaneous umbilical blood sampling (PUBS), 83, 230, 243–244, 426, f 427, 428, 445
261–263, 510 chromosome 1 abnormalities, t231, 246, 251, t255, 349–350,
benefits, 243 t373, 447
history, 230 chromosome 13, deletion/loss of, 348–349, f 348
limitations, 243–244 complex karyogram in, f 350
risks, 243–244 del(17p), t313, 315, t317, 319, t336, 342, 343, 351, t353
specimen requirements, 244 FISH, 346, 350, t423, 426
548 Index
Plasma cell myeloma (PCM) (cont.) Post-zygotic

hyperdiploid karyogram in, f 349 errors, 282
hyperdiploidy, 347–348 non-disjunction, 126, 186, 281
hypodiploid karyogram in, f 348 Potassium chloride (KCl), 57
hypodiploidy, 347–349 pPNET. See Peripheral primitive neuroectodermal tumor (pPNET)
IGH@ rearrangements, 350–351 PPP1R9A, 484
MYC rearrangement(s), 351, f 351 Prader–Willi syndrome (PWS), 129, f 151, 164, 242, 417, f 418,
Karyogram in patient with, f 351 474–478, t477, f 478, 486, 505
Plasmacytoma, 426–428. See also Plasma cell myeloma etiology, t477
Pleomorphic adenoma (PA), t385, 387, f 387 genetic counseling, 505
PLL. See Prolymphocytic leukemia (PLL) and genomic imprinting, 476, 477, 486
Ploidy laboratory diagnostic approach, 478, f 478
analysis in prenatal, 7, 417, t421 phenotype, t418
aneuploidy, 113–130, 160, 164, 177, 187–188, 190, 198, 219, 223, recurrence risk of, t477
242, 247, 250, 251, 253–254, t255, 256–258, 278, pRCC. See Papillary renal cell carcinoma (pRCC)
280–282, 287, 289, 293, t311, 330, 416, 421, 422, 431, Pre-analytical testing components, 78
480, 503, 506–509 Precursor B-cell lymphoblastic leukemia/ lymphoma
changes and clonal evolution, 443 (pre-B-ALL/LBL), 327
definition, 32 Precursor T-cell lymphoblastic leukemia/lymphoma
diploidy, 223, 280 (pre-T-ALL/LBL), 327
euploidy, f 482 Pregnancy-associated plasma protein A (PAPP-A), 246, 251, 257, 508
hyperdiploidy, 32, 187, 327, t327, 330–331, t336, 347–348, f 349, Pregnancy loss. See also Fetal loss; Spontaneous abortion
425, 428, 447 after chorionic villus sampling, 229
hypodiploidy, 293, t293, 327, 329–330, t336, 347–348, 443, 447 and chromosomal abnormality, 276
numerical abnormalities and, 32 chromosomally normal, 219, 245, 286
polyploidy, 113, 124, 219, 263, 287, 289, 293 evaluation of, 276, 287–289
tetraploidy, 126, 231, 257, 282 and familial rearrangements, 142–145
triploidy, 125–126, 231, 252, 257, 283, t398, 421, 442, 473, 511 and gestational age, 240, 244, 276, t276, t277
Plus sign (+) in nomenclature, t28, 33, 37, 41, 43, t43 outcome in early amniocentesis, t236
PLZF, 324 recurrent, 285–288, 505
PMF. See Primary myelofibrosis (PMF) specimens for cytogenetic studies, 286–287
PML, 324, 423, t423 spontaneous, 143, 178, 224, 234, 241, 276, 278, 280, 286, 288
PML-RARA, 324, 371 structural rearrangements in, 284–286
PNET, supratentorial, t373, 376, 378 and unbalanced chromosome rearrangements, 505, 507
POF. See Premature ovarian failure (POF) Preimplantation genetic diagnosis (PGD), 224, 259, 422, 462
POI. See Premature ovarian insufficiency (POI) Preliminary reports, 79, 88, 230
Pokeweed, 55 Premature aging syndrome. See Werner syndrome
Polar body(ies), 114, 422 Premature centromere division (pcd), 187
first, 20, f 21 definition, t49
second, 20, f 21, 125, 126 Premature centromere separation (PCS), 300, f 300
Polycystic ovary syndrome (PCOS), f 214 Premature chromosome condensation (pcc), definition, t49
Polycythemia vera (PV), 316–317, 319, t317, f 319 Premature ovarian failure (POF), 188–191, f 215
Polymerase chain reaction (PCR), 86, 99, 180, 181, 197, 220, 302, Premature ovarian insufficiency (POI), 183, 461, 462
328, 331, 338, 350, 351, 416, 434, 447, 464, 479 Premature separation of sister chromatid, 114–116, f 115
Polymorphisms Prenatal detection of chromosomal abnormalities via FISH, 421
analysis using linked, 454–455 Prenatal detection of preimplantation/embryo studies via FISH, 422
based single nucleotide microarrays, 39, 46, 442–443, 511 Prenatal ploidy analysis via FISH, 7, 64, 421–422, t421, t422
C-banding for clarification of, f 61 sex chromosome abnormalities, 422–423
cytogenetic, 64 Prenatal cytogenetics, 7, 223, 229–265, 282, 511
DNA, 119, 125, 126, 454, 486, 488 Prenatal diagnosis, 229–264
identification of, 61 amniocentesis, 214, 233, 454
inversion, 147, 154, 155, 162 chorionic villus sampling, 214, 237–238, 282, 454, 503, 505
linked, for fragile X diagnosis, 454–455 early amniocentesis, 64, 235, 237–238
microsatellite, 39 incidence of de novo structural rearrangements, 141, 143, 231,
notation of, on analysis worksheet, 86 t232, t233
single nucleotide, 6, 39, 46, 81, 264, 287, 330, 385, 421, 441, 511 indications for, 53, 230, 244, 264, 465
Y chromosome, 198 noninvasive, 233, 265
Polyploidy, 113, 124, 219, 263, 287, 289, 293, t311 percutaneous umbilical blood sampling, 64, 83, 243–244, 510
chromosome number in, 32 ploidy analysis/assessment, 421–422, t421
definition, 124 role of arrays in, 511
karyogram, f 118 Prenatal FISH, 421, 508
nonmosaic and mosaic, 113 Prenatal identification of a chromosome abnormality, 509–511
tetraploidy, 126, 231, 257, 282 Prenatal screen, abnormal, 503, 508–509
triploidy, 125–126, 231, 252, 257, 283, t398, 421, 442, 473, 511 Pre-T-ALL/LBL. See precursor T-cell lymphoblastic leukemia/
Position effect, 143, 188–190, 199 lymphoma (Pre-T-ALL/LBL)
Post-analytical testing components, 87–88 Pretreatment, in FISH procredure, 99, f 100
Index 549
Primary amenorrhea, 188, 213, 215, t216 Pseudomosaicism, 83, 235, 236, 238, 244, 510
Primary constriction, 23, 24, f 26, t48, 156, 157. See also and true mosaicism, 55, 260–262
Centromeres (cen) psu dic. See Pseudodicentric chromosomes (psu dic)
Primary myelofibrosis (PMF), 316, t317, 319 psu dic(20;20)(p13;p13), in upd(20)pat, 488
Primary oöcyte, 20, f 21, 283 psu idic. See Pseudoisodicentric chromosomes (psu idic)
Primary ovarian failure, 184, f 215 +psu idic(9)(q21.1), f 156
Primary spermatocyte, 20, f 21 PTCL-NOS. See Peripheral T-cell lymphoma, not otherwise specified
Primed random in situ hybridization (PRINS), 113, 432, (PTCL-NOS)
434, f 435 PTEN, t373, 374, 375, t379, 381, 382, t385, 389
Principle of nondirectiveness, 499 PTH1R, t379, 380
PRINS. See Primed random in situ hybridization (PRINS) PUBS. See Percutaneous umbilical blood sampling (PUBS)
Printer, imaging systems, 101, f 101, 102 Punctuation, ISCN, t28
PRKX, 154 Pure erythroid leukemia, t323
PRKY, 154 PV. See Polycythemia vera (PV)
Probes PWRN1, 476
alpha satellite DNA, 14, 129, 157, 300, 415, 420, f 420 PWS. See Prader–Willi syndrome (PWS)
break-apart, 43, 44, 329, 341, t423, 424–426, 428, 429, 431
centromere-specific, 157, 415
control, f 418, f 420, 432 Q
directly labeled, 417 11q23, abnormalities, t313
dual color, 340, f 420, 423, t423, f 425, 428, f 429 14q32, 34, 36, 428, 486
dual fusion, 44, 423, t423, 424, f 425 QA. See Quality assurance (QA)
interphase, 85–86 QA/QC program, 77
metaphase, 85 Q-bands/banding (quinacrine banding), t25, f 59, 74, 198, f 481
painting, 181, 416, f 416 idiograms of, f 27
panel of, 426, f 427 QC. See Quality control (QC)
radioactive, 415 QF-bands/banding, t25
repetitive sequence, 415–417 QFH-bands/banding, t25
single copy, 417, 421, 434 QFQ bands/banding, t25
subtelomere, f 154 QM. See Quinacrine mustard (QM)
unique sequence, 415–417 qr. See Quadriradial (qr)
whole chromosome, 85, 416 Quad/quadruple screen, 256–257, 302, 509
Proficiency testing, 91–92 Quadriradial (qr), definition, t49
Prolymphocytic leukemia (PLL), t336 Quadriradial
B-cell, t335, 345, f 346 configurations in Bloom syndrome, symmetric, 5, 38, 296,
T-cell, 353, t353, f 354 302, 506
Prometaphase, 17, 59, 62, 142 formation, 302
PROP1, t217 Quality assurance (QA)
Prophase accreditation and inspections, 91–92
chromosomes, 62 in Canada, 92
meiotic, 19, f 19, 20, f 115, f 116, 160 compliance training, 94
mitotic, 17, f 18 culture failure, 62–63, 89, 263, 448
Prostate cancer, 381–382 external proficiency testing, 91–92
Protected health information (PHI), 93 HIPAA, 93, 94, 101
Protein synthesis, 10–11, f 15, 17, 457–459 international, 92–94
mRNA processing, 10, 222 labeling errors, 78–80, 88, 90
protein release, 11 laboratory staff qualifications, 92
transcription, 10 misdiagnosis, 90–91
translation, 11 post-culturing errors, 89, 90
Proto-oncogene, 380, 424 reference laboratories, 93
P2RY8, 333 safety, 77, 93
Pseudoautosomal regions. See also PAR1; PAR2 specimen failures, 89
of X chromosome, 19, 175, f 176 state licensure, 92
X/Y, 19, 175, f 176 Quality control (QC)
of Y chromosome, 19, 175–177, f 176 amniotic fluid, 54, 83, 262, 263
Pseudodicentric chromosome (psu dic), nomenclature, 36 analysis worksheets, 86, 90
Pseudodicentric(s) analytical testing components, 78, 79, 87–88
chromosome 9, t156 banding and staining, 25, 58, 59, 80–82, 90, 95
chromosome 15, t150 bone marrow, 84, 89, 90
detection of, 60 cell culture, 79, 80
Xp chromosomes, 194 chorionic villus samples, 83
Yp chromosome, 18 equipment maintenance, 80–81
Yq chromosome, 18 harvesting, 77, 80, 81, 89–90
Pseudodiploid, t311, 426 imaging systems, 86–87
Pseudoisodicentric chromosome 9, 156, f 156 international, 92
Pseudoisodicentric chromosomes (psu idic), nomenclature, 36 karyogram production, 77, 86–87
550 Index
Quality control (QC) (cont.) Reciprocal translocation(s), 160–161, f 160, f 161, 167, 220, t218, 258,
laboratory reports, 87–88 259, 284, 345, 383, 473, 480, 481, f 481. See also
lymph nodes, 84 Balanced translocation(s)
microscopy, 82 autosomal, 159–161, 189, 219, 220
percutaneous umbilical blood sampling, 83 de novo in amniocentesis, t232
PHA-stimulated blood, 83 de novo incidence in prenatal diagnosis, t233
post-analytical testing components, 87–88 in hepatic mesenchymal hamartoma, 386
pre-analytical testing components, 78 in infertile men, t218, t221
retention of case materials, 87 involving MYC in Burkitt lymphoma, 336
slide making, 81, 90 risk for abnormality in de novo, 510
solid tissue, 83 X;autosome, 189, f 189
solid tumors, 84–86 Y;autosome, 196
specimen accessioning, 79 Recombinant chromosome(s), 35, f 152, 153, f 154, 155, t155, 156,
specimen analysis, 82 166, 167, 193, 220
specimen submission, 78 description, t30
test validation, 77, 78, 85 nomenclature of, 30, 34
Questionable karyotypic results, 39 Recombinant chromosomes (rec)
Question mark (?) in nomenclature, t28, 39 definition, 35, t49
Quinacrine bands/banding. See Q-bands/banding Recombinants. See Recombinant chromosomes (rec)
Quinacrine mustard (QM), f 5, 6 Recombination, 115–117, 140–141, 293, 294, 296
in ataxia telangiectasia, 298
between homologous sequences in jumping translocations, 165
R in Bloom syndrome, 302
r. See Ring chromosome (r) errors and IGH@ rearrangements, 350
+r(12), f 394 in formation of Robertsonian transloction, 163
r(18), f 158 in generation of intrachromosomal insertions, 166
r(18)(p11.2q23), f 158 in interchromosomal insertions, 167
r(22)t(17;22), t385, 388, 389 intrachromatid, 140
in DFSP and variants, t385, 388 in meiosis, 19, 152, t158
r(X), 181, 182 nonallelic homologous, 139–141, t140, 147, 152, 155, 157, 162,
r(Y), 198 194, 417
RAEB-1. See Refractory anemia with excess blasts-1 (RAEB-1) in the pseudoautosomal regions, 175
RAEB-2. See Refractory anemia with excess blasts-2 (RAEB-2) and uniparental disomy (UPD), 480
RANBP2, 396 in Werner syndrome, 300
RANBP2-ALK, 396 within paracentric inversion loop, 155, 156
RAP1GDS1-NUP98, t334 within pericentric inversion loop, 154, f 154
RARS. See Refractory anemia with ring sideroblasts (RARS) Recurrent abnormality
RB1, 44, 342, 348, 349, t373, 374, 381, 390, t391, 394, 395, in AML, 322
t398, 403, 480 definition, t311
RBA-bands/banding, t25 in hepatosplenic T-cell lymphoma, 355
R-bands/banding, 24, 25, t25, f 27, 59–60, f 60, 87, f 298 in MDS, 315
RB-bands/banding, t25 in T-ALL, 334
RBG-bands/banding, t25 Recurrent chromosome abnormalities
RCC. See Renal cell carcinoma (RCC) in AML, 317
RCMD. See Refractory cytopenia with multilineage dysplasia in B-All, t327
(RCMD) CLL, 342, f 342
rcp. See Reciprocal (rcp) in CML, 321
RCUD. See Refractory cytopenia with unilineage dysplasia in lymphoid neoplasms, 327
Rearrangement(s) in mature B-cell lymphoid neoplasms, 335, t336
chromosome, definition, t311 in mature T-cell lymphomas, 352
complex chromosome, 167, f 354, 432 in MDS, t313, 314–317
nomenclature of, 32, 46–47 in MDS/MPN, t322
cryptic, 310, 317 in natural killer cell leukemia, 353
de novo, 143, 224, 258 in primary MDS and t-MDS, t313
differenting between balanced and unbalanaced, 86, 142 in splenic marginal zone B-cell lymphoma, 341
familial, 142–145, 285 in T-ALL, t334
formation of chromosomal, 139–142, f 140 Recurrent miscarriage, 188, t259, 284, 504. See also Recurrent
of IGH@ (14q32.3), 333, 343, 345, 350–351 pregnancy loss; Recurrent spontaneous abortion;
interchromosomal, 106 Spontaneous abortion
intrachromosomal, 151, 433 Recurrent pregnancy loss, 285–288, 505. See also Recurrent
of MLL (11q23), 15, t327, 328–329, f 329, 335, 424, 425 miscarriage; Recurrent spontaneous abortion;
structural, 139–168, t233, 287, 384–387 Spontaneous abortion
subtelomere/subtelomeric, 419, 506 Recurrent spontaneous abortion, 288, 485, 504, 507. See also
rec. See Recombinant chromosomes (rec) Recurrent miscarriage; Recurrent pregnancy loss;
rec(5)dup(5q)inv(5)(p15.33q33.1), t154 Spntaneous abortion
Reciprocal (rcp), definition, t49 Reed, Sheldon, 499
Index 551
Reed-Sternberg cells, 352 Retinoic acid receptor alpha (RARA), 324, 371, 423, t423, 424
Reference laboratories, 93 Reverse bands/banding. See R-bands/banding
Refractive index (RI), 70, 71, 73 Reverse FISH, 434–436, f 436
Refractory anemia with excess blasts-1 (RAEB-1), 313, t313, 315 RFA-bands/banding, t25
Refractory anemia with excess blasts-2 (RAEB-2), 313, t313, 315 RF-bands/banding, t25
Refractory anemia with ring sideroblasts (RARS), 313, t313 Rhabdoid tumor, atypical teratoid/(AT/RT), 371, t373, 378
Refractory cytopenia with multilineage dysplasia (RCMD), t313 Rhabdoid tumor of kidney (RTK), t379, 381
Refractory cytopenia with unilineage dysplasia (RCUD), t313 Rhabdomyosarcoma
Region alveolar, 397–399, t398, f 399
definition, t49 embryonal, 398–400, t398, f 400, 479
pseudoautosomal, 19, 43, 175, f 176, 178, 179, 186, 191, 196, 197, RH-bands/banding, t25
199, 200, 333 RHG-bands/banding, t25
Rejection criteria (for specimens), 79 RI. See Refractive index (RI)
Renal cell carcinoma (RCC), 378, t379 Ribonucleic acid (RNA), 176, 222, 458, 461, 462, 476, 480
chromophobe, 378–381, t379 mRNA, 10, 11, f 12, f 13, 14, f 14, f 15, 343, 457, 458, 461, 462,
clear cell, 378–380 464–465
oncocytoma, 378, t379, 381 processing, f 13
papillary, 378, t379, 380, f 380 polymerase, 10, f 12
t(Xp11.2), t379 rRNA, 10, 13, 14, f 15, 25, 26, 61, 163, 168
with TFEB translocations, 380 tRNA, 11, f 14, f 15
with TFE3 translocations, 380 Richter syndrome (RS), 342, 343
Renal pyelectasis, 251–252, t255 Ring chromosome (r)
Renal tumors, 381, 397. See also Renal cell carcinoma (RCC) definition, 33, t49, t311
Repetitive DNA sequence, t49, 164, 165 description, t30
Repetitive/repeated sequence DNA, 13 nomenclature of, 33, 35
Repetitive sequence probes, 415 Ring chromosome(s)
Replication acentric, f 140
abnormal, 141 autosomal, 158–159
asynchronous, in Robert syndrome, 300 carriers of, 159
bubble, 10 in Cat eye syndrome, t150
DNA, 9–10, f 11, 13, 141, 294, 455 in dedifferentiated liposarcoma, 393, f 394
early, 17, 189 in dermatofibrosarcoma protuberans, 388
fork(s), 10, 141, 302, 456 detection with microarrays, 444
and instability of the CGG repeat, 455–456 determination of origin with FISH, 181–182, 421
lagging, strand, 141 in hematologic neoplasms, 312
late, 176, 189, 190 instability of, 159, 198
semiconservative, 10, f 11 interlocking, 159
timimg, 190, 475–476 mechanism of formation, 158
in xeroderma pigmentosum, 302, 303 and neocentromeres, 157, 158
and X-inactivation, 176, 189, 191 size of, 59
Reports in spontaneous abortions, 285
final, 88 supernumerary, 130, 158, 159, 388, 393
preliminary, 79, 88, 230 and uniparental disomy, 159
Reproductive risks, 142–144, 152, 153, 156, 167, 278, 504, 507 X chromosomes, 180–182
for carriers of Robertsonian translocations, 164–165 Y chromosome, f 194, 198
Reproductive system, tumors of, Ring syndrome, 159
endometrial stromal tumors, 382–383 RNA. See Ribonucleic acid (RNA)
endometrial stromal nodules (ESN), 382 Robertsonian translocation (rob), 163–165, f 163
endometrial stromal sarcomas (ESS), 382 definition, 36, t49, 163
undifferentiated endometrial sarcomas (UES), 383 nomenclature of, 30, 36, 164
Requisition forms, 78–79, 90 Robertsonian translocation(s)
Resolution carrier(s), 163–165, 219
of arrays, 264, 436, 443, 444, 447, 448, 511 de novo balanced, in amniocentesis, t232
band/banding, 25, 26, 33, 58, 59, 64, 82, 83, 88 de novo incidence in prenatal diagnosis, t233
in brightfield microscopy, 67, 69, 72 de novo nonhomologous, parental origin, 141–142
in fiber FISH, 434 dicentric, 156
of mBAND, 433 homologous, 164–165
in M-FISH, 105–106 involving chromosomes 13, 14, or 15 and 21, t259
levels of, f 27 in liveborn babies, t231
vs. magnification, 70 loss of short arms in formation of, 146
ultrasound, 245, 254 in male infertility, 218, t218, 219, t221
Results from microarray testing (arr), t43 mechanisms for formation of, 141
nomenclature, 46–47 nonhomologous, 141–142, 163–165
RET, 259 prenatal results for, t259
Retention of case materials, 87 reproductive risks for carriers of, 164–165
Retinoblastoma, 225, t398, 403, 479, 480 risk associatd with de novo, 510
552 Index
Robertsonian translocation(s) (cont.) rhabdomyosarcoma, 378, 381, 397, 398, t398, 429, 477,
and risk of unbalanced chromosomes in the fetus, 258 479–480
in spontaneous abortuses, 285 skeletal myxoid chondrosarcoma, 395
and uniparental disomy, 481, 488, 489 synovial sarcomas, t391, 392, f 392, 429, f 430
and upd(14)mat, 486 undifferentiated endometrial sarcomas, 383, 386
and upd(14)pat, 486 well-differentiated liposarcoma, t391, 393, f 394, 429
upd(21)mat, 488 Wilms tumor, 201, 296, t379, 381, 477, 479–480
upd(21)pat, 488 Satellite(s)
upd(22)pat, 488 alpha (a)
Robert syndrome (RS), t49, 296, 300, f 300, 506 DNA, 14, 25, 129, 157, 300, 415
premature centromere separation in, 300, f 300 oligonucleotides, f 435
Robotic harvesters, 95–96, f 96 probes, 416, 420, t420, 421, 430, 431
problems with, 89–90 sequences, 60, 415, 434
RPN1, 325 beta, sequences, 416
RRC. See Renal cell carcinoma (RCC) classical
RS. See Richter syndrome (RS); Robert syndrome (RS) DNAs, 299
RTK. See Rhabdoid tumor of kidney (RTK) probe, 421
RTL1, 486 sequences, 416
Rubinstein-Taybi syndrome, t148 DNA, 14, 25, 139–141, 158
RUNX1, 313, 321, 322, t323, t327, 331, 332, 423, t423, 425 stalks, 13, 61, f 61
amplification, t327 and NORs, 13, 61, f 61
rearrangements, t323 Satellited
RUNX1-RUNXT1, 322 marker, t232, 420, 511
RUNX1T1, 423, t423 supernumerary markers, 129
X chromosome, 199
Y chromosome, 198–199
S SBC. See Secretory breast carcinoma (SBC)
S phase. See Synthesis (S) phase SCA1. See Spinocerebellar ataxia type 1 (SCA1)
Safety, 93 SCA12. See Spinocerebellar ataxia type 12 (SCA12)
Salivary gland tumors, 341, 387–388 SCE. See Sister chromatid exchange (SCE)
carcinoma ex pleomorphic adenoma, t385, 387 Scoring
mucoepidermoid carcinoma/cancer, 387, f 388, 389 rules, 105
pleomorphic adenoma, t385, 387, f 387 of signal patterns, 432
Warthin’s tumor, t385, 387, 389 SDHD, 479
Sample requirements, 53–54 sdl. See Sideline (sdl)
Sarcoma Secondary oöcyte, 20, f 21
alveolar rhabdomyosarcoma, 397, 398, t398, f 399 Secondary spermatocyte, 20, f 21
alveolar soft part, t379, t391, 395 Second trimester maternal serum screen/screening, 508, 509
clear cell sarcoma, 395, t398, 403 Second-trimester screening, 257. See also Combined screening;
clear cell sarcoma of the kidney, t379, 381, 383 Integrated screening
congenital fibrosarcoma/infantile fibrosarcoma, t379, Secretory breast carcinoma (SBC), t385, 390
390–392 Segregation
dedifferentiated liposarcoma, t391, 393, f 394, 429 2:2, f 161, 160, 166
dermatofibrosarcoma protuberans, t385, 388, f 389 3:1, 161, f 160, 163
dermatofibrosarcoma protuberans with fibrosarcoma-like 4:0, 161
changes, 388 abnormal, 145, 293
embryonal rhabdomyosarcoma, 398–400, t398, f 400 adjacent, 161
endometrial stromal sarcomas, t379, 381–383 alternate I, 161
Ewing sarcoma, 162, t398, 401–402, f 402, 429 alternate II, 161, 219, 285
extraskeletal myxoid chondrosarcoma, t391, 395 of balanced rearrangement, 510
fibrosarcomas, t379, 381, f 382, 388, 390, 392 in complex chromosome rearrangements, 167
giant cell fibroblastoma, t385, 388 and crossing-over, 152
granulocytic sarcoma, 326 mechanism assuring correct, 116
infantile fibrosarcoma, t379, f 382, 390, t391 normal, of dicentric chromosomes, 156
leiomyosarcomas, t391, 394–39 in recurrent spontaneous abortions, 507
liposarcoma, t391, 393–394, f 394, 429 unbalanced, 505, 506
malignant peripheral nerve sheath tumors, 392 in unbalanced chromosome rearrangements, 505
malignant rhabdoid tumor, t391, 397 in unbalanced translocations, 190
myeloid sarcoma, t391, 397 Semen analysis, 217–218
myxofibrosarcoma, t391, 394 Semicolon (;) in nomenclature, t28, 30, 33, t43
myxoid/round cell liposarcoma, t391, 429 Semiconservative replication, f 11
osteosarcoma, t391, 395, 480 Seminomatous GCTs (gonadal germ cell tumors), 383
peripheral primitive neuroecto-dermal tumor, t398, 401–402 sep. See Separated signal (sep)
pigmented Bednar tumor, 388 Separated signal (sep) in nomenclature, t43, 44, 45
pleomorphic liposarcoma, t391, 394 Sequential screening, 257. See also Combined screening; Integrated
rhabdoid tumor of the kidney, t379, 381 screening; Serum, integrated screening
Index 553
Serum Sex chromosome aneuploidy/aneuploidies, 5, 175, 177, 224, 230, 280,

alpha-fetoprotein (AFP), 509 289, 421, 506
elevated/high, 121, 122, 233, 253, 256, 262, 297 and age, 187–188
low, 256 loss and aging, 188
normal, 233, 234, 299 low level, 287
fetal bovine, 54, t233 spontaneous abortion, 280
hCG, elevated, 122 at mid-gestation, f 281
IgA in ataxia telangiectasia, 297 mitotic nondisjunction, 281
IgG2 in ataxia telangiectasia, 297 monosomy, 281
integrated screening, 257 Sex chromosome complement, 4, 23, 82–86, 175
leptin, elevated, t217 in nomenclature, 26, 28, 30–32, 37, 46
marker(s), 198, 238, 247, 250, 254 opposite, in bone marrow transplant, 429
maternal, 232, 253, 254, 503 in sex mis-matched bone marrow or stem cell transplant,
positive maternal, marker screen, 256 428–429
screen/screening, 232, 238, 247, 252 in tetraploidy, 126
abnormal, 251 in trploidy, 125
first-trimester, 245 Sex chromosome component, 41
maternal, 232, 234, 251, 253, 254 Sex chromosome constitution, 64
second-trimester, 247, 508, 509 Sex chromosome disorder(s), 175, 177, 182–184, 186, 506
Sex chromatin, 4. See also Barr body; X chromatin Sex chromosome loss, 187, 315
body, 176, 229 in AML with t(8;21)(q22;q22.3), 322
Sex chromosome(s) in AML with t(9;11)(p22;q23) and other translocations involving
loss, and age, 187–188 MLL, 325
markers, origin of, 421 in hepatosplenic T-cell lymphoma (HSTL), 355
mosaicism, 179, 180 Sex chromosome trisomies, t277, 280–281
normal, and disorders of sex development, 199–201 Sex-determining region of Y chromosome (SRY), f 176, 177,
numerical abnormalities of, acquired, 41 199, 217
origin of extra chromosomes, 186 and mosaicism of the Y chromosome, 180
probes for marker chromosome assessment, 420, t420 and 46,XX disorders of sex development, 200–201
ring, origin of, 198 and 46,XY disorders of sex development, 191, 201
structural abnormailities of, 177 Sex development, disorders of, 175–201
structurally abnormal, 179 with “normal” sex chromosomes, 196, 199–200
Sex chromosome aberration(s), 32, 46 46,XX testicular disorders of sex development, 200
Sex chromosome abnormality/abnormalities/anomalies, 219, disorders of androgen excess, 201
t221, 506 disorders of gonadal (ovarian) development, 200
acquired, 41 ovotesticular disorders of sex development,
de novo, 224 200–201
fetal, 177, 256 46,XX testicular disorders of sex development, 200
FISH analysis for, 422 46,XY disorders of sex development, 201
and genetic counseling, 506 disorders of gonadal (testicular) development, 201
i(X)(q10), 181 Sex, genetic 175, 199, 217
incidence in newborn screening, 219 and SRY gene, 217
and infertility, 506 Sex-mismatched bone marrow transplant, 45, 428–429
and intracytoplasmic sperm injection (ICSI), 222–223 Sex vesicle, 19, 196
in live births, and spontaneous abortions, t277 Sexual differentiation
in male infertility, 218, t221 role of X and Y chromoasomes, 175
and maternal serum marker screens, 256 and Yq deletions, 196
mosaic, 83 Sézary syndrome (SS), t353, 355
prenatal detection/identification of, 190, 509 SHC1, t398, 403
and short stature, 213 Sherman paradox, 453, 455, 463
in spontaneous abortions and live births, t277 SHMT1, t418
and 45,X and 45,X mosaicism, 213 Short frmur, 253, t255
X/monosomy X, 178, 187 Short humerus, 253, t255
XX males, 422 Short interspersed elements (SINEs), 14
XXX, 4, 179, 230, 231, t231 SHOX, 175, 178, 186, 191, 192, 194–196
XXXX, 126 Shprintzen syndrome, t149. See also Deletion 22q; Velocardiofacial
XXXXX, 186 syndrome; DiGeorge syndrome
XXXXY, 185 Sideline (sdl)
XXXY, 185, 186 analysis requirements, 84–85
XXXYY, 186 definition, 40
4XXY/XY, 219 in nomenclature, 40–41
XXY, 219, 230, 231, t231, 244, 252 SINEs. See Short interspersed elements (SINEs)
XXYY, 185–187, 506 Single-copy
XY,i(X)(q10), 186 DNA, 13
XYY, 5, 185, 186, 219, 230, 231, 244 probe(s), 417, f 420, 421, 434
XYY/XY, 219 target, 418
554 Index
Single nucleotide polymorphism (SNP) non-neoplastic, studies, 83

analysis, 6 samples/specimens, 56–57, 63
based arrays/microarrays, 264, 287, 385, 421, 441–443, 511 Solid tumors (STs)
for diagnosis of unbalanced karyotypes, 287 analysis requirements, 84
for identification of uniparental disomy (UPD), 39 in ataxia telangiectasia, 297
in nomenclature, 46 of bone and soft tissue origin (see Bone and soft tissue tumors)
Sister chromatid(s), 12, 13, t15, 17, 18, 36, t48, 57, 114, f 115, 116, of the breast (see Breast cancer)
f 116, 139, 140, f 140, 147, 149, 157, f 158, 181, of the central nervous system (CNS) (see Central nervous system
194, 197 tumors)
Sister chromatid cohesion, 293, 300 culture and analysis, 371–372
Sister chromatid exchange (sce) of dermal origin (see Dermal tumors)
definition, t49 of epithelial origin (see Epithelial cnacer)
Sister chromatid exchange (SCE) factors affecting growth of, 372
in Bloom syndrome, 302, f 302 FISH, 84, 85, 417, t423, 429
definition, 302 of gastrointestinal origin (see Gastrointestinal tumors)
and ring instability, 15 of genitourinary origin (see Genitourinary tumors)
in xeroderma pigmentosum, 303 of germ cell origin (see Germ cell tumors)
SKY. See Spectral karyotyping (SKY) growth affecting factors, 372
Slant line, double (//), in nomenclature, t28, 39, 45 of the lung (see Lung cancer)
Slant line, single (/), in nomenclature, t28, 30, 31 of salivary gland origin (see Salivary gland tumors)
Slide small round cell (see Small round cell tumors (SRCT))
aging, 81 SOP manual. See Standard operating procedure (SOP) manual
chambers, 55 Specimen
drying chambers, 58 accessioning, 77, 79
making, 40, 81, 90, 97, 99 analysis, 82
markers, 96–98 assessing condition of, 79
microscope, 72 collection
preparation, 58, 77, 80, 81, 101 amniotic fluid, 54
printing, for microarrays, 107 bone marrow, 53–54
processing system, controlled, f 100 peripheral blood, 53
scan/scanning, 103, f 103 protocol, 78
SM. See Systemic mastocytosis (SM) solid tissue, 54, 56, 57
Small round cell tumors (SRCT), 397–404, t398 controls in SNP-based arrays, 442, 446
adrenal cortical carcinoma, t398, 403 culture, 80
alveolar rhadbomyosarcoma (RMS), 397, t398 for cytogenetic study, 286–287
clear cell sarcoma, 395, t398, 403 failure, 89
desmoplastic small round cell tumor (DSRCT), t398, 402 handling
embryonal rhabdomyosarcoma, 398–400, t398, f 400 amniotic fluid, 54, 83
Ewing sarcoma (EWS)/peripheral primitive neuroectodermal bone marrow, 54, 64
tumor (pPNET), 162, t398, 401–402 peripheral blood, 53
medulloblastoma, 376, 378 solid tissue, 54, 57
neuroblastoma, t398, 400–401, f 401 labeling, 78–80, 90
peripheral primitive neuroecto-dermal tumor small round cell preparation for culture, 56
tumors, t398, 401–402 rejection, 79
retinoblastoma, t398, 403 requirements
rhabdomyosarcoma, 397, t398 CVS, 83
and suspension culture, 372, t398 PUBS, 83
SMARCB1-INI1, t373, 378, 381, 385, t391, 397 submission, 78, 79
SMC proteins. See Structural maintenance of chromosomes types, 53, 57, 78, 83, 99, f 100, 288, 417
(SMC) proteins Spectral karyotyping (SKY), 74, 105, 310
Smith-Lemli-Opitz syndrome, 201 Spermatid, 20, f 21
Smith-Magenis syndrome, t148, 150, 151, 418, 505–506 Spermatocyte(s), 20, f 21, 117, t222
sMZBCL. See Splenic marginal zone B-cell lymphoma (sMZBCL) Spermatogenesis, 20, f 21, 144, 162, 167, 177, 184, 197, 219, 222,
SNORD116, 476 278, 475, 480
SNP. See Single nucleotide polymorphism (SNP) Sperm/spermatozoa/spermatozoan, 20, 113, f 21, 125, 126, 161, 175,
SNRPN, 417, f 418, t418, 420, f 420, t420, 475–479 184–186, 198, 217–220, t218, 222, 223, t223, 280, 283,
SNURF-SNRPN, 476 285, 462
Sodium citrate, 57 mitochondria of, 225
Soft tissue tumors, 390–397, t391, 403, 429–430 Spina bifida, 121, 187, 224, t249, 258. See also Neural tube defects
Software Spinocerebellar ataxia type 1 (SCA1), 455, t455
imaging systems, 102, 107 Spinocerebellar ataxia type 12 (SCA12), 455, t455
interface of metaphase finding system, 103, f 104 Splenic marginal zone B-cell lymphoma (sMZBCL), 335, 340–342
Solid stained chromosomes, 58, 453. See also Unbanded sPNET. See Supratentorial primitive neuroectodermal tumors (sPNET)
chromosomes Spontaneous abortion, 231, 275. See also Fetal loss; Recurrent
Solid tissue miscarriage; Spontaneous pregnancy loss;
biopsies, 53, 54, 448 Spontaneous loss
culture failure of, 63 chimerism, 39, 282–283
Index 555
chromosomal abnormalities in, 131, t277, 280, 288 of meiosis, 219

chromosomally normal pregnancy loss, 286 of mitosis, 18
evaluation, of pregnancy loss Stains/staining
developmental age, 287 and banding errors, 58
embryo/fetus, 288, t288 Cd, 61
morphology, 288 chromosome, 58
phenotype, t288 DAPI, t25, f 436
preimplantation assessment, 289 DAPI/DA, t25, 61–62
fertilization, errors in, 283 G-banding, 24, f 24, t25, 26, f 27, 59, f 59
gestational age, 276–277, t276 NOR, t25, 26, 61, 87
intrauterine mortality, 276, t276 Q-banding, t25, 59, f 59
meiosis, errors in, 114, t114, 278–280, 282 solid, 58, 300, 453
mitosis, errors in, t114, 278, 282 T-banding, t25, 26, 60–61
and preimplantation genetic disgnosis, 259 techniques, 15, t25, t28, 58
prenatal loss, 277, t277 Standard operating procedure (SOP) manual, 77
recurrent, 288, 485, 504, 507 Standards and Guidelines for Clinical Genetics Laboratories, 77, 432
risk of, following chorionic villus sampling (CVS), 240 STC2, 380
risk of, following conventional amniocentesis, 233 Stem cell
risk of, following earlyamniocentesis (EA), 235–236, t236, 237 disorders, 316
sex chromosome aneuploidies hematopoietic, neoplasms 312
at mid-gestation, f 281 line, 260
mitotic nondisjunction, 282 myeloid and lymphoid neoplasms, 320
monosomy, 280, 281, f 281 neoplasm(s), 312, 316, 321, 325
specimens Stem call transplant(s)/transplantation, 428–429. See also Bone
CGH, 287 marrow transplant(s)/transplantation
flow cytometry, 287 in nomenclature, 39, 45
fluorescence in situ hybridization, 286 reporting mosaicism, chimerism, and chimerism secondary to, t28
in situ method, direct preparations, 286 Stemline (sl), 84–85
microarray, 287 definition, 40–41, t48, t49
structural chromosome rearrangements, 506, 507, 510 in nomenclature, t40
trisomies Stillbirths and neonatal deaths, 231–232, 236. See also Fetal loss
distribution of individual, t279 frequencies of chromosomal abnormalities in, 233, t234
oöcytes examination, 280 String, nomenclature, 26, t29, 32, 37–42, 44, t48, t49
recurrence risk, 276 Structural abnormality, 5, 56
spermatocytes evaluation, 280 associated risks, 142
trisomy 21, 278, f 279 mosaicism of an autosomal, 236
Spontaneous loss, 237, 277, t281. See also Fetal loss; Recurrent most common, in Turner syndrome, 157
miscarriage; Spontaneous abortion; Spontaneous most common, of the X chromosome, 194
pregnancy loss; Spontaneous loss most common recurrent, in hepatoblastoma, 384
Spontaneous pregnancy loss. See also Fetal loss; Recurrent rate of in stillbirths, 277
miscarriage; Spontaneous abortion; Spontaneous loss of the Y chromosome, 180, 218
chromosomally normal, 286 Structural chromosome rearrangements
evaluation of, 287–289 and advanced maternal age, 507
following chorionic villus sampling (CVS), 233, t233, 236, 237, associated risks of, 142
240–241 balanced, 142, 167, 258, 507
following conventional amniocentesis, 233–236, 238 in B-ALL, 331
following early amniocentesis(EA), 235–238 complex, 141, 167, 434
following percutaneous umbilical blood sasmpling (PUBS), 244 cryptic subtelomeric, 419
in pregnancies with combined placental mosaicism (CPM), 242 de novo, 141, 142, 167, 258
and sex chromosome aneuploidy, 280–282 incidence of, in prenatal diagnoses, t233
45,X, f 281 differentiating between balanced and unbalanced, 142
Sporadic rearrangements, 139 errors leading to, 278
SPTBN1-FLT3, t321 familial, 142, 167
Square brackets ([ ]) in nomenclature, t28, 30, 31, 38, 40, 41 most common, 167, 331
SRCT. See Small round cell tumors (SRCT) nonrecurring, 141
SRY. See Sex-determining region of Y chromosome (SRY) paternally derivation, 141
SS. See Sézary syndrome (SS); Synovial sarcoma (SS) and prenatal loss of chromosomally abnormal fetuses, t277
SS18, f 392, 429 and risk for recurrent spontaneous abortion, 507
FISH for rearrangement of, f 430 subtelomere, 506
SSX1-SS18, t391, f 392 unbalanced, 142, 167, 258, 507
SSX2-SS18, t391, f 392 of the X chromosome, 258, 507
Staff qualifications, laboratory, 92 of the Y chromosome, 167
Stage, microscope Structural maintenance of chromosomes (SMC) proteins, 116, 299
and coordinate location, 71 STs. See Solid tumors (STs)
motorized, 104 Sub-bands, 25, f 26, t28, 58, 59
Stages Submetacentric, 13, f 17, 23, 163, t311
of the cell cycle, 15, 16 subtel. See Subtelomere FISH (subtel)
556 Index
Subtelomere Langer-Giedion, t148

probe, f 154 Li-Fraumeni, 385, 395, 403
rearrangements, 506 microdeletion, 149–151, t150, 154, 258, t418, 504, 505, 508
Subtelomere FISH (subtel), t43 microduplication, 140, 147–149, t148, t149, 151
nomenclature, t43, 44 microduplication-microdeletion, t149
Subtelomeric recurring, t150
rearrangements, 419 Miller-Deiker, t150, t418, 506
region, 13, 43, 44, t49, 146, 419, 443, 506 monosomy 1p36, t148
Supernucleosome, 474 myelodysplastic, 46, 312–313, 321, 322, 326, 445
Supernumerary Nijmegen breakage, 299
chromosome(s), 126 Pallister-Killian, 123, 127, 128, t150, 263, 511
with associated clinicsl festures, 511 Patau, 5, 278
isochromosome(s), 142 pentasomy X, 184
of 8p, 162 Phelan-Mcdermid, t149
of 9p, 127, 157 polysomy X, 186
of 12p, f 384 Potocki-Shaffer, 141, t148
marker chromosomes, 113, 129–130, 231, t232, 489, 510 Prader-Willi, 129, t148, f 151, 164, 242, 417, f 418, t418, 474,
rings, 158, 393 476–478, t477, t478, 486, 505
Supratentorial primitive neuroectodermal tumors (sPNET), 376, 378 proximal 15q11.2 microduplication, 150
SYK-ETV6, t321 pseudodicentric 15, t150
Symbols 5q, t313, 314
in nomenclature, 24, 26, 38, 42, 43, t43 7q11.23 microduplication, t150
in pedigrees, f 501 15q13.3 microdeletion, t148
Synapsis, 13, 19, f 152, 153, 166, 196 17q21.3 duplication, t150
Synaptonemal complex, 19 17q21.3 microdeletion, t150, 154
Syndrome 22q11.2 microdeletion, 258
Alagille, t148 Reichter, 342, 343
androgen insensitivity, 201, f 214, t217 Robert, t49, 296, 300, 506
Angelman, 129, 149–151, t150, f 151, 164, 225, 417, 474, Rubinstein-Taybi, t148
476–478, t477, f 478, 486, 487, 505, 511 Russell-Silver, 225
Asherman, f 214 segmental aneusomy, 146, 147
Beckwith-Wiedemann, 149, 225, 381, 385, 386, 404, 419, Sézary, 353, 355
477–479 Shprintzen, t149
Bloom, 5, 38, 296, f 302, 506 Smith-Magenis, t150, 418, 505–506
cat-cry, 5, f 146 Sotos, t148, 154
Charcot-Marie-Tooth 1A, t150 STS, 195
contiguous gene, 146, 147, 149, 190, 191, 417, 419 tetrasomy X, 184
cri du chat, 5, 146, 148, 252 trichorhinophalangeal syndrome type II, t148
deletion, 505 triple X, 182
recurring, 140, 141, 147–151, 154, 159, 162, 163, 325, trisomy 13, 505
381, 385 trisomy 18, 503–505, 508, 509
deletion/duplication, t148, t149, 505 trisomy X, 182
DiGeorge, 43, 124, t150, 151, 418, 419, 505 Turner, 4, 35, 157, 178–182, f 179, f 181, 186–188, 191, 194–198,
Down, 4–6, f 4, 37, 41, 114, 117–119, f 118, f 119, f 163, 184, 214, 215, 222, 231, 244, 245, 251, 279–282, f 279, t420, 506,
230, 245, 247, 256, 278–280, f 279, t323, 325, 507, 509
326, 333, 503–505, 508, 509 UPD, 489
duplication 3q, t150 velocardiofacial, t148
duplication/triplication, 476–477, 484 Werner, 296, 300–301, f 301
recurring, 147, t148, t150, 154 Williams, 43, 142, 147, 148, 154, 419, 505
Edwards, 5, 278 Wolf-Hirschhorn, t148, 162, 350
familial adenomatous polyposis (FAP), 385 X-linked ichthyosis, 191
fragile X, 38, 62, 83, t216, 259, 294, 453–456, 458–460, f 459, XYY, 185
462, 463, 502 Synovial sarcoma (SS), 353, 355, t391, f 392, 429, f 430
fragile X tremor ataxia syndrome, 461, 462 Synthesis (S) phase, 17
FRAXE, t455, 465 Systemic mastocytosis (SM), 316, 317, 320
hereditary neuropathy with liability to pressure palsies, t148, SYT, 429, f 430
151, 418
ICF(see Syndrome, immunodeficiency, centromere instability, and
facial anomalies (ICF))ichthyosis (X-linked), t149, 191, T
194, 195 t(1;2) in
immunodeficiency, centromere instability, and facial anomalies ALCL-ALK+, t353
(ICF), 296, 299–300, f 299 T-PLL, t353
Jacobsen, 295, t465 t(1;2)(p22.3;p12), in MZBCL, 341
Kallman, 195 t(1;2)(q21.3;p23.2), in inflammatory myofibroblastic (IMT), t391
Klinefelter, 4, 41, 184–186, 194, 200, 219, 222, 244, t379, 383, t(1;2)(q21.3;p23.2)/TPM3-ALK, in inflammatory myofibroblastic
503, 506, 507 (IMT), 396
Index 557
t(1;7)(p32;q34), in T-ALL, t334 t(2;22), 396–398, in ALCL-ALK+, 353, t391

t(1;9)(p32.3;p21), f 160 t(2;22)(q33.3;q12.2), in angiomatoid fibrous histiocytoma (AFH),
t(1;12), AML (megakaryoblastic) with, t323 t391, 396, f 397
t(1;12)(q25;p13.2), t321 t(2;22)(q35;q12.2), in Ewing sar-coma/peripheral primitive
t(1;13)(p36;q14), in soft tissue tumors, 429 neuroectodermal tumor (EWS/pPNET), t398
t(1;13)(p36.13;q14.11), in alveolar rhabdomyosarcoma (RMS), t(3;3), in AML with inv(3) or t(3;3), t323
t398, f 399 t(3;3)/inv(3) in
t(1;14), in MZBCL (MALT type), t336 AML (megakaryoblastic), t323
t(1;14)(p22.3;q32.3), in MZBCL, 341 AML with injv(3) or t(3;3), t323
t(1;14)(p32;q11.2), in T-ALL, t334 erythroleukemia (erythroid/ myeloid), t323
t(1;14)(q32;q32), f 481 t(3;3)(q21.3;q26.2) in
t(1;17)(p34.3;p13.2), in aneurysmal bone cyst, t391, 395 acute AML with recurrent genetic abnormalities, 315, 322, t323
t(1;19) in AML with inv(3) or t(3;3), t323
ALL, 327, 331, 425 AML with inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2), 325
B-ALL, 327, 329, 331, 425 MDS with other chromosome abnormalities, 315
t(1;19)(q23.3;p13.3) in t(3;8)(p22.1;q12.1), in pleomorphic adenoma, t385, 387, f 387
B-ALL, 327, t327, 329 t(3;12)(q28;q14.3), in conventional lipoma, t391, 393
B-CLL, 426 t(3;14)(p13;q32.2), in MALT lymphoma, 341
t(1;22)(p13;q13) in t(3;17)(q21.3;p13.2), in aneurysmal bone cyst, t391, 395
AML (megakaryoblastic), in children, t323 t(3;20)(q27;q12), in dedifferentiated liposarcoma, t394
AML (megakaryoblastic) with t(1;22), t323 t(3;21)(q26.2;q22.3), 325
t(1;22)(p13.3;q13.1) in t(4;11)(q21.3;q23), rearrangements involving MLL
acute megakaryoblastic leukemia with t(1;22)(p13.3;q13.1), rearraggements, 328
325–326 t(4;11)(q22.3;p15.4), in T-ALL, t334
megakaryoblastic, t323, 325–326 t(4;12)(q12;p13.2), in myeloid and lymphoid neoplasms associated
t(1;22)(p36;q11.2), in malignant rhabdoid tumor (MRT), t391 with PDGFRA, PDGFRB, and FGFR1, f 320
t(2;2)(p23.2;q12.3) in t(4;14), in plasma cell myeloma (PCM), t336, 351, 426, f 427
epithelioid variant, t391 t(4;14)(p16.3;q32.3), in plasma cell myeloma (PCM), translocations
inflammatory myofibroblastic (IMT), t391 involving IGH@, 347, 350
t(2;2)(p23.2;q12.3)/ALK-RANBP2, inflammatory myofibroblastic t(5;9)(q33.3;q22.2), in peripheral T-cell lymphoma, not otherwise
(IMT), 396 specified (PTCL, NOS), 355
t(2;2)(p23.3;q36.1), in alveolar RMS, t398 t(5;12)(q33;p13.2), in myeloid and lymphoid neoplasms associated
t(2;3), in anaplastic large T-cell lymphoma, ALK-positive with PDGFRA, PDGFRB, and FGFR1, f 320, 321
(ALCL-ALK+), t353 t(5;14)(q31.1;q32.3), in B-ALL with recurrent IGH@
t(2;3)(q11.2;q25), in aneurysmal bone cyst, f 396 rearragngements, 333
t(2;5), 428 t(5;14)(q35.1;q32.2) (cryptic), in T-ALL, t334
t(2;5)(p23.1;q35.1) in t(5;17) and response to ATRA in in acute promyelocytic leukemia
anaplastic large T-cell lymphoma, 356, f 356 (APL), 324
anaplastic large T-cell lymphoma, ALK-positive (ALCL-ALK+), t(5;17)(q35.1;q21.2), in acute promyelocytic leukemia (APL) with
t353, 356 t(15;17)(q24.1;q21.2), 324
t(2;8) in t(6;7)(p12.3;p15.2)/JAZF1-PHF1, in endometrial stromal tumors
B-cell lymphoma, unclassifiable, with features intermediate (EST), 383
between diffuse large B-cell lymphoma and Burkitt t(6;7)(p21.3;p15.2), in endometrial stromal tumor (EST), t379
lymphoma, 338 t(6;9)(p23;q34.1), in acute myeloid leukemia (AML) with recurrent
B-cell prolymphocytic leukemis (B-PLL), 345 genetic abnormalities, 322, t323, 325
t(2;8)(p11;q24), in non-Hodgkin lymphoma, 428 t(6;10)(p12.3;p11.22)/EPC1-PHF1 and variants, in endometrial
t(2;8)(p11;q24.1), in plasma vell lymphoma (PCM), f 351, f 352 stromal tumors (EST), 383
t(2;8)(p12;q24.1), in plasmablastic lymphoma, f 352 t(6;10)(p21.3;p11.22), in endometrial stromal tumor (EST), t379
t(2;8)(p12;q24.2), in Burkitt lymphoma, 336, f 338 t(6;11)(p21.1;q13.1) in
t(2;12) (p12;p13), in mantle cell lymphoma, 340, f 340 papillary renal cell carcinoma (pRCC), 380
t(2;13)(p16;q12.2), t321 t(6;11) renal cell carcinoma (RCC), t379
t(2;13) (q35;q14), in soft tissue tumors, 429 t(6;11)(q27;q23), in AML with t(9;11)(p22;q23) and other
t(2;13)(q36.1;q14.1), in alveolar rhabdomyosarcoma (RMS), f 399 translocations Involving MLL, 325
t(2;13)(q36.1;q14.11), in alveolar rhabdomyosarcoma (RMS), t398 t(6;13)(p21;q14.1), in CLL with non-IGH@ reciprocal
t(2;14), in CLL/SLL, t336 translocations, 345
t(2;14)(p16.1;q32.3), in CLL with IGH@ abnormalities, 343 t(6;14), in mantle cell lymphoma (MCL), t336
t(2;14)(p24;q32.3), in mantle cell lymphoma, 340 t(6;14)(p21;q32), in plasma cell myeloma (MCL)/plasmacytoma, 426
t(2;17), in ALCL-ALK+, t353 t(6;14)(p21;q32.3), in mantle cell lymphoma, 340, f 340
t(2;17)(p23.2;q23.1)/ALK-CLTC, in inflammatory myofibroblastic t(6;14)(p21.1;q32.3) in plasma cell myeloma (PCM) with IGH@
tumor (IMT), 396 rearrangements, 350
t(2;17)(p23.2;q23.1), in inflammatory myofibroblastic, t391 t(6;14)(p22.3;q32.3), in B-ALL with IGH@ rearrangements, 333
t(2;18)(p12;q21.3), in follicular lymphoma, 335 t(7;7), in T-ALL, t334
t(2;19), in ALCL-ALK+, t353 t(7;7)(p14;q35), in ataxia telangiectasia, f 298
t(2;19)(p23.2;p13.12)/ALK-TPM4, inflammatory myofibroblastic t(7;9)(q34;q31.2), in T-ALL, t334
(IMT), 396, t398 t(7;9)(q34;34.3), in T-ALL, t334
t(2;19)(p23.2;p13.12), in inflammatory myofibroblastic, t391 t(7;10)(q34;q24.3), in T-ALL, t334
558 Index
t(7;11)(q34;p13), in T-ALL, t334 in B-cell prolymphocytic leukemia (B-PLL), 345

t(7;12)(q34;p13.3), in T-ALL, t334 the, recurring translocation, 162–163
t(7;12)(q36.3;p13.2), in primary MDS and t-MDS, t313 t(8;22)(q24;q11)
t(7;14), in T-cell prolymphocytic leukemia (T-PLL), t353 in Burkitt lymphoma, 428
t(7;14)(p14;q11), in ataxia telangiectasia, f 298 t(8;22)(q24.2;q11.2), in Burkitt lymphoma, 336
t(7;14)(p14;q32), in ataxia telangiectasia, f 298 t(9;11), prognostic outcome of MLL translocations, 324
t(7;14)(q34;q32.1) t(9;11)(p22;q23)
in T-ALL, t334 in AML with recurrent genetic abnormalities, 322
in T-cell prolymphocytic leukemia (T-PLL), 353 in AML with t(9;11), t323
t(7;14)(q35;q11), in ataxia telangiectasia, f 298 with translocations involving MLL, 324–325, 329
t(7;14) (q35;q32), in ataxia telangiectasia, f 298 t(9;12)(p24.1;p13.1), in T-ALL, t334
t(7;17)(p15.2;q11.2), in endometrial stromal tumor (EST), f 379 t(9;12)(p24.1;p13.2), t321
t(7;17)(p15.2;q11.2)/JAZF1-SUZ12, in endometrial stromal nodules t(9;12)(q22;p13.2), t321
(ESN), endometrial stromal sarcomas (ESS), and t(9;12)(q34;p13.2), t321
undifferentiated endometrial sarcomas (UES), 383 t(9;14)(p13.2;q32.3), in B-ALL with IGH@ rearrangements, 333
t(7;19)(q34;p13.2), in T-ALL, t334 t(9;15)(q22;q21.3), in extraskeletal myxoid chondrosarcoma,
t(7;22)(p21.2;q12.2), in Ewing sar-coma/peripheral primitive t391, 395
neuroectodermal tumor (EWS/pPNET), t398 t(9;17)(q22;q11.2) in extraskeletal myxoid chondrosarcoma, 395
t(8;8), in T-PLL, t353 t(9;17)(q22;q12), in extraskeletal myxoid chondrosarcoma, t391
t(8;9)(p22;p24), in atypical chronic myeloid leukemia (aCML), 322 t(9;17)(q22;p13.2), in in aneurysmal bone cyst (ABC), 395
t(8;9)(p22;p24.1), t321 t(9;17)(q22.31;p13.2), in aneurysmal bone cyst, t391
t(8;9)(q24.2;p13), in B-cell lymphoma, unclassifiable, with features t(9;22)
intermediate between diffuse large B-cell lymphoma in B-ALL, 331
and Burkitt lymphoma, 338 in CML, 317
t(8;13)(p12;q12), in myeloid and lymphoid neoplasms associated with testing for, to rule out, 319
PDGFRA, PDGFRB, and FGFR1, f 320 t(9;22)(p24.1;q11.2), t321
t(8;14) in t(9;22)(q22;q12.2), in extraskeletal myxoid chondrosarcoma,
B-cell lymphoma, unclassifiable, with features intermediate t391, 395
between diffuse large B-cell lymphoma and Burkitt t(9;22)(q34;q11.2)
lymphoma, 338 in acute B-cell lymphoblastic leukemia/lymphoma, 327
B-cell prolymphocytic leukemia (B-PLL), 345 with atypical FISH pastern, 424
Burkitt lymphoma, t336 in B-ALL, t327, 328, f 328
t(8;14) and variants, in unclassifiable— diffuse large B-cell lymphoma in chronic myeloid leukemia (CML), 316–317, t317, t318, 424
(DLBCL/Burkitt), t336 in CNL, 320
t(8;14) or variants, in CLL/SLL, t336 FISH for hematologic malignancies, t423
t(8;14)(q11.2;q32.3), with IGH@ rearrangements in B-ALL 333 in mixed phenotype acute leukemia (MPAL), t327
t(8;14)(q24;q32) and the Philadelphia (PH) chromosome, 328
in Burkitt lymphoma, 428 with loss of DNA on chromosome 9 and adverse prognosis, 424
FISH for hematologic malignancies, t423 t(10;11)(q21.3;q23), in AML with t(9;11)(p22;q23) and other
t(8;14)(q24.2;q11.2) in translocations involving MLL, 325
CLL with non-IGH@ reciprocal translocations, 345 t(10;13)(q24;q14), in CLL with non-IGH@ reciprocal
T-ALL, t334 translocations, 345
t(8;14)(q24.2;q32.3), in Burkitt lymphoma, 335, 336 t(10;14)(q24.3;q11.2), in T-ALL, t334
t(8;14;18)(q24.2;q32.3;q21.3), in aggressive “double-hit” t(10;17), in clear cell sarcoma of the kidney (CCSK), 381, 383
lymphoma, f 339 t(10;17)(q22.3;p13.3)
t(8;16)(p12;p13.3), AML (monoblastic/monocytic), t323 in clear cell sarcoma of the kidney (CCSK), t379, 381
t(8;21) in in endometrial stromal tumor (EST), t379
AML, 423 t(10;17)(q22.3;p13.3)/FAM22-YWHAE, in high-grade endometrial
AML with, t323 stromal sarcoma (ESS), 336, 383
AML with (8;21)(q22;q22.3), 322 t(*;11)(*.*; q23), t423
AML with KIT mutation, t323 t(11;14)
systemic mastocytosis (SM), 320 in B-cell prolymphocytic leukemia (B-PLL), 345, f 346
t(8;21)(q22;q22) detection by FISH in plasma cell myeloma (PCM), 338–340
in AMML, 322 in mantle cell lymphoma (MCL), 336, 340, 350
FISH for hematologic malignancies, t423 in plasma cell myeloma (PCM), t336, 349, 351, 426
t(8;21)(q22;q22.3) in with IGH@ rearrangements, 350
acute myeloid leukemia (AML), t323 in unclassifiable–diffuse large B-cell lymphoma
acute myeloid leukemia with gene mutations, 326 (DLBCL)/Burkitt, t336
chronic myeloid leukemia (CML), 317 t(11;14) and other CCND1 variants, in mantle cell lymphoma
cute myeloid leukemia (AML) with recurrent genetic (MCL), t336
abnormalities, 322 t(11;14)/CCND1-IGH@ in mantle cell lymphoma (MCL), 340
myeloid sarcoma, 326 t(11;14)(p13;q11.2), in T-ALL, t334
t(8;22) t(11;14)(p15.4;q11.2), in T-ALL, t334
in B-cell lymphoma, unclassifiable, with features intermediate t(11;14)(q13;q32)
between diffuse large B-cell lymphoma and Burkitt in non-Hodgkin lymphoma, 428
lymphoma, 338 in plasma cell myeloma (PCM)/plasmacytoma, 426
Index 559
t(11;14)(q13;q32.3), in liposarcoma with myxoid histology only or myxoid and round cell
blastoid variant of mantle cell lymphoma, t341 histology, 393
chronic lymphocytic leukemia (CML) with IGH@ myxoid or myxoid round cell liposarcoma, t391
rearrangements, 343 t(12;21)
mantle cell lymphoma (MCL), 338, 347, f 339 associated AML, 331
plasma cell myeloma (PCM), f 351 in B-ALL, cryptic, 331, f 332
with IGH@ rearrangements, 350 t(12;21)(p13;q22), FISH for hematologic malignancies, t423
t(11;14)(q13;q34), FISH for hematologic malignancies, t423 t(12;21)(p13.2;q22.3), in B-cell lymphoblastic leukemia/ lymphoma
t(11;17) (B-ALL), 327, t327, 331
in acute myeloid leukemia (AML), 423 t(12;22), in angiomatoid fibrous histiocytoma, atypical presentation in
and response to ATRA in APL, 324 organs, t391
t(11;17)(q23.2;q21.2), in acute promyelocytic leukemia (APL) with t(12;22)(q13.3;q12.2) in
t(15;17)(q24.1;q21.2), 324 angiomatoid fibrous histiocytoma (AFH), t391, 396
t(11;18), in marginal zone B-cell lymphoma (MZBCL) clear cell sarcoma (CCS), t398, 403
(MALT type), t336 myxoid liposarcoma, 393
t(11;18)(q21;q21.3), in mucosa-associated lymphoid tissue (MALT) myxoid or myxoid round cell liposarcoma, t391
lymphoma, 335 t(12;22)(q13.12;q12.2), in angiomatoid fibrous histiocytoma (AFH),
t(11;19) t391, 396
in AML with t(9;11)(p22;q23) and other translocations involving t(12q14.3), in pleomorphic adenoma, t385
MLL, 324–325 t(*;14)(*.*;q32), t423
in B-ALL with MLL rearrangements, 328–329 t(14;14), in T-cell prolymphocytic leukemia (T-PLL), f 353, t353
in mucoepidermoid cancer, t385 t(14;14)(q11;q32), in ataxia telangiectasia, f 298
negative cases in mucoepidermoid cancer/carcinoma (MEC), 387 t(14;14)(q11.2;q32.1), 334, t334, f 353, t353
in Warthin’s tumor, 387 t(14;14)(q11.2;q32.3), in B-ALL with IGH@ rearrangements, 333
t(11;19)/CRTC1-MAML2 t(14;15)(q32.2;q11.2), in diffuse large B-cell lymphoma
in hidradenoma, 389 (DLBCL), 338
in Warthin’s tumor, 387, 389 t(14;16), in plasma cell myeloma (PCM), t336, 351, 426
t(11;19)(q13;q13.4), in hepatic mesenchymal hamartoma (HMH), t(14;16)(q13;q21), in plasma cell myeloma (PCM), 426
t385, 386 t(14;16)(q32.3;q23.1), in plasma cell myeloma (PCM) with
t(11;19)(q21;p13.11) translocations involving IGH@, 347, 350, 351
in hidradenoma, t385, 389 t(14;18) in
in mucoepidermoid cancer/carcinoma (MEC), t385, 387, f 388 chronic lymphocytic leukemia (CLL), 343
in Warthin’s tumor, t385, 387 chronic lymphocytic leukemia/small lymphocytic lymphoma,
t(11;19)(q23;p13.3), in B-ALL with MLL rearrangements, 328–329 (CLL/SLL), t336
t(11;22)(p13;q12.2), in desmoplastic small round cell tumor diffuse large B-cell lymphoma (DLBCL), t336, 337
(DSRCT), t398 follicular lymphoma, 335
t(11;22)(q13;q11.2) in marginal zone B-cell lymphoma (MZBCL)
desmoplastic small round cell tumor (DSRCT), t398 (MALT type), t336
mantle cell lymphoma (MCL), 340, f 340 mucosa-associated lymphoid tissue lymphoma, 341
t(11;22)(q23.3;q11.2), the, recurring constitutional unclassifiable–diffuse large B-cell lymphoma
translocsation, f 161 (DLBCL)/Burkitt, t336
t(11;22)(q24;q12), in soft tissue tumors, 429 t(14;18) and variants, in follicular lymphoma, t336
t(11;22)(q24.3;q12.2), in Ewing sarcoma/peripheral primitive t(14;18)(q32;q21)
neuroectodermal tumor (EWS/pPNET), t398, 402, f 402 FISH for hematologic malignancies, t423
t(11;V)(q23;V), in MDS, t317 in non-Hodgkin lymphoma, 428
t(12;13)(p13.2;q12.2), t321 t(14;18)(q32.3;q21.3) in
t(12;14), in mantle cell lymphoma (MCL), t336 atypical CLL, f 344
t(12;14)(p13;q32.3), in mantle cell lymphoma (MCL), 340 CLL with IGH@ rearrangements, 343
t(12;14)(p13.3;q11.2), in T-ALL, t334 follicular lymphoma (FL), 335, t337, 338
t(12;14)(q14.3;q24), in leiomyoma, t391, 394 mucosa-associated lymphoid tissue lymphoma, 341
t(12;15), in congenital mesoblastic nephroma (CMN), 381 t(14;19), in CLL/SLL, t336, f 344
t(12;15)(p13;q26)/ETV6-NTRK3 in t(14;19)(q32.3;q13), in CLL, 343, f 344
congenital (infantile) fibrosarcoma, 390 t(14;19)(q32.3;q13.1) in B-ALL with IGH@ rearrangements, 333
secretory breast carcinoma, 390 t(14;20), in plasma cell myeloma (PCM), t336, 426
t(12;15)(p13.2;q25.3), t321 t(14;20)(q32.3;q12), in plasma cell myeloma (PCM), 347
in cellular congenital mesoblastic nephroma, grade III, f 382 with IGH@ rearrangements, 350
in congenital fibrosarcoma (CFS)/infantile fibrosarcoma (IFS), t(14;20)(q32.3;q13.1), in B-ALL with IGH@ rearrangements, 333
390, t391, 392 t(15;17), 324, 371, 423
in congenital mesoblastic nephroma (CMN), t379, 381 t(15;17)(q22;q21.1), t423
in secretory breast carcinoma, t385 t(15;17)(q24.1;q21.2)
t(12;16), in angiomatoid fibrous histiocytoma, t391 in acute myeloid leukemia (AML) with recurrent genetic
t(12;16)(q13;p11), in soft tissue tumors, 429 abnormalities, 322
t(12;16)(q13.12;p11.2), in angiomatoid fibrous histiocytoma (AFH), in acute promyelocytic leukemia (APL) with t(15;17)
t391, 396 (q24.1;q21.2), 324
t(12;16)(q13.3;p11.2) in, in AML with t(15;17), t323
angiomatoid fibrous histiocytoma (AFH), t391, 396 in chronic myeloid leukemia (CML), 317
560 Index
t(16;16) in hepatosplenic, 355

AML with inv(16) or t(16;16), t323, 324 peripheral, not otherwise specified, 355–356
AML with KIT mutations, t323 T-cell prolymphocytic leukemia (T-PLL), 353, f 354
myeloid sarcoma, 326 TCF3, 329, 425
systemic mastocytosis, 320 TCF3-PBX1, 425
t(16;16)(p13;q22), FISH for hematologic malignancies, t423 TDF. See Testis-determining factor (TDF)
t(16;16)(p13.1;q22.1) in tel. See Telomere (tel)
acute myeloid leukemia (AML) with inv(16) or t(16;16), Telomerase, 13, 146, 301, 382
t323, 324, 424 Telomere (tel), definition, t49
AML with gene mutations, 326 Telomere(s), 12, 13, f 15, 26, f 26, 60, 146
t(16;17) in aneurysmal bone cyst (ABC), 395 banding, 60–61
t(16;17)(q21;p13.2), in aneurysmal bone cyst (ABC), t391 elongation/extension, 301
t(16;17)(q22;p13), in aneurysmal bone cyst (ABC), 395 shortening/loss, 187, 297, 301
t(16;21)(q24.3;q22.3), in primary MDS and t-MDS, t313 Telomeric association (tas), t49
t(17;17) variants, 395 Telophase
t(17;17)(q22;p13.2), in aneurysmal bone cyst (ABC), 395 in meiosis I, 19–20, f 19, f 115
t(17;19)(q22;p13.3), in B-ALL, 329 in meiosis II, 20, f 20, f 116
t(17;22)(q21.3;q13.1), in dermatofibrosarcoma protuberans in mitosis, 18, f 18
(DFSP), 388 Teratogen exposure, 287, 503
t(17;22)(q21.31;q12.2), in Ewing sarcoma/peripheral primitive Teratozoospermia, 218, 223
neuroectodermal tumor (EWS/pPNET), t398 Terminal deletion(s), t29, f 146, 191, f 442
t(17;22)(q22;q13.1) and variants, in dermatofibrosarcoma protuberans in nomenclature, 33, 44
(DFSP), t385 Tertiary aneuploidy, f 160
t(18;22)(q21.3;q11.2), in follicular lymphoma (FL), 335 Testis-determining factor (TDF), 177, 195, 215
t(21;22)(q22.3;q12.2), in Ewing sarcoma/peripheral primitive Testosterone
neuroectodermal tumor (EWS/pPNET), t398 insufficiency, 505, 506
t(X;1)(p11.23;p34.3), in t(Xp11.2) RCC, t379 replacement therapy, 506
t(X;1)(p11.23;q23.1), in t(Xp11.2) RCC, t379 Test validation, 77, 78, 85
t(X;1)(p11.23;q23.1)/TFE3-PRCC, in RCC with Xp11.2 TET1, 325
translocations/TFE3 gene fusions, 380 TET2, 319, 321
t(X;1)(p11.23;p34.3)/TFE3-SFPQ, in RCC with Xp11.2 12-0-tet-radecanoylphorbol-13-acetate (TPA), 55
translocations/TFE3 gene fusions, 380 Tetralogy of Fallot (TOF), 186, 247–249, t249
t(X;2), in anaplastic large cell lymphoma, ALK-positive Tetraploidy, 126
(ALCL-ALK+), 398 mosaic, 126
t(X;2) (q13.1;q36.1), in alveolar rhadbomyosarcoma (RMS), nonmosaic, 126
398, t398 origin of, 126, 282
t(X;3)(p11.3;p21.2), f 189 and spontaneous abortion, 231
t(X;14), in T-cell prolymphocytic leukemia (T-PLL), t353 Tetrasomy
t(X;14)(p22.3;q32.3), cryptic translocation in B-ALL with IGH@ 5p, 127
rearrangements, 333 8, in myelodysplastic syndrome with trisomy 8, 315
t(X;14)(q28;q11), in ataxia telangiectasia, f 298 8p, 127
t(X;14)(q28;q11.2), in T-cell prolymphocytic leukemia 9p, 127
(T-PLL), 353 12p, 127–128, f 128, 150 (see also Isochromosome 12p;
t(X;17) in Pallister-Killian syndrome)
balanced in renal cell carcinoma (RCC), t379 mechanism for formation, 158
unbalanced in alveolar soft part sarcoma (ASPS), t379 of X chromosome, 181
t(X;17)(p11.23;q23.1), in t(Xp11.2) RCC, t379 of Y chromosome, 181
t(X;17)(p11.23;q25.3), in t(Xp11.2) RCC, t379 15pter-15q13, t150
t(X;17)(p11.23;q25.3)/TFE3-ASPSCR1, in RCC with Xp11.2 18p, 129
translocations/TFE3 gene fusions, 380 21, in hyperdiploid B-ALL, 330, f 331
t(X;18)(p11.2;q11.2), in soft tissue tumors, 429 22q, t150 (see also Cat-eye syndrome)
t(X;18)(p11.23;q11.2), in synovial sarcoma (SS), t391, 392, f 392 22q11.2, 130, t150, 511 (see also Cat-eye syndrome)
t(Xp11.2) renal cell carcinoma (RCC), t379 partial, 157
t(Y;14)(p11.3;q32.3), in B-ALL with IGH@ rearrangements, 333 X syndrome (XXXX), 184
TAL1-TRA@, t334 TFE3, 395
TAM. See Transient abnormal myelopoiesis (TAM) TFE3 and TFEB translocation RCC, 380
Tandem repeats, 13, 294, 475 TFE3-ASPSCR1, t379, 380
tas. See Telomeric association (tas) TFEB, 380
TAT. See Turnaround time (TAT) TFEB-ALPHA, t379
T-banding (telomere banding), t25, 26, t49, 60–61 TFE3-CLTC, t379
T-cell ALL/LBL. See Acute T-cell lymphoblastic leukemia TFE3-NONO, 380
T-cell lymphoma, t321, t353 TFE3-PRCC, t379, 380
anaplastic large, 356, t356 TFE3-SFPQ, t379, 380
angioimmunoblastic, 356 Therapy-related MDS/AML, 322, 325, f 325
biologic and genetic characteristics of mature, t353 Therapy-related myelodysplastic syndrome (t-MDS), t313, 314,
cutaneous, 355 315, 445
Index 561
Therapy-related myeloid neoplasms, t323 unbalanced, f 189, 190, t312, 314, 315, 319, 343, 350, f 416, 441,
Therapy-related neoplasms, 324 443, 444, 505
THRAP3, 395 3-way, nomenclature of, 34
THRAP3-USP6, t391 whole-arm, t30, 34, 431
TKI. See Tyrosine kinas\ inhibitor(s) (TKI) of X chromosomes, 188, 215–216
TLX2, 384 of Y chromosomes, 215
TLX3-BCL11B, t334 (X;Y), 194–196
TLX1-TRD@, t334 Translocation/inversions, in AML partial karyogram, f 324, f 325
TM. See Transmitting male (TM) Transmitted light source, 68–69
t-MDS. See Therapy-related myelodysplastic syndrome Transmitting male (TM), 453
(t-MDS) TRA@-TCL1A, t334
TMPRSS2, 381 TRB@-CCND2, t334
TMPRSS2-ERG, t379, 382 TRB@-LMO2, t334
TMPRSS2-ETS, 381 TRB@-LYL1, t334
TMS. See Triple marker screening (TMS) TRB@-NOTCH1, t334
TOF. See Tetralogy of Fallot (TOF) TRB@-TAL2, t334
TOP3A, t418 TRB@-TCL1A, t334
Topoisomerase II inhibitors, 313 TRB@-TLX1, t334
Total quality management (TQM), 77 trc. See Tricentric chromosome (trc)
TP53, 45, 314, 315, 334, 338, 341–343, 345–347, 349, 373–375, t373, TRD@, 333, t334
t379, 381, 382, 387, 390, t391, 394, 395, t398, 403, TRG@, 333, t334
t423, 426–428, t426, f 427 Tricentric chromosome (trc), description, t30
TPA. See 12-0-tet-radecanoylphorbol-13-acetate (TPA) Trichorhinophalangeal syndrome type II. See Langer-Giedion
T-PLL. See T-cell prolymphocytic leukemia (T-PLL) syndrome
TPM3-ALK, t391, 396 Trinucleotide repeat
TQM. See Total quality management (TQM) disorders/diseases, 455, t455, 456
TRA@, 333 expansion, 294, 455, 463
Transabdominal chorionic villus sampling (CVS), 229–230, 237–240, regions, 141
f 241, 243 Triple marker screen/screening (TMS), 252, 253, 256, 257
Transabdominal vs. transcervical CVS, 241 Triple X /trisomy X (XXX)
Transcervical chorionic villus sampling (CVS), 229–230, 239–241, development, 183
f 241, 243 incidence, 117, 119, 120
Transcription, 10, f 12, t48, 79, 201, 301, 321, 331, 333, 338, 380, in liveborn babies, 224, 230, t231
390, 392, 393, 395, 402, 403, 424, 428, 456, f 459, 465, mosaicism, 164, 260, t260, 263
474, 480 origin, 182
Transfer RNA (tRNA), 11, f 14 phenotype, 183
Transient abnormal myelopoiesis (TAM), 117, 323 Triploid(s)/triploidy
Translation, 11, f 14, f 15 active X chromosomes in, 176
mRNA, and FMRP, 457–458 androgenic, 125
nick, 417 in chromosomally abnormal losses, 278
Translocation(s) in chromosomally abnormal miscarriges, 283
the (4;8), 140, 162 diandric, 126
the (8;22), 162–163 digynic, 126, 473
the (11;22), 161 maternal, 277, 283, f 284
balanced, 144, f 145, 159–161, 165, 188–190, f 189, 220, t232, and maternal age, 244
259, 285, 301, 312, 313, 315, 333, 447, f 501, 510 (see mosaic(s), 126, 283
also Reciprocal translocation(s)) in neuroblastoma, 400
balanced, carrier(s), 144, f 144 nonmosaic, 126
complex, 324 origin of, 126, 284
cryptic, 333, f 422 and partial moles, 287
definition, t312 paternal, 283, f 284
de novo, 161, 189 prenatal loss, t277
de novo reciprocal, 510 in recognized pregnancies, 283
description, t29 in spontaneous abortions, t233, t277
jumping, 165 69,XYY, 126
nomenclature of, 34 Trisomy/trisomies
balanced, 37 autosomal, 117–124, 504–505
unbalanced, 35, 44 in abortuses, 113
whole arm, 36 frequency and gestational age, 278
reciprocal, 159, 160, f 160, 161, f 161, 167, 189, f 189, 196, frequency for each chromosome among aborted
218–220, t218, t232, t233, 258, 259, 284, 325, 336, specimens, t234
345, 383, 386, 473, 480, 481, f 481, 510(see also in liveborn babies, t231
Balanced translocation(s)) loss rate, 277
reciprocal, carrier(s), 473 and maternal age, 281
recurring, 161, 325 meiotic/meiotic origin of autosomal, 455
Robertsonian (see Robertsonian translocations) mosaicism diagnosed in amniocytes, t260
562 Index
Trisomy/trisomies (cont.) mosaic/mosaicisms, 123, 243

mosaicism/discrepancies in CVS tissue, t242 parental and meiotic/mitotic origin, t114
origin of, 279 Trisomy 8
parental and meiotic/mitotic origin of, t114 acquired, in myeloid neoplasms, 121
parental origin of, 455 constitutional, 121
prenatal loss of, t277 craniofacial dysmorphism in, t121
rare/uncommon/unusual, 123–124, 263, 278 in distribution of individual trisomies in spontaneous
in spontaneous abortions, 231, 278 abortions, t279
in spontaneous abortions and live births, t277 FISH for, in AML, 423
in unselected spontaneous abortions, t233 FISH for, in CML, 423
in villus mesenchyme, 242 frequency and gestational age, 278
caused by errors in meiosis, 114, 278–280 in hematologic neoplasms, 315
caused by errors in mitosis, 278, 282 in MDS with trisomy 8, 315
double, 278, 425 mosaic, 121, 127, 243, 282
mechanism and etiology of, 114–117 mosaicism diagnosed din amniocytes, t260, t263
partial, 121, 149, 158, 159, 161, 166–167, f 189 mosaicism/discrepancies in chorionic villus tissue, t242
pregnacy loss and, 234, 238, 278, 286, 288 phenotype, 121
rescue, 119, 164, 481, f 482, 485, 486 probes for, in myeloid disorders, 416
sex chromosome, t277, 280–281 significance of single cell with, in prenatal cases, 260
in spontaneous abortion, 113, 224, 231, 278 Trisomy 9
Trisomy 1, 13, 278 and choroid plexus cysts, 252
in distribution of individual trisomies in spontaneous with confined placental mosaicism, 485
abortions, t279 in distribution of individual trisomies in spontaneous
partial, 159, f 160 abortions, t279
Trisomy 2 full, 258
associated with cofined placental mosaicism, 483 mosaicism diagnosed in amniocytes, t260, t263
in direct vs. long-term chorionic villus sampling, 243 mosaicism/discrepancies in chorionic villus tissue, t242
in distribution of individual trisomies in spontaneous mosaic/mosaicism, 122, 252, 260
abortions, t279 nonmosaic, 121–122
mosaicism diagnosed in amniocytes, t260, t263 partial, t160
mosaicism/discrepancies in chorionic villus tissue, t24 Trisomy 10
mosaic/mosaicism, 123, 483 in distribution of individual trisomies in spontaneous
parental and meiotic/mitotic origin, t114 abortions, t279
Trisomy 3 and maternal UPD 10, 485
complete, in splenic marginal zone B-cell lymphoma mosaic, 123, 243
(sMZBCL), 431 mosaicism/discrepancies in chorionic villus tissue, t242
detected by CVS in confined placental mosaicism, 483 Trisomy 11
in direct vs. long-term chorionic villus sampling, 243 in distribution of individual trisomies in spontaneous
in distribution of individual trisomies in spontaneous abortions, t279
abortions, t279 mosaicism detected in amniocytes, t260
mosaic, 123 mosaicism diagnosed in amniocytes, t260, t263
mosaicism diagnosed in amniocytes, t260, t263 mosaicism/discrepancies in chorionic villus tissue, t242
mosaicism/discrepancies in chorionic villus tissue, t242 and paternal UPD 11, 485
Trisomy 4 Trisomy 12, 121
in confined placental mosaicism, 483 in CLL, 342, 343, f 344, t426
in distribution of individual trisomies in spontaneous complete in splenic marginal zone B-cell lymphoma
abortions, t279 (sMZBCL), 341–342
mosaicism diagnosed in amniocytes, t260, t263 in distribution of individual trisomies in spontaneous
Trisomy 5 abortions, t279
in distribution of individual trisomies in spontaneous FISH for, in CLL t423
abortions, t279 mosaicism diagnosed in amniocytes, t260, t263
mosaic, 123 mosaicism/discrepancies in chorionic villus tissue, t242
mosaicism diagnosed in amniocytes, t260, t263 mosaic/mosaicism, 261
mosaicism/discrepancies in chorionic villus tissue, t242 probes for, in chronic lymphocytic leukemia, 416
Trisomy 6 Trisomy 13. See also Patau syndromein association with absence of
in AML with t(8;21)(q22;q22.3), 322 nasal bone, 251
in distribution of individual trisomies in spontaneous in association with choroid plexus cysts, 252, t254
abortions, t279 in association with echogenic intracardiac foci, 250
mosaic, 123 complete, 121
mosaicism diagnosed in amniocytes, t260, t263 description, 120
Trisomy 7 in distribution of individual trisomies in spontaneous
in distribution of individual trisomies in spontaneous abortions, t279
abortions, t279 in fetal deaths subsequent to amniocentesis, t232
false-positive, 243 in fetuses with nuchal folds or membranes and/or cystic
mosaicism diagnosed in amniocytes, t260, t263 hygromas, 246
mosaicism/discrepancies in chorionic villus tissue, t242 frequency and gestational age, 278
Index 563
full, 260, 261 full, 260

incidence, 120 incidence, 119
and increased risk for spina bifida, 258 as an indication for genetic counseling, 504, 505
as an indication for genetic counseling, 504, 505 in liveborns, 231
in liveborn(s) babies, t231 and low maternal serum AFP, 256
mosaic, 121, 260, 261 and maternal age, 119
nonmosaic, 121 mosaic, 120, 243, 260, 261
parental and meiotic/mitotic origin, t114 nonmosaic, 243
partial, 121 parental and meiotic/mitotic origin, t114
phenotype, 120–121 partial, 120, 129
recurrence of trisomy with previous, 119, 121 phenotype, 119–120, f 120
resulting form unbalanced translocation, 505 and previous pregnancy or child with open neural tube defect, 258
Trisomy 14 prolonged survival of, 243
in distribution of individual trisomies in spontaneous recurrence of trisomy with previous, 119–121
abortions, t279 and recurrent pregnancy loss, 288
mosaic, 123–124, 258 and spontaneous abortion, 243
mosaicism diagnosed in amniocytes, t260, t263 Trisomy 19
mosaicism/discrepancies in chorionic villus tissue, t242 in distribution of individual trisomies in spontaneous
parental and meiotic/mitotic origin, t114 abortions, t279
partial, 486 mosaic, 124
Trisomy 15 mosaicism diagnosed in amniocytes, t260, t263
in distribution of individual trisomies in spontaneous mosaicism/discrepancies in chorionic villus tissue, t242
abortions, t279 Trisomy 20
hematologic neoplasms, 315 in distribution of individual trisomies in spontaneous
liveborn, 124 abortions, t279
in MDS with other chromosome abnormalities, 316 and maternal UPD 20, 487–488
and association with loss of the Y chromosome, 316, f 317 mosaicism diagnosed in amniocytes, t263
mosaicism diagnosed in amniocytes, t260, t263 mosaicism/discrepancies in chorionic villus tissue, t242
mosaicism/discrepancies in chorionic villus tissue, t242 mosaic/mosaicism, 122, 244, 261
parental and meiotic/mitotic origin, t114 nonmosaic, 122, 161
Trisomy 16 and paternal UPD 20, 488
in abortuses, 122 pseudomosaic, 244
in distribution of individual trisomies in spontaneous Trisomy 21. See also Down syndrome
abortions, t279 and absent nasal bone, 251–252, t255
frequency in spontaneous abortuses or liveborns, 113 acquired, 41, 362
full, 122 in acute myeloid leukemia (AML), t323
maternal origin of, 114, 261 in AML with t(8;21)(q22;q22.3), 322
mosaic, 122, t242, 261, 262, 487 in association with choroid plexus cysts, 252–253
mosaicism diagnosed in amniocytes, t260, t263 in association with echogenic intracardiac foci, 250
mosaicism/discrepancies in chorionic villus tissue, t242 in association with nuchal fold/thickening, 246, 247
nonmosaic, 261 in association with structural heart abnormalities, 248
parental and meiotic/mitotic origin, t114 and confusion with Turner syndrome in spontaneous
and reduced recombination, 116 abortion, f 279
Trisomy 17 detection by
confined mosaic, 124 BAC array, 444, f 444
in distribution of individual trisomies in spontaneous first-trimester screening, 257
abortions, t279 integrated and combined screening, 257
and maternal UPD 17, 487 oligonucleotide array, 444
mosaicism diagnosed in amniocytes, t260, t263 SNP array, f 444
mosaicism/discrepancies in chorionic villus tissue, t242 in distribution of individual trisomies in spontaneous
Trisomy 18, 120. See also Edwards syndrome abortions, t279
and abnormal prenatal screen, 503, 508–509 in fetal deaths subsequent to amniocentesis, t232
and absence of nasal bone, 251 in fetuses with nuchal folds or membranes and/or cystic
in association with choroid plexus cysts, 252–253, t255 hygromas, 246
in association with echogenic intracardiac foci, 250 frequency and gestational age, 278
in association with hyperechoic bowel, 253 in hyperdiploid B-ALL, 330–331, f 331
detection by first-trimester screening, 257 incidence, 117, 230
detection by triple marker screening, 256 karyogram of, f 118
detection with interphase FISH, in confined placental in liveborn(s) babies, t231
mosaicism, 243 and low maternal serum AFP, 256
in distribution of individual trisomies in spontaneous maternal age-specific risks for, 118
abortions, t279 maternal origin of, 117
in fetal deaths subsequent to amniocentesis, t232 and maternal serum marker screening, 251
in fetuses with nuchal folds or membranes and/or cystic mosaic, 116, 119, 261
hygromas, 246 mosaicism/discrepancies in chorionic villus tissue, t242
frequency and gestational age, 278 myeloid leukemia associated with, 117
564 Index
Trisomy 20 (cont.) genetic counseling, 188, 506–507

nonmosaic, 117, 119, 504 incidence, 181
parental and meiotic/mitotic origin, t114 infertility in, 506–507
partial, 117–118 isochromosome X, 157, 181, f 181, 186, 188, 194
phenotype, 117–119, f 119 with isochromosome Y, 197
and possible association with hyperechoic bowel, 253, t254 isodicentric Xq in, 194
and possible association with renal pyelectasi, 251 in liveborn babies, t231
and possible paternal age association, 503 marker chromosomes in patients with, 182
prenatal detection with interphase FISH, f 422 and maternal age, 231, 244
quadruple screening for detection of, 256 mosaicism in, 179–180
recurrence of trisomy with previous, 119, 120 detection of Y chromosome sequences in, 215
and reduction of recombination, 116 45,X/47,XXX, 182
risk by maternal age at term, t503 with a Y chromosome, 180–182
secondary to a translocation or tan-dem duplication of nuchal thickening, 245
chromosome 21, 159 origin of the X chromosome in, 178
and short femur, 253, t255 ovarian degeneration in, 507
and short humerus, 253, t255 phenotype, 178–179, f 179
in spontaneous abortion(s), 278, f 279 ring X in, 181–182
and transient abnormal myelopoiesis, 117, t323 risk of gonadoblastoma, with presence of Y chromosome, 182
ultrasound markers for, t255 SHOX
Trisomy 22 deletion in, 191, 195
in distribution of individual trisomies in spontaneous haploinsufficiency in, 178, 191
abortions, t279 spontaneous loss, 178, 281
in maternal UPD 22 ultrasound abnormalities in, 178
mosaicism detected in amniocytes, t260 variants, 178, 179
mosaic/mosaicism, 122, 262, 488 47,XY,i(X)(q10), 186
nonmosaic full, 122, 279, 488 with X;Y translocations and 45,X cell line, 195
parental and meiotic/mitotic origin, t114 Y chromosomes, 180, 194
phenotype, 122 with Y chromosome sequences, 215
secondary to a Robertsonian translocation, 164 Tyrosine kinase
in spontaneous abortions, 278 aberrant, fusion protein, 431
survival, 122 abnormal, t317, 431
Trisomy 22q. See Cat-eye syndrome; Tetrasomy 22q activity/activation, 356, 392, 424
Trisomy rescue in acute myeloid leukemia with gene rearrangements, 326
and reproductive risks for carriers of Robertsonian chimeric, fusion product, 390
translocations, 164 genes, 384
in trisomy 21, 119 inhibitor(s) (TKIs), 319–321, 371, 384, t385, 388, 390, 428
in uniparental disomy (UPD), 164, 481, f 482, 485, 486 myeloid neoplasms with translocations involving, 321
Trisomy X/triple X (XXX), 182 rearrangements involving genes that code for, t321
development, 183 receptor, 384, 390, 395
incidence, 117, 119, 120
in liveborn babies, 224, 230, t231
mosaicism, 164, 260, t260, 263 U
origin, 182 UBE3A, 417, 477, t477
phenotype, 183 UDS. See UV-induced unscheduled DNA synthesis (UDS)
tRNA. See Transfer RNA (tRNA) uE3. See Unconjugated estriol (uE3)
trp. See Tricentric chromosome (trp) U-loop exchange, 155, 156
Trypsin, t25, 55, 56, 59, 230 Ultrasound
Trypsin-Giemsa banding method, 310 abnormal fetal/findings on, 245
TSPAN31, 391–393, t391 abnormal, findings, 261, 262, 264, 445
TSPY1, 199 and advanced paternal age, 260
TTAGGG, 13, 26, t49, 146, 416 and amniocentesis, 237–238
Tubulin, 57 choroid plexus cysts, 252
Tumor suppressor gene(s), 295, 312, t315, 375, 378, 385, 389, 403, 480 clinical findings amenable to detection by, t249
Turnaround time (TAT), 56, 101, 102, 232, 233, 237–240, 448, 511 clinical outcome of second-trimester finding of isolated bright
Turner syndrome (45,X and variants). See also Monosomy X; 45,X hyperechoic bowel, t254
and confusion with trisomy 21 in spontaneous abortion, f 279 for couples with a previous pregnancy or child with a chromosome
deletions of Xp, 190–191, 195 abnormality, 257–258
deletions of Yp, 196 for couples with a previous pregnancy or child with open neural
detection of Y chromosome sequences, 215 tube defect, 258
development, 179 for detection of
and estrogen replacement therapy, 507 absent or hypoplastic nasal bone, 250–251, t255
features with isochromosome Yp, 196 association with trisomy 21, 250
features with isochromosome Yq, 198 choroid plexus cysts, 252, t255
FISH for marker chromosome assessment, 420 clinical findings consistent with 22q11.2 deletion, t249
FISH with suspected low-level Y chromosome mosaicism in, 281 cystic hygroma and/or hydrops, 507
Index 565
echogenic bowel, 250, 253 (see also Hyperechoic bowel; in polycythemia vera, 319
Hyperechogenic fetal bowel) trisomy 13 resulting from, 505
echogenic foci/focus, 250, 256 trisomy 18 resulting from, 505
echogenic intracardiac foci/focus, 250, t255, 256 X;autosome, 190
echogenic lesions, 250 Unbanded chromosomes, 463. See also Solid stained chromosomes
extracardiac abnormalities, 248 Unclassifiable, B-cell lymphoma, with features intermediate
with features seen in known UPD syndromes, 489 between diffuse large B-cell lymphoma and Burkitt
hyperechogenic fetal bowel, 253 (see also Epigenic bowel; lymphoma, 338
Hyperechoic bowel) Unclassifiable, B-cell lymphoma, with features intermediate
hyperechoic bowel, 253, f 254 (see also Epigenic bowel; between diffuse large B-cell lymphoma and Hodgkin
Hyperechogenic fetal bowel) lymphoma, 338
intracardiac echogenic foci, 248, 250, t255 Unclassifiable-DLBCL/Burkitt, t336
isolated bright hyperechoic bowel, t254 Unclassifiable-DLBCL/Hodgkin, t336
isolated IUGR, 242 Unconjugated estriol (uE3), 256, 509
left ventricular echogenic foci, 248 Underline, single (_) in nomenclature, t28
markers of fetal aneuploidy, t255 Uniparental disomy (UPD), 39, t49, 86, 114, 116, 123, 124, 129, 144,
nasal bone, 250–251, t255 147, 159, 164, 181, 191, 242, 261, 262, 278, 282, 287,
nuchal thickening/fold/translucency, 245–247, t246, f 255, t255 385, 386, 417, 420, 473–489, f 482, 505, 508, 510, 511
septated, t255, 508 in Angelmann syndrome, 129, 164, 417, 474, 476–477, t477, 486
simple, t255 in Beckwith-Weidemann syndrome, 385, f 419, 477–479
22q11.2 deletion syndrome, 505 in chromosome 15 nondisjunction, 116
rare trisomy mosaicism determined by, and/or physical in confined placental mosaicism, 242, 483
examination at termination or birth, t263 definition, t49
renal pyelectasis, 251–252, t255 detection by SNP array, 511
right ventricular echogenic foci, 248, 250 formation by gamete complementation, f 481
septated nuchal membrane, t255 monosomy rescue, 159, 481
short femur, 253 nomenclature of, 39
short humerus, 252 in Prader-Willi syndrome, 129, 164, 417, 474, 476–477, t477, 486
simple nuchal membrane, t255 reproductive risk for carriers of Robertsonian translocations,
structural heart defect, t255 164–165
trisomy 18, 246, 250, t255, 503 risk of, in a phenotypically normal parent with a balanced
trisomy 21 (Down syndrome), 246–248, 503 translocation, 510
and early amniocentesis, 235, 237 trisomy rescue and, 164, 481, f 482, 485, 486
effect of BMI on detection of, abnormalities, 256 Uniparental isodisomy, detection by SNP array, t445
first trimester, 245–247, 257 Unique sequence
in genetic counseling, 509, 510 DNA, 13
guidance for chorionic villus sampling, 508 DNA probes, 415–417
high-resolution, 261, 262 Unknown origin (add), additional material of,
interpretation of, rusults on obese patients, 256 definition, t48
numerical abnormalities of the sex chromosomes, 177 description, t29
and percutaneous umbilical blood sampling (PUBS), 244, 510 in nomenclature, 37, 39
in predicting phenotypic sex, 198 Unknown origin, materal of/chromosome of
real-time, 230 in nomenclature, 33, 35
second trimester, 246, 250, 252, 253, f 246, t254 reverse FISH for identification of, 434–435
targeted, 264 Unknown rec, description, t30
for Y-chromosome mosaicism in 45,X patients, 180 Unrelated clones,
Ultraviolet (UV) light karyograms of, 84
emission, 76 in nomenclature, 40, 41
excitation, 76 UPD. See Uniparental disomy (UPD)
excitement of a fluorochromes, 415 upd(1)mat, 482
exposure, in xeroderma pigmentosum (XP), 302 upd(1)pat, 482
irradiation, in xeroderma pigmentosum (XP), 302 upd(2)mat, 483
sensitivity, in xeroderma pigmentosum (XP), 302 upd(2)pat, 483
and slide aging, 58 upd(3)mat, 483
and transmitted light source, 69 upd(3)pat, 483
Unbalanced rearrangement(s), 142, 143, 284, 285, 315, 325, upd(4)mat, 483
332, 425, 506 upd(5)pat, 484
Unbalanced translocation(s) upd(6)mat, 484
in CLL with deletion of 17p, 343 upd(6)pat, 484
detected with “painting” probe, 416 upd(7)mat, 484
Down syndrome resulting from, 505 upd(7)pat, 484
in MDS with deletion of 5q, 314 upd(8)mat, 484–485
in MDS with other chromosome abnormalities, 315 upd(8)pat, 485
microarray analysis, 441, 443, 444 upd(9)mat, 485
in nomenclature, 35, 44 upd(10)mat, 485
in plasma cell myeoloma (PCM), 349, 350 upd(11)pat, 485
566 Index
upd(12)mat, 485 t(1;19), in B-ALL, 329

upd(13)mat, 485 t(2;8)(p11;q24), in Burkitt lymphoma, 428
upd(13)pat, 485–486 t(8;14)
upd(14)mat, 486 in B-cell prolymphocytic leukemia (B-PLL), 345
upd(14)pat, 486 in Burkitt lymphoma (BL), t336
upd(15)mat, 486–487 in CLL/SLL, t336
upd(15)pat, 487 in follicular lymphoma,(FL), t336
upd(16)mat, 487 in unclassifiable—DLBCL/Burkitt, t336
upd(16)pat, 487 t(8;22)(q24;q11), in Burkitt lymphoma, 428
upd(17)mat, 487 t(12;22)(q13.3;q12.2), in lipoblastoid, 393
upd(20)mat, 487–488 translocation(s), 317
upd(20)pat, 488 in AML with t(8;21)(q22;q22.3), 322
upd(21)mat, 488 in conventional lipoma, t391
upd(21)pat, 488 in follicular lymphoma, 335
upd(22)mat, 488 involving 2p23 (ALK), in anaplastic large cell lymphoma,
upd(22)pat, 488 ALK-positive (ALCL-ALK+), t353, 428
upd(X)mat, 488 that rearrange PAX3, in alveolar rhabdomyosarcoma, 398
upd(X)pat, 488–489 of unknown significance
upd(XY)pat, 489 xeroderma pigmentosum (XPV), 303
Urothelial cancer, 431. See also Bladder cancer Variegated translocation mosaicism (VTM), 301
UroVysion, f 105, 382, 431, f 431 VCAN, 380
US Food and Drug Administration (FDA), 78, 258, 382, 430 VCD. See Ventricular septal defect (VCD)
USP6, t391, 395 VCF. See Velocardiofacial (VCF) syndrome
USP6-COL1A1, t391, f 396 v-CLL, t335
USP9Y, 197, 222 Velocardiofacial (VCF) syndrome, 418, t149, 505. See also Deletion
UTY, 222 22q syndromes; DiGeorge syndrome; Shprintzen
Uveal melanoma, t385, 389 syndrome
UV. See Ultraviolet (UV) light Ventricular septal defect (VCD), 117, 120, 123, 127, 186, 224,
UV-induced unscheduled DNA synthesis (UDS), 303 247–249, f 248, t249
Vernier grids, 72
von Hippel-Lindau (VHL), 378–380
V VTM. See Variegated translocation mosaicism (VTM)
Variable expressivity, 145, 168, 502
Variant(s),
BCR-ABL1 transcript, 320 W
blastoid, of mantle cel lymphopma, f 341 Warthin’s tumor, t385, 387, 389
breakpoints, of MLL translocation, 325 Watson and Crick, 9
CCND1, in mantle cell lymphoma (MCL), t336, 340 Watson-Crick base pairs, 456
CCND2, in mantle cell lymphoma (MCL), t336, 340 Watson, James, 9
CCND3, in mantle cell lymphoma (MCL), t336, 340 WCP. See Whole chromosome probes (WCP)
cellular, of congenital mesoblastic nephroma (CMN), 390 Werner syndrome (WS), 296, 300–301, f 301
chromosomal, 167 clinical features, 300
chromosomes, 167 prevalence, 301
fetal deaths subsequent to amniocentesis, 232 WRN, 301
clear cell, of Hidradenoma, t385, 389 WHO. See World Health Organization (WHO)
CML, 317 Whole-arm translocations,
copy number, 192, 511 description, t30
dermatofibrosarcoma protuberans (DFSP) and, t385 nomenclature, 34–36
detected in CGH, 264 Whole chromosome probes (WCP), 85, 416
and microarrays, 264 WHSC1, 350
epitheliod, t391 Wilkins, Maurice, 9
ETV6-RUNX1 fusion patterns, 331 Williams syndrome (WS), 147, 154, 419, 505
forms, 152 Wilms tumor (WT), 201, 296, 371, t379, 381, 402, 477,
granular, of ccRCC, 381 479–480, 485
hairy cell leukemia (HCL), 346 Wolf-Hirschhorn syndrome, t148, 350
histologic, of endometrial stromal tumors (EST), 383 Worksheets
inversions, 152 analysis, 86, 90
Klinefelter syndrome, 185 culture, 90
leukemic, of mantle cell lymphoma (MCL), 345, f 346 World Health Organization (WHO)
mosaic, 213 classification of acute myeloid leukemia (AML), t323
normal, 152, 198, 199, 253 classification of brain tumors, 378
polymorphic, 167 classification of endometrial stromal tumors, 382
population, 258 classification of mature B-Cell lymphoid neoplasms, t366
recurrent, 151 classification of MDS, 312–313, 423
t(1;2) (p22.3;p12), in mucosa-associated lymphoid tissue (MALT) subdivision of, t313
lymphoma, 341 translocations and inversions that define, f 324
Index 567
CVS registry, 230, 240 ring, 181–182

gliomas of, grade II or III, 374 lacking XIST, 182
guidelines for hematologic neoplasms, 312 role of, in sexual differentiation, 175
and infertility, 213, 217 satelitted, 199
study on limb defects and CVS, 239 translocations of, with Y chromosome, 195
WRN, 301 unbalanced translocations with, and autosomes, 190
WS. See Werner syndrome (WS) variants of Klinefelter syndrome with additional, 184–185
WS. See Williams syndrome (WS) XCI (see X-chromosome inactivation (XCI))
WT. See Wilms tumor (WT) X-chromosome inactivation (XCI), 175–176
WT1, 201, 321, 326, 402 Xeroderma pigmentosum (XP)
WT1-EWSR1, t398, 402 chromosome breakage in, 303
clinical features, 302
complementation groups (XPA to XPG, and XP-V), 303
X diagnosis, 303
45,X and 45,X mosaicism, 213–215 DNA repair, 303
and in vitro fertilization, 214–215 frequency, 303
Y chromosome sequences in, 215 genes involved in, 303
X;autosome translocations, 143, 188–190, 215–216 ocular involvement in, 303
X chromatin. See Barr body; Sex chromatin body skin cancer in, 303
X chromosome(s) XIC. See X-inactvation ceneter (XIC)
abnormalities in niomenclature, 28, 30–31 X-inactivation ceneter (XIC), 176, 177
additional, 184 X-inactive-specific transcript, 176, f 176
gonadal dysgenesis in patients with, 184 XIST
androgen excess, disorders of, 200, 201 FISH probe for, 180, t420
aneuploidy and age, 177, 187–188 ideogram of X chromosome showing, f 176
balanced reciproclal translocation of, f 189 inactivation and size of rings, 180
balanced translocations with in i(Xp) chromosomes, 194
and autosomes, 188–190 lack/absence of, in ring X, 180, 182
autosomes in males, in 190 negative marker chromosomes, 420
and ovarian failure, 188 positive marker chromosomes, 42
deletions promotor mutation, 177
of Xp, 191 X-linked dominant inheritance, 502
of Xp: gonadal dysgenesis in, 191 X-linked ichthyosis, t149, 191
of Xp: ichthyosis (X-linked), t149 X-linked mental retardation, 193, 453, 454, 460
of Xp: Kallman syndrome, 195 X-linked recessive inheritance, 502
of Xq, 191 xma. See Chiasma/chiasmata (xma)
in disorders of gonadal (ovarian) development, 200–201 XP. See Xeroderma pigmentosum (XP)
in disorders of gonadal (testicular) development, XP genes, 303
177, 201 45,X, 231
duplications in female infertility due to ovarian dysfunction, 214
of Xp, 191, 192 in liveborn babies, t231
of Xq, 192 45,X/46,X,i(X)(q10)/46,XX, 181
ideogram of, f 176, f 215 45,X/46,X,r(Y), 198
inactivation (XCI), 175–176 45,X/46,XX
escape of, 175, 191 in choroid plexus cysts (CPC), 252
establishment of, 182 in deaths subsequent to amniocentesis, t232
extent of, 182 difference between fetal and newborn rates of, 231
random, 189, 192, 193, 474 in female infertility due to ovarian dysfunction, 214
skewed, 192 origin of, 260
inactive, 17, 143, 176 in upd(X)pat, 488
inversions of 45,X/46,XX/47,XXX, in female infertility due to ovarian
paracentric, 30, 33, 193, 258, 435 dysfunction, 214
pericentric, 29, 193, t218, 231, t259 45,X/46,XX/47,XXX/48,XXXX, in female infertility due to ovarian
pericentric: gonadal dysgenesis in, 193 dysfunction, 214
isochromosomes of 45,X/46,XY
t(X;14)(p22.3;q32.3) in B-ALL, 333 chimerism, 200
Xq, 181, 186, 188 with deletions of the Y chromosome, 222
isodicentric difference between fetal and newborn rates of, 231
identification by mBAND, 433, f 435 in female infertility due to ovarian dysfunction, 214
Xp, 193–194 genetic counseling of, 509–510
Xp: gonadal dysgenesis in, 194 gonadoblastoma in, 177
Xq, 194 and ICSI in men with, 223
Xq: gonadal dysgenesis in, 194 in men with infertility, t218
loss of, in bone marrow of female patients, 315 in mixed gonadal dysgenesis, 200
origin of, in Turner syndrome, 178 mosaicism, 180–181
pseudoautosomal regions of, 43, f 176, 178, 186, 191 45,X/47,XXX, 182, 214
568 Index
45,X/69,XXY, 126, 283 and sex chromosome disorder of sex development, 200
45,X[20]/46,X,i(X)(p10)[20], secondary to bone marrow stem plant in Turner syndrome variants, 179
transplantation, t28 47,XXY/46,XY, 219, t221, 506
46,X,del(Y)(q11q22), in men with infertility, t218 and ICSI in men with, 223
46,X,del(Y)(q12), in men with infertility, t218 47,XXY/48,XXXY;46,XX, t218, 219
46,X,dic(X;Y)(Xqter®Xp22::Yp11®Yqter), 195 47,XY,i(X)(q10), 186
46,X,i(Xq), in female infertility due to ovarian dysfunction, 213 47,XY,+psu idic(9)(q21.1), f 256
46,X,idic(Yp), 197 47,XYY
46,XX and advanced maternal age, 244
complete hydatidiform mole with, t285 chromosome abnormalities occurring in pregnancies
and disorders of androgen excess, 201 of men with, 259
in disorders of gonadal (ovarian) development, 200 development, 186–187
with disorders of sex development (DSD), 200, 201 diagnosis of, 186, 219
in female infertility due to ovarian dysfunction, 214 difference between fetal and newborn rates of, 231
in infertile men, t218, t221 fertility of men with, 259
with sex reversal, 199 in fetal deaths subsequent to aamniocentesis, t232
46,XX/46,XY and high maternal serum alpha-fetoprotein, 256
chimerism, 200, 283 incidence of, 219, 280
hermaphroditism, 283 in infertile men, 218, t221
and ovotesticular disorders of ovarian development (DSD), Klinefelters variants with, 185
200–201 in liveborn babies, 231
46,XX/47,XX,r(Y), 198 in men with infertility, t218, 219
46,XX disorders of sex development, (DSD) 200 origin of, 186
46,XX DSD, 200. See also Disorders of sex phenotype, 186, 507
development (DSD) in spontaneous pregnancy loss and in liveborn infant, 280
46,XX male(s), 141, 154 in triploidy, 125
46,XX newborns with sex reversal, 199 47,XYY/46,XY
46,XX sex-reversed patients/males, 200, f 422 in men with infertility, 219
46,XX (SRY+), 195 in renal pyelectasis, 251
46,XX testicular disorders of sex development, 200 48,XXXX, 184
46,XY 48,XXXY, 185, 186, t218
complete gonadal dysgenesis, 201 48,XXYY, 185–186, 506
disorders of gonadal (testicular) development, 201 48,XYYY, 187
disorders of sex development (DSD), 200, 201 49,XXXXX, 184, 186
female(s) 177, 199, 201 49,XXXXY, 185–186
in female infertility due to ovarian dysfunction, 213–214 49,XXXYY, 186
in male infertility, t218 49,XYYYY, 187
male with female phenotype, t217 69,XXX, f 125, 126, 258, 283
sex reversal, 201 69,XXY, 126, 258, 280, 283, f 284
46,XY,inv(1), t218 69,XXY, paternal origin of, 283
46,XY,inv(9)(p11q12), t218 92,XXXY, 126
46,XY,inv(9)(p23q33), t218 XX male(s), 141, 154–155, 195, 200. See also 46,XX testicular
47,XXX, 179, 182, 186, 213, 231, 246, t254, 256, 280, 507 disorders of sex development
development, 183 XX women, “drumstick” in, 176
in female infertility due to ovarian dysfunction, 213–214 XXX, 4, 125–126, 183, 230, 231
in fetal deaths subsequent to amniocentesis, t232 XXXX, 126
and high maternal serum alpha-fetoprotein, 256 in alveolar rhadbomyosarcoma, t399
in liveborn babies, t231 XXX:XXY:XYY, in spontaneous abortuses, 125
origin of, conceptions, 182 XX/XY, admixture/mixture of cells in maternal cell contamination
phenotype, 183 (MCC), 243, 263
47,XXY XXXY, 126
and choroid plexus cysts (CPC), 252 XXXYY, 186
diagnosis od, 219 XXY, 125, 126, 183
difference between fetal and newborn rates of, 231 XXYY, 126, f 384, 506
in fetal deaths subsequent to amniocentesis, t232 XY
genetic counseling of, 506 aneuploidy sperm, 198
and high maternal serum alpha-fetoprotein, 256 cells in amniotic fluid, 263
and ICSI in men with, 184, 222–223 females, 155, 177, 195, 201 (see also Disorders of gonadal
incidence of, 230 (testicular) development)
in infertile males, t221 fetus with trisomy 21, using unique copy probes, f 422
and Klinefelter syndrome, 506 gonadal dysgenesis, 177
in liveborn babies, t231 with microdeletions of Y, 222
and maternal age, 244 paternal UPD, 489
in men with infertility, t218, 219 sex reversal, 199, 201
and oligospermia, non-obstructive azoospermia, and sperm, 186
teratozoospermia, 218 translocation, 141
Index 569
X-Y bivalent, 219, 220 in patients with Turner syndrome, 180–182

XYY polymorphisms of
incidence of, 230 distal Yq, staining for, 61, 62
in infertile men, t221 heterochromatin length, 198
XYYY, 126, 187 inverted, 199
XYYYY, 187 length, 198–199
satellited, 198–199
pseudoautosomal regions, 19, 175–177, f 176, 186, 197,
Y 199, 200, 333
Y;autosome translocations, 195, 196 ring
Y chromosome(s), 177 FISH for detection of, 198
age associated loss of, 188 microarray for detection of, 198
deletions of the, 196 origin of, 198
dicentric, 197 and sexual differentiation, 175, 196
and disorders of sex development, 191, 196, 199–201 structural abnormalities of, 194–198
gonadoblastoma, risk of in females with, 177, 181, 182, 194, and susceptibility to gonadoblastoma, 177
196–197, 281, 423, 510 translocations involving, 141, 194–196, t195, 200
heterochromatic region of, 177 Yq11®Yqter onto Xp22, 194
ideogram of, f 176 Y sequences, 200
inactivation, 176, 177 YWHAE, 381
isochromosome(s) of
dicentric, 197
mosaicism involving, 197, 198 Z
origin of, 198 ZAC, 484
Yp, 197–198 ZAP70, 345
Yq, 197–198 Zeta-chain (TCR)-associated protein kinase (ZAP70), 345
isodicentric(s) of, 197–198 ZFY, 199
marker chromosomes derived from, and risk ZNF9, 395
of gonadoblastoma, 421 Zygote, 18, 20, 39, 119, 126, 159, 261, 480, f 482
mosaicism involving, 179–181 Zygotene, 19

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Steven L.

The Principles of Clinical

ISBN 978-1-4419-1687-7 ISBN 978-1-4419-1688-4 (e-Book)

Library of Congress Control Number: 2012947405

© Springer Science+Business Media New York 2013

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

Some cytogeneticists took offense at this.

Part I Basic Concepts and Background

14 Chromosome Instability .......................................................................................... 293

Marilyn L. Slovak, Ph.D. Cytogenetics Laboratory, Palo Verde Laboratory/Sonora Quest

Fig. 1.1 Jérôme Lejeune

Fig. 1.2 The first photograph of

Introduction guanine are purines with a double-ring structure, whereas

enzyme DNA primase uses ribonucleotides to form a ribo-

5’ Okazaki DNA and RNA

5’ DNA and RNA

Fig. 2.3 Transcription. A DNA

rRNA. In humans, there are theoretically ten nucleolar

The telomeres are the physical ends of chromosomes.

Fig. 2.6 Overview of protein

Constitutive heterochromatin consists of simple repeats of Facultative Heterochromatin

The Cell Cycle

Fig. 2.8 The functional and Metacentric Submetacentric Acrocentric

Long arm (q)

Chromosomes are at their greatest elongation and are not

Fig. 2.10 Mitosis. Schematic

Anaphase of mitosis are two genetically identical daughter cells, each

Meiosis I is comprised of several substages: prophase I,

The cells move directly from telophase I to metaphase II

Spermatogenesis and Oögenesis

The steps of spermatogenesis and oögenesis are the same in

Fig. 2.13 Spermatogenesis and Spermatogenesis Oögenesis

Spermatids Ovum (egg) &

ISCN [2] is one of several “dog-eared” books within an arm’s

Table 3.2 ISCN punctuation and significance

Table 3.4 (continued)

del(5)(p15.3) Inversions (inv)

Insertions (ins) The material distal to 9q34.1 is translocated to chromosome

A translocation is an abnormality resulting from an exchange Derivative Chromosome Generated by One

46,XX,+1,der(1;7)(q10;p10) The arrows in the detailed system describe the derivative

Derivative Chromosome Generated by Isochromosomes (i)

or detailed system: 46,X,i(X)(q10)

These are structurally altered chromosomes with two 46,XY,ider(22)(q10)t(9;22)(q34.1;q11.2)

long arm of chromosome 21, resulting in Down syndrome in Neocentromeres (neo)

45,XY,dic(14;21)(p11.2;p11.2) For example, a derivative chromosome containing a neo-

The triplet “add” is used to describe material of unknown

46,Y,t(X;22;2)(q22.1;q22.2;p21) Marker Chromosomes (mar)

Ten cells were examined; for cancer karyotypes, this is

Fragile Sites (fra) Additional Symbols

Examples of using cancer nomenclature follow:

Unrelated Clones This indicates a tumor from a patient with constitu-

Non-clonal or single cell aberrations should not be reported

Table 3.5 Common symbols and abbreviations used in molecular cytogenetics

ish 22q11.2(HIRA×2) Subtelomere FISH (subtel)

ish.del(2)(q13q13)(NPHP1− −) ish subtel(41×2)

Two copies each of RB1, D18Z1, and DSCR were

This FISH study is positive for a biallelic 13q deletion. In

Gene Ampliﬁcation Detected by FISH

single nucleotide polymorphism (SNP)-based arrays deter- Oncology Microarrays