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2011.diagnostic Implications of Excessive Homozygosity Detected by SNP Based Microarray

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17 views19 pages

2011.diagnostic Implications of Excessive Homozygosity Detected by SNP Based Microarray

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rpina.gen
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Diagnostic Implications of

Excessive Homozygosity
Detected by SNP-Based
Microarrays: Consanguinity,
Uniparental Disomy, and
Recessive Single-Gene Mutations
Hutton M. Kearney, PhDa,*, Joseph B. Kearney, PhDa,
Laura K. Conlin, PhDb

KEYWORDS
• Microarray • Single nucleotide polymorphism
• Homozygosity • Autozygosity • Consanguinity
• Uniparental disomy

Single nucleotide polymorphism (SNP)-based microarray analysis provides detection


of copy number variations (CNVs) as well as genotype information at multiple
polymorphic loci throughout the genome. Although the clinical utility of CNV detection
is well accepted,1,2 information derived from SNP-based microarray analysis has only
more recently been utilized in the constitutional cytogenetics laboratory setting.3,4 In
addition to specific genotype data, analysis of SNP allele patterns can provide
(1) confirmation of CNV calls, (2) sensitivity for detection of mosaicism, and (3)
detection of excessive homozygosity, which is the focus of this review.
Several different SNP-based microarray platforms are currently in clinical use, and
this review illustrates similar data obtained from Affymetrix- and Illumina-based
microarrays (Affymetrix Inc, Santa Clara, CA, USA; Illumina Inc, San Diego, CA, USA).
It is generally not necessary to derive specific genotypes from the microarray data to
view the SNP probes as present in a homozygous or heterozygous state. For both
platforms, biallelic SNPs are denoted as “A” or “B.” Relative allele distribution is
typically provided by either allele difference plots or B-allele frequency plots (Fig. 1).

Fullerton Genetics Center: data derived from the CytoScan HD was supported in part by
Affymetrix, Inc. (arrays and reagents provided in kind for platform R&D).
a
Fullerton Genetics Center, Mission Health System, 267 McDowell Street, Asheville, NC 28803, USA
b
Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, 3615
Civic Center Boulevard, Abramson Research Building, Room 1007B, Philadelphia, PA 19146, USA
* Corresponding author.
E-mail address: [email protected]

Clin Lab Med 31 (2011) 595– 613


doi:10.1016/j.cll.2011.08.003 labmed.theclinics.com
0272-2712/11/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
596 Kearney et al

Fig. 1. Allele plots. (A) Screenshot of a chromosome 13 from a patient DNA sample run on the
Illumina Quad610 array, and visualized in BeadStudio software. Top: Log R ratio plot shows
a normal copy number state for the chromosome. Bottom: B-allele frequency plot shows a
large region of homozygosity from bands 13q12.11 to 13q14.3. (B) Screenshot of a chromo-
some 13 from a patient DNA sample run on the Affymetrix CytoScan™ HD array and
visualized in Affymetrix Chromosome Analysis Suite (ChAS) software. Top: Weighted log2
ratio plot shows a normal copy number state for the chromosome. Bottom: Allele difference
plot shows 2 regions of homozygosity from bands 13q13.1 to q13.3 and 13q33.2 to q33.3.
Panels C and D represent magnified views of the allele plots for each software, from the
boxed regions in panels A and B. (C) B-allele frequency, as plotted in Illumina BeadStudio
software. The Y-axis value is determined by the formula given on the right. Alleles are plotted
as a frequency, determined by the number of B alleles compared with the total number of
alleles (A⫹B). B-allele frequency of 1 indicates homozygosity for the B allele (2/2), a frequency
of 0 indicates homozygosity for the A allele (0/2), and a frequency of 0.5 indicates a
heterozygous genotype (1/2). (D) Allele difference, as plotted in Affymetrix Chromosome
Analysis Suite (ChAS) software. The Y-axis value is determined by the formula given on the
right. Alleles are plotted as the difference between the estimated number of A alleles and the
estimated number of B alleles (A-B). Each allele corresponds to a value of 0.5. Values near 1
indicate homozygosity for the A allele (AA: [0.5⫹0.5] – [0] ⫽ 1), values near –1 indicate
homozygosity for the B allele (BB: [0] – [0.5 ⫹ 0.5] ⫽ –1), and values near 0 indicate a
heterozygous genotype (AB: [0.5] – [0.5] ⫽ 0).

Multiple terms are used to describe regions of homozygosity, and each conveys a
slightly different meaning (eg, loss of heterozygosity, absence of heterozygosity, runs
of homozygosity). Here we use the term long-contiguous stretch of homozygosity
(LCSH) to describe an uninterrupted region of homozygous alleles with genomic copy
number state of 2. The term LCSH excludes loss of heterozygosity caused by single
copy deletions because these regions exist in a hemizygous state. Minimal thresholds
for LCSH calls are generally set around 0.5 to 1 Mb in population genetic analyses5–7
and more conservatively at 3 to 10 Mb in clinical analyses,4(Kearney H, Kearney J,
and Conlin L, personal communication).
Detection of excessive homozygosity, in and of itself, is not diagnostic of any
underlying condition and may be clinically benign. The first step in assessing the
clinical relevance of observed LCSH is to distinguish between excessive homozygos-
ity found in multiple regions throughout the genome versus LCSH restricted to a
single chromosome. When multiple LCSH regions are found throughout the genome,
the findings are generally assumed to represent regions identical by descent (IBD),
Diagnostic Implications of Excessive Homozygosity 597

Table 1
Correlation between percentage of LCSH and degree of parental relationship
Coefficient of LCSH (IBD) Predicted
Parental Relationship Degree Inbreeding (F) in Child (⬃%)a
Parent/child First 0.25 25
Full siblings First 0.25 25
Half siblings Second 0.125 12.5
Uncle/niece or aunt/nephew Second 0.125 12.5
Double first cousins Second 0.125 12.5
Grandparent/grandchild Second 0.125 12.5
First cousins Third 0.0625 6
First cousins once removed Fourth 0.03125 3
Second cousins Fifth 0.015625 1.5
Third cousins Seventh 0.0039062 ⬍ 0.5
a
Assuming outbred population.

with the associated concerns for recessive disorders mapping to the homozygous
intervals. When the genomic homozygosity is sufficiently excessive, the finding may
trigger a suspicion for parental consanguinity or incest (Table 1; Fig. 2).8 One or more
regions of LCSH found only on a single chromosome can be a hallmark of uniparental
disomy (UPD), either whole-chromosome UPD or segmental UPD,3,4,9 and may
warrant further clinical investigation, particularly when involving chromosomes asso-
ciated with imprinted gene disorders (Table 2). Regardless of mechanism or size, all
LCSH segments have the potential to harbor homozygous recessive mutations.

CONSANGUINITY

When LCSH is found distributed throughout the genome, this observation is pre-
sumed to represent homozygosity caused by inheritance of genomic regions of IBD.
When the parents of a proband share a recent common ancestor, their union is
defined as consanguineous. The closer the parental relationship, the greater the
proportion of shared alleles and, therefore, the greater the risk of the child (proband)
inheriting 2 copies of a deleterious gene mutation from his parents.10 –12 Clinical
laboratories that perform SNP-based microarray analysis will encounter many cases
of presumed parental consanguinity, occasionally with suspicion of abuse/incest
because of the degree of consanguinity estimated (Fig. 2).8
Although this has no immediate clinical utility and represents a pursuit largely of
academic or social/ethical/legal interest, an estimate of the total proportion of the
LCSH in the genome can be used as a rough assessment of degree of parental
relationship (see Table 1). A simple method to grossly estimate parental relationship
is to add all homozygous regions greater than a defined threshold (eg, 3 Mb),
excluding the sex chromosomes (because males are always hemizygous, and X
chromosomes in females are often observed with increased LCSH because of more
limited recombination). The total autosomal LCSH can then be divided by total
autosomal length (2,867,733 kb for hg18) to estimate the percentage of IBD. This
estimation can then be correlated with the predicted percentage of IBD for various
degrees of relationship (see Table 1). It should be noted that this crude calculation is
likely to represent an underestimate of the actual homozygous proportion given that
598 Kearney et al

Fig. 2. LCSH patterns seen in consanguinity. (A) Male patient with LCSH on multiple chromo-
somes (Affymetrix Genome-Wide Human SNP 6.0 array data visualized in Affymetrix Chro-
mosome Analysis Suite [ChAS] software). Note that the sex chromosomes are excluded in
LCSH modeling. Calculations of total percentage IBD with different minimal LCSH thresholds
in this case result in the following estimations: LCSH threshold ⱖ 0.5 Mb: 868,341 kb (30%
IBD); ⱖ 1 Mb: 868,341 kb (30%); ⱖ 3 Mb: 866,089 kb (30%); ⱖ 5 Mb: 858,676 kb (30%); ⱖ 10
Mb: 797,445 kb (28%); ⱖ 15 Mb: 700,140 kb (24%), and ⱖ 20 Mb: 577,279 kb (20%). These
data and those of additional cases suggest that minimal LCSH thresholds between 0.5 and 10
Mb have negligible impact on estimation of percentage of IBD. These data are consistent
with first degree consanguinity, as illustrated in panel B (and Table 1). (B) Pedigree illustrat-
ing first-degree consanguinity and predicted percentage IBD. The mother and father of the
proband share 50% of their genome (because they are first-degree relatives). There is a
one-half probability that the proband will inherit an identical genomic region from both of
his parents, making the total estimate of the proband’s percentage of IBD in this scenario
roughly 25%. Note that other first-degree parental relationships (eg, full siblings) would also
yield an estimated 25% IBD in the proband.

(1) only LCSH long enough to be detected by with the array platform/applied
threshold will be included and (2) the denominator includes regions of the genome
that may not be covered on the microarray (eg, acrocentric short arm and centromeric
regions). Although in theory, the background level of population-specific homozygos-
ity in any genome may artificially inflate this observation, in practice, this does not
complicate the calculated percentage to any measurable degree, particularly if the
threshold used to calculate total LCSH is greater than 1 Mb.6,7
Estimating percentage of IBD can be illustrative and suggestive of degree of
parental relationship, but if this inference is made at all, it should be accompanied by
an appropriate measure of uncertainty. Most importantly, this estimate cannot be
taken as evidence of a specific parental relationship. Additionally, this inference
assumes a standard 50% distribution of parental alleles in each meiotic division, and
unpredictable recombination and chromosome segregation patterns may result in
significant deviation from this distribution.7 Furthermore, consanguinity is very
common practice in some populations13,14; therefore, it is likely that individuals from
Table 2
UPD chromosomes associated with imprinting disorders
UPD Maternal vs Known/Proposed
Chromosome Imprinted Locus Paternal UPD Associated Disorder Major Clinical Features Dysregulated Gene(s)
6 q24 Paternal Diabetes Mellitus, 6q24- Intrauterine growth retardation, PLAG1, HYMAI
related Transient neonatal hyperglycemia
Neonatal52
7 p11.2–p12 and Maternal Russell-Silver syndrome53 Intrauterine and postnatal GRB10, MEST
q32.2 growth retardation, triangular
facies
11 p15.5 Paternal Beckwith-Wiedemann Macrosomia, macroglossia, IGF2, H19, CDKN1C,

Diagnostic Implications of Excessive Homozygosity


(segmental) syndrome54 visceromegaly, omphalocele KCNQ1, KCNQ1OT1
Maternal Russell-Silver syndrome55 Intrauterine and postnatal IGF2, H19, CDKN1C,
growth retardation, triangular KCNQ1, KCNQ1OT1
facies
14 q32.2 Maternal Maternal UPD14 Precocious puberty, hypotonia, RTL1, DLK1
syndrome56–58 joint laxity
Paternal Paternal UPD14 Skeletal abnormalities, joint RTL1, DLK1
syndrome56–59 contractures, intellectual
disability
15 q11–q13 Paternal Angelman syndrome60 Severe intellectual disability, UBE3A
speech impairment
Maternal Prader-Willi syndrome61 Hypotonia, hypogonadism, SNRPN, MKRN,
obesity MAGEL2, NDN,
U5snoRNAs
20 q13.3 Paternal Pseudohypoparathyroidism Neonatal hyperbilirubinemia, GNAS
type 1b62,63 parathyroid hormone
resistance

599
600 Kearney et al

these populations share not just a single recent ancestor but also multiple common
ancestors (eg, total genomic homozygosity near or exceeding that seen with
first-degree consanguinity, yet the parents have a fairly distant relationship). The
laboratory generally has limited or no information regarding the family/social/ethnic
situation of the proband; therefore, inferences regarding suspected abuse involving a
parent are generally poorly supported; any communications regarding suspicion of
abuse should follow appropriate professional guidelines and take place under
advisement of one’s institutional legal/ethics consult. Currently, there are no profes-
sional guidelines for whether concerns for abuse/incest should be revealed after
SNP-array analysis and, if so, under what circumstances, although guidance from the
American College of Medical Genetics is forthcoming. Regardless of whether con-
sanguinity is suspected, estimated, or even revealed, simply informing the referring
physician of increased suspicion for recessive disorders has clinical utility (see
Autozygosity Mapping section).

UNIPARENTAL DISOMY

Generally, when isolated LCSH (involving only a single chromosome) is detected,


particularly when longer than 10 Mb,4 uniparental disomy is considered a likely
mechanism. UPD is defined as the inheritance of both homologues from a single
parent.15,16 Many excellent reviews have been devoted to UPD,17–24 and there are
informative Web-based resources available as well (Lier lab site, Jena University
Hospital25; Morrison lab site, University of Otago26; Robinson lab site, University of
Diagnostic Implications of Excessive Homozygosity 601

British Columbia27; Jirtle lab site, Duke University28). This review will, therefore, not
cover the history, exhaustive mechanisms, or specific clinical features of UPD
syndromes (see Table 2 for summary), but will instead focus on the laboratory
detection of UPD through SNP-based microarray analysis and appreciation of
associated data complexities. Historically, UPD was only suspected when accompa-
nied by a hallmark cytogenetic finding (mosaic trisomy, marker chromosome, or other
structural rearrangement, such as a Robertsonian translocation), by clinical manifes-
tation of a disorder of imprinting,29 or finding homozygosity for a recessive allele with
only a single carrier parent.16 Through the use of SNP-based microarrays, clinical
laboratories may now have serendipitous detection of unanticipated UPD events by
recognition of hallmark patterns of homozygosity.4 To appreciate the expected
patterns of homozygosity encountered with UPD-involved chromosomes, it is useful
to review the various mechanisms known to generate UPD. It is of fundamental
importance to first appreciate the basic concepts of meiosis and meiotic recombina-
tion, which are summarized in Fig. 3.
There are 2 primary mechanisms by which UPD involving a whole chromosome
may be generated: (1) trisomy rescue, the most frequently observed mechanism and
(2) monosomy rescue.23 Gamete complementation has also been proposed as a UPD
mechanism,15 but this is thought to be a very rare event. Additionally, segmental UPD
(involving only part of a chromosome) may also be generated through somatic
events.17 Given that the majority of these mechanisms generate UPD with full or
partial homozygosity of parental markers (Figs. 4 – 6), SNP-based microarrays are
very useful for detecting LCSH patterns that may be predictive (but not diagnostic) of
UPD.3,4,9,24 Uniparental isodisomy refers to inheritance of 2 identical chromosomes
from a single parent, whereas uniparental heterodisomy refers to inheritance of 2
homologous chromosomes from the same parent. Because of meiotic recombination,
even UPD events involving an entire chromosome are usually not purely isodisomic or
heterodisomic, but instead often have a mixture of both types of segments.

Trisomy Rescue
Zygotes with nonmosaic trisomy for most chromosomes are usually not compatible
with life (exceptions are trisomies involving chromosomes 13, 18, 21, and sex
chromosomes). Those zygotes that do survive have typically either lost the trisomy
entirely, have experienced a structural reduction of the trisomic chromosome (usually
conversion to a marker or ring chromosome), or are mosaic. These events are
collectively known as trisomy rescue.21 Restoration to euploid state can occur
through mitotic nondisjunction, which produces a normal (euploid) daughter cell and
an aneuploid daughter cell (with monosomy, which presumably does not survive) or
by anaphase lag, in which one of the aneuploid chromosomes is not incorporated into
the new-forming nucleus and is subsequently lost. By chance alone, two-thirds of the

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™
Fig. 3 Normal meiosis. (A) A pair of homologous chromosomes illustrated before chromo-
some replication; shading differences represent general heterozygosity. (B) Chromosome
replication resulting in identical sister chromatids. (C) Recombination between nonsister
chromatids (note that involvement of only 2 chromatids is depicted for clarity, yet crossover
events may collectively involve 2, 3, or 4 chromatids). (D) Resulting genetic exchange from
meiotic recombination. (E) First division (meiosis I), with separation of chromosome homo-
logues into 2 daughter cells. (F) Second division (meiosis II), with each daughter cell (gamete
or polar body) containing 1 chromatid.
602 Kearney et al

Fig. 4. Uniparental disomy resulting from meiotic errors. Columns A–E each represent 1 type
of meiotic error, including (A) trisomy resulting from a meiosis I error without recombination
and (B) with recombination, (C) a meiosis II error without recombination and (D) with
recombination, and (E) a monosomy rescue resulting from either meiosis I or II error. Rows
1–3 model the chromosome involved in the uniparental disomy cells through various stages
of development. Row 4 shows a theoretical allele plot for each of these scenarios as plotted
in Illumina BeadStudio software. Row 5 shows a theoretical allele plot involving mosaicism
for a uniparental disomy cell line and a trisomic cell line for each of the mechanisms.
Predicted chromosomal compositions in the sampled tissue are illustrated below the allele
plots. Plot A4 shows array data resulting from complete uniparental heterodisomy. This
chromosome does not contain any LCSH and cannot be distinguished from a chromosome
with normal biparental inheritance. Plots C4 and E4 show array data resulting from complete
uniparental isodisomy; there are no heterozygous SNP alleles. These 2 plots are indistinguish-
able from one another; therefore, unless mosaicism is present (C5), the mechanism cannot be
inferred. Plots B4 and D4 show chromosomes with mixed isodisomy and heterodisomy. The
placement of the detected LCSH can be used to infer meiosis I errors (B4) from meiosis II errors
(D4). Note that not all of the data modeled in this illustration represent confirmed cases of
UPD; some of the data are modeled to illustrate concepts.
Diagnostic Implications of Excessive Homozygosity 603

time a “rescue” event results in a disomic line with biparental inheritance, whereas
one-third of the time UPD occurs. For topical relevance, the remainder of the
discussion of trisomy rescue will presume that the rescue results in UPD. Depending
on the origin of the trisomy (eg, meiosis I or II) and the stochastic arrangement of
recombination events, the UPD chromosomes may be completely heterodisomic,
completely isodisomic, or mixed hetero- and isodisomic (see Fig. 4). Importantly, the
location of isodisomy (homozygosity) relative to any imprinted loci has no clinical
relevance when considering UPD involving an entire chromosome. For example, as
illustrated in Fig. 5, an individual with Prader-Willi syndrome caused by maternal
UPD15 may have uniparental heterodisomy (heterozygous allele calls) across the
critical region at 15q11.2– q13. This concept will be further supported in the following
discussions of UPD mechanism.
First, consider trisomy rescue in the absence of recombination (see Fig. 4A). If the
original segregation error occurred in meiosis I, the resulting gametes would either be
nullisomic or contain both chromosome homologues (heterodisomic). Fertilization of
the heterodisomic gamete followed by trisomy rescue to generate UPD would result
in uniparental heterodisomy for the entire chromosome. This is a very important
scenario to understand, given that this UPD event would not generate any regions of
homozygosity detectable by SNP-based microarray yet would be pathogenic if
involving an imprinted chromosome. If the initiating segregation error originated in
meiosis II (see Fig. 4C), the resulting gametes would be normal, nullisomic, or contain
2 copies of identical chromosomes (isodisomic). Fertilization of the isodisomic
gamete followed by trisomy rescue to generate UPD would result in uniparental
isodisomy (homozygosity) for the entire chromosome and would be readily detectable
by SNP-based microarray. When whole chromosome homozygosity is encountered,
UPD can be reasonably presumed.
Now, let us consider trisomy rescue involving chromosomes with meiotic recom-
bination events (crossing over) (see Fig. 4B, D). As before, a trisomic zygote can be
corrected by a subsequent mitotic event (either nondisjunction or anaphase lag).
Those rescued trisomies resulting from meiosis I errors will result in heterodisomy
around the centromere, whereas those from meiosis II errors will result in isodisomy
around the centromere. The remaining distribution of hetero- and isodisomy is
dependent on the number and position of meiotic crossover events.

Monosomy Rescue
Zygotes with monosomy for a chromosome (other than the X chromosome) are not
compatible with life and can be rescued only by duplication of the monosomic
chromosome. This can occur through a mitotic nondisjunction, resulting in a daughter
cell with nullisomy and a daughter cell with restored euploidy. This mechanism can
only result in whole-chromosome uniparental isodisomy (see Fig. 4E).

Mitotic Errors of Chromosome Segregation to Generate UPD


In addition to uniparental disomy as a result of meiotic error, UPD can be a
consequence of somatic events. When nondisjunction or anaphase lag occurs in a
euploid somatic cell, the resulting daughter cells would be aneuploid. A second
nondisjunction or anaphase lag could correct this event, resulting in a euploid cell
again; however, this may result in daughter cells with incorrect distribution of the
parental chromosomes, with UPD as a result (see Fig. 6B). This UPD would always be
isodisomic for the entire chromosome (LKC, unpublished data).30
604 Kearney et al
Diagnostic Implications of Excessive Homozygosity 605

Segmental UPD
Segmental isodisomy, in which only a portion of a chromosome shows uniparental
inheritance with the remainder of the chromosome showing biparental inheritance,
can also occur somatically. One mechanism to generate segmental UPD is nonsister
(homologous) chromatid exchange during mitosis (Fig. 6A). This would result in 2
daughter cells containing complementary segmental uniparental isodisomies. Be-
cause most cases of segmental UPD have only one abnormal cell line detected
(reviewed in Kotzot17), one of the resulting daughter cells may not be compatible with
survival. Alternatively, these events may represent chromosomal breakage and repair
(break-induced replication) using the homologous chromosome as a template.31 The
resulting UPD from either mechanism (assuming a single break or exchange) would
affect only one arm of the chromosome and should extend from the break/exchange
to the telomere. Segmental UPD is unlikely if the homozygosity is interstitial or present
at the termini of both arms, as these findings are not consistent with a single mitotic
break/exchange event.18 In rare cases, if 2 mitotic exchanges occurred, the segmen-
tal UPD would be interstitially located.32 Because segmental UPD mechanisms
generate only uniparental isodisomy, the location of the LCSH (isodisomy) relative to
any imprinted loci should be considered.28 In contrast to whole-chromosome UPD,
only the loci involved in the isodisomic segment are at risk for imprinted disorders if
segmental UPD is confirmed as the mechanism underlying the LCSH (see Follow-up
testing to confirm UPD).

Whole-genome UPD
Chimeric individuals with whole-genome UPD have been reported in a handful of
cases, with both maternal and paternal whole-genome UPD.3,33–37 These individuals
are chimeric for 2 cell lines: one cell line with normal biparental inheritance and one
cell line with uniparental isodisomy of the entire genome. The presence of paternal
whole-genome UPD (androgenetic chimerism) can result in the clinical presentation of
known paternally imprinted disorders, such as Beckwith-Wiedemann syndrome,
whereas the presence of maternal whole-genome UPD (parthenogenetic chimerism)
can present with Prader-Willi or other maternally imprinted disorders (Table 2).
Although several mechanisms have been hypothesized for these chimeric whole-
genome UPD observations, the most plausible is endoreduplication of 1 parental
genome before pronuclei fusion in the fertilized egg.18,38

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™
Fig. 5. Trisomy rescue resulting in a mosaic marker chromosome and UPD. SNP-array data
(Illumina Quad610 array, visualized in BeadStudio software) seen in a patient with Prader-
Willi syndrome, with proposed mechanisms illustrated. (1) Gametes, with an ovum contain-
ing 2 chromosomes 15 as a result of a meiosis I error. (2) Zygote, with trisomy for chromosome
15. (3) A trisomy rescue event resulting in a small marker derived from the paternal
chromosome 15. (4) Subsequent mosaic loss of the marker, resulting in whole-chromosome
maternal UPD. (5) SNP array results show heterodisomy for the majority of the chromosome,
with isodisomy only at the q terminus. A mosaic duplication is detected near the centromere,
showing a pattern consistent with a mosaic marker. The imprinted region at 15q11.2 to q13
associated with Prader-Willi syndrome is indicated on the chromosome ideogram. Note that
the marker chromosome does not contain this genomic region; therefore, both cell lines
exhibit UPD of the relevant chromosomal region. Note also that the imprinted region is
within a region of heterodisomy, but is fully uniparental (see also Fig. 4-A5).
606 Kearney et al
Diagnostic Implications of Excessive Homozygosity 607

Inferences Regarding Origin of UPD Derived from Detection of Mosaicism


Mosaicism for a trisomic cell line, a cell line with a marker/derivative chromosome, or
a cell line with normal biparental inheritance is an additional line of evidence by which
one may infer the origin of the UPD (see Figs. 4 – 6). If a trisomic cell line is present
either in the same tissue as the UPD or in other tissues (such as placenta), the origin
of the UPD is most likely a trisomy rescue (see Fig. 4A–D). Mosaicism involving a
normal biparental cell line with a segmental UPD cell line or a whole chromosome
uniparental isodisomy cell line most likely indicates a mitotic origin (see Fig. 6),
although other more rare and esoteric chromosomal arrangements are possible.17,18
SNP arrays have been used successfully to detect mosaicism for segmental UPD in
cases of Beckwith-Wiedemann syndrome3,38 and in revertant skin in patients with
ichthyosis.39
The presence of homozygosity for an entire chromosome is most likely associated
with a monosomy rescue (see Fig. 4E-4); however, chromosome segregation errors in
meiosis II without the presence of crossovers (see Fig. 4C-4) and mitotic segregation
errors (see Fig. 6B-2) may also result in uniparental isodisomy for the whole
chromosome. The finding of mosaicism for a monosomic cell line (consistent with
monosomy rescue), a trisomic cell line (consistent with trisomy rescue), or a normal
biparental cell line (consistent with mitotic nondisjunction) may clarify the mechanism
of the UPD event (see Figs. 4E, 4C-5, and 6B-3). Note that the presence of a
monosomic cell line in a monosomy rescue event would have to be ascertained by
decreased log2 values across the chromosome (suggesting low-level monosomy), or
traditional cytogenetic methods. SNP allele patterns would be unaffected in this
scenario.

Follow-up Testing to Confirm UPD


As outlined previously, failure to detect LCSH by SNP microarray analysis does not
exclude the possibility of UPD (see Fig. 4A). For this reason, SNP microarrays cannot
be considered a diagnostic assay for UPD when analyzing the proband alone.
Similarly, the finding of LCSH involving a single chromosome should not be consid-
ered diagnostic of UPD. Large isolated regions of LCSH (even those larger than 10
Mb) may still represent IBD with distant consanguinity. Regions of LSCH greater than
1 Mb have been found in outbred populations, typically attributed to recombination
“cold spots” leading to SNP markers in linkage disequilibrium.4,6,7 Additionally,

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™
Fig. 6. Uniparental disomy resulting from mitotic errors. Row 1 illustrates a potential model
of somatic events consistent with the SNP data shown. Row 2 shows patient allele plots
(Illumina Quad610 array, visualized in BeadStudio software). Row 3 shows patient allele plots
involving mosaicism for a UPD cell line and a normal cell line. Models for chromosome
composition responsible for the data shown are illustrated below the allele plots. (A)
Segmental uniparental disomy resulting from mitotic recombination. Homologous chroma-
tid exchange during somatic development is illustrated with crossing over on the short arm of
chromosome 7. The 2 potential daughter cells resulting from this exchange are shown;
however, only one of these cell lines is detected in this individual (the shaded cell is not
detected). Alternatively, the SNP data shown are consistent with break-induced replication at
the site of exchange (not illustrated). (B) Mitotic nondisjunction resulting in whole chromo-
some isodisomy. Sequential mitotic nondisjunction and selection events can result in cells
with isodisomy for an entire chromosome. Note that here only trisomy rescue is illustrated,
but one could also model rescue of the monosomic cell line created by the first mitotic error.
608 Kearney et al

structural elements such as translocation or inversion breakpoints are known to inhibit


recombination40; therefore, it is possible that some families may be cosegregating
very large (even ⬎10 Mb) contiguous haplotypes surrounding these rare elements.
When an LCSH detected by SNP-based microarray suggests UPD, further molec-
ular analysis is necessary for UPD confirmation and determination of parent of origin.
LCSH involving a chromosome associated with an imprinting disorder (summarized in
Table 2) should be strongly considered for UPD confirmatory testing, particularly
when the clinical features of the patient are consistent with the syndrome of interest.
In principle, definitive UPD testing requires the demonstration of uniparental
inheritance of multiple informative markers for the chromosome in question. Testing
typically involves genotyping microsatellite polymorphisms distributed along the
length of the putative UPD chromosome in the mother, father, and proband (trio
analysis).41,42 In cases of suspected UPD for imprinted chromosomes, specific
diagnostic testing of methylation patterns for the corresponding imprinting disorder
may also be available.43
Established clinical guidelines for whole-chromosome UPD molecular testing44
require at least 2 fully informative markers to establish either uniparental or biparental
inheritance of a chromosome. Informative markers distinguish maternal and paternal
alleles such that the parent (or parents) of origin for a chromosome segment in the
proband can be unambiguously determined. Because nonpaternity can confound
UPD test results, it is recommended that parentage be established by demonstrating
paternal inheritance of at least 1 marker on a non-UPD chromosome. Guidelines for
the analysis of segmental UPD have not been established. Although numerous
well-defined microsatellite markers exist, this technique may not be suited for
detection of segmental UPD, given that the established (clinically validated) assay
may not have sufficient markers within the specific region of isodisomy to confirm a
small segmental UPD. Microarray-based SNP genotyping of trios has been used to
detect both whole-chromosome and segmental UPD.45,46 This option seems espe-
cially suited for follow-up of incidental LCSH findings detected by SNP-based
microarray analysis, given that this analysis already genotypes thousands of SNPs
distributed along the proband’s putative UPD chromosome. Were it not cost
prohibitive, this technique might be used to replace the relatively inexpensive
microsatellite UPD analysis.
AUTOZYGOSITY MAPPING

Regardless of whether LCSH is present as a result of consanguinity, UPD, or


undetermined origin, the potential for recessive disease may be significant. The term
autozygosity refers to homozygosity of alleles that are identical by descent (inherited
from a common ancestor), as contrasted with homozygosity identical by state
(random inheritance). Autozygosity mapping has been used successfully to discover
the genetic basis of numerous recessive disorders in consanguineous families.47– 49
Although it is generally outside the scope of a routine diagnostic laboratory investi-
gation to pursue gene discovery efforts, autozygosity mapping restricted to known
disease genes remains a feasible and useful diagnostic tool.50,51 When the clinical
indication for study is very specific and the differential diagnosis is limited to
genetically well-characterized recessive disorders, mapping candidate genes to
determine location relative to LCSH segments is relatively straightforward.48,50
Finding a gene of interest in an LCSH, although certainly not diagnostic of a
homozygous mutation in that gene, may help to prioritize follow-up testing for
multigenic disorders. The utility of this exercise is illustrated in the following case
(Kearney H , unpublished data). SNP microarray testing for a patient with a clinical
Diagnostic Implications of Excessive Homozygosity 609

diagnosis of Usher syndrome (unknown type) revealed 4 regions of LCSH (each 3–10
Mb in length, totaling approximately 0.8% of the autosomal length). Given that Usher
syndrome can result from mutations in multiple genes, all known disease-causing
genes were investigated, and only one, USH2A, was found in an LCSH. This finding
was followed by referral for USH2A gene sequencing, which resulted in successful
identification of a homozygous pathogenic mutation in the patient (Fig. 7).
For cases of excessive homozygosity in which the genetic basis for the patient
features is unclear, or in which limited phenotypic information is communicated to the
laboratory, the referring physician can be provided with a list of candidate genes in the
homozygous intervals. Further consideration of those genes associated with reces-
sive disorders may prompt the physician to consider disorders that may not have

Fig. 7. Autozygosity mapping used to identify a USH2A homozygous mutation. (A) Screen
shot of a 10-Mb LCSH (purple bar) on chromosome 1q seen in a patient with clinical features
of Usher syndrome. This segment spans 85 genes, including USH2A, one of several known
genes involved in the etiology of Usher syndrome. Vertical lines denote area of zoom
highlighted in lower panel. (B) Zoomed view shows that the 1q LCSH spans only a portion of
the USH2A gene (underscoring the utility of high-density SNP arrays for autozygosity
mapping). Subsequent referral for USH2A gene sequencing identified a pathogenic homozy-
gous recessive mutation (position of mutation shown as an asterisk).
610 Kearney et al

been obvious during the initial clinical evaluation. Personal laboratory experience
suggests that causative recessive gene mutations are detected through autozygosity
mapping and sequence confirmation in approximately 10% of the cases reported of
LCSH (Kearney and colleagues unpublished data).
SUMMARY

Given its widespread clinical use and high sensitivity for CNV detection, chromosomal
microarray analysis has become an indispensible component of constitutional cyto-
genetic laboratory testing. SNP-based microarrays, with their added ability to detect
genome-wide allele states, can also detect LCSH. Although a stand-alone finding of
LCSH rarely results in a definitive diagnosis, it can trigger additional testing,
potentially leading to a diagnosis of an imprinting syndrome or rare genetic recessive
disorder not recognized in the original clinical differential. In addition, SNP data allow
for inferences regarding complex cytogenetic mechanisms, furthering our under-
standing of uniparental disomy. As SNP-based arrays gain more widespread diag-
nostic use and more patient series are published, the clinical impact of LCSH
detection will be more fully realized.
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