ELSEVIER’S INTEGRATED REVIEW

GENETICS
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ELSEVIER’S INTEGRATED REVIEW

GENETICS
SECOND EDITION

Linda R. Adkison, PhD

Professor of Genetics
Associate Dean for Curricular Affairs
Kansas City University of Medicine and Biosciences
Kansas City, Missouri
1600 John F. Kennedy Blvd.
Ste 1800
Philadelphia, PA 19103-2899

ELSEVIER’S INTEGRATED REVIEW GENETICS ISBN: 978-0-323-07448-3

Copyright © 2012, 2007 by Saunders, an imprint of Elsevier, Inc.

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Notices

Knowledge and best practice in this field are constantly changing. As new research and
experience broaden our understanding, changes in research methods, professional practices, or
medical treatment may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in
evaluating and using any information, methods, compounds, or experiments described herein. In
using such information or methods they should be mindful of their own safety and the safety of
others, including parties for whom they have a professional responsibility.

With respect to any drug or pharmaceutical products identified, readers are advised to check the
most current information provided (i) on procedures featured or (ii) by the manufacturer of each
product to be administered, to verify the recommended dose or formula, the method and duration
of administration, and contraindications. It is the responsibility of practitioners, relying on their
own experience and knowledge of their patients, to make diagnoses, to determine dosages and the
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Previous edition copyrighted 2007.

Library of Congress Cataloging-in-Publication Data

Adkison, Linda R.
Elsevier’s integrated review genetics / Linda R. Adkison.—2nd ed.
p. ; cm.—(Elsevier’s integrated series)
Integrated review genetics
Rev. ed. of: Elsevier’s integrated genetics / Linda R. Adkison, Michael D. Brown. c2007.
Includes bibliographical references and index.
ISBN 978-0-323-07448-3 (pbk. : alk. paper) 1. Medical genetics. I. Adkison, Linda R. Elsevier’s
integrated genetics. II. Title. III. Title: Integrated review genetics. IV. Series: Elsevier’s integrated
series.
[DNLM: 1. Genetics, Medical. QZ 50]
RB155.A2565 2012
616′.042—dc22 2011004253

Acquisitions Editor: Madelene Hyde

Developmental Editor: Andrea Vosburgh
Publishing Services Manager: Pat Joiner-Myers
Project Manager: Marlene Weeks
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Last digit is the print number: 9 8 7 6 5 4 3 2 1

I have been fortunate to have excellent mentors during my career in academics. I have
learned a great deal about my own learning through this journey. My goals as a teacher
are to help students become challenged by the fascination of learning, visualize what
they cannot necessarily see, and describe what they see with integration of thought
broadly across disciplines. This textbook is dedicated to the many wonderful students
and colleagues who constantly challenge the boundaries of learning – theirs and mine.
Finally, without the support and understanding of my family, especially my children,
Emily and Seth, this project could not have been completed.
Linda R. Adkison, PhD
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 vii

Preface
Though the youngest of all the medical specialties, genetics explains better the morphologic variation observed in embry-
embodies the essence of all normal and abnormal develop- ologic development and anatomic presentation. It provides
ment and all normal and disease states. Perhaps because of better insight into susceptibility to infection and disease. It
its recent recognition as a discipline and perhaps because of offers insight into neurologic and behavioral abnormalities. It
its derivation from research in several areas, it is easier for is defining the strategies for gene therapy and pharmacoge-
genetics to be an “integrated” discipline. Approaching genet- nomics. For these reasons, it has been exciting to put this
ics as “a particular gene located on a specific chromosome book together.
and inherited in a specific manner” loses the appreciation of This text focuses on well-known and better described dis-
spatial and temporal dimensions of expression and the many, eases and disorders that students and practitioners are likely
many factors affecting every single aspect of development, to read about in other references. Many of these do not occur
survival, and even death. at a high frequency in populations, but they underscore major
Every medical discipline is connected to human well-being mechanisms and major concepts associated with many other
through the mechanisms of gene expression, environmental medical situations. It is my hope that this text will be as
influences, and inheritance. Genetics underscores the many stimulating to read as it was to write.
biochemical pathways, physiologic processes, and pathologic
mechanisms presented in other volumes of this series. It Linda R. Adkison, PhD
 viii

Editorial Review Board

Chief Series Advisor James L. Hiatt, PhD
J. Hurley Myers, PhD Professor Emeritus
Professor Emeritus of Physiology and Medicine Department of Biomedical Sciences
Southern Illinois University School of Medicine; Baltimore College of Dental Surgery
President and CEO Dental School
DxR Development Group, Inc. University of Maryland at Baltimore
Carbondale, Illinois Baltimore, Maryland

Anatomy and Embryology Immunology

Thomas R. Gest, PhD Darren G. Woodside, PhD
University of Michigan Medical School Principal Scientist
Division of Anatomical Sciences Drug Discovery
Office of Medical Education Encysive Pharmaceuticals, Inc.
Ann Arbor, Michigan Houston, Texas

Biochemistry Microbiology
John W. Baynes, MS, PhD Richard C. Hunt, MA, PhD
Graduate Science Research Center Professor of Pathology, Microbiology, and Immunology
University of South Carolina Director of the Biomedical Sciences Graduate Program
Columbia, South Carolina Department of Pathology and Microbiology
University of South Carolina School of Medicine
Marek Dominiczak, MD, PhD, FRCPath, FRCP(Glas) Columbia, South Carolina
Clinical Biochemistry Service
NHS Greater Glasgow and Clyde Neuroscience
Gartnavel General Hospital Cristian Stefan, MD
Glasgow, United Kingdom Associate Professor
Department of Cell Biology
Clinical Medicine University of Massachusetts Medical School
Ted O’Connell, MD Worcester, Massachusetts
Clinical Instructor
David Geffen School of Medicine Pathology
UCLA; Peter G. Anderson, DVM, PhD
Program Director Professor and Director of Pathology Undergraduate
Woodland Hills Family Medicine Residency Program Education
Woodland Hills, California Department of Pathology
University of Alabama at Birmingham
Genetics Birmingham, Alabama
Neil E. Lamb, PhD
Director of Educational Outreach Pharmacology
Hudson Alpha Institute for Biotechnology Michael M. White, PhD
Huntsville, Alabama; Professor
Adjunct Professor Department of Pharmacology and Physiology
Department of Human Genetics Drexel University College of Medicine
Emory University Philadelphia, Pennsylvania
Atlanta, Georgia
Physiology
Histology Joel Michael, PhD
Leslie P. Gartner, PhD Department of Molecular Biophysics and Physiology
Professor of Anatomy Rush Medical College
Department of Biomedical Sciences Chicago, Illinois
Baltimore College of Dental Surgery
Dental School
University of Maryland at Baltimore
Baltimore, Maryland
 ix

Contents
1 BASIC MECHANISMS 1

2 CHROMOSOMES IN THE CELL 12

3 MECHANISMS OF INHERITANCE 28

4 GENETICS OF METABOLIC DISORDERS 51

5 CANCER GENETICS 65

6 HEMATOLOGIC GENETICS AND DISORDERS 93

7 MUSCULOSKELETAL DISORDERS 114

8 NEUROLOGIC DISEASES 133

9 CARDIOPULMONARY DISORDERS 159

10 RENAL, GASTROINTESTINAL, AND HEPATIC DISORDERS 177

11 DISORDERS OF SEXUAL DIFFERENTIATION AND DEVELOPMENT 192

12 POPULATION GENETICS AND MEDICINE 209

13 MODERN MOLECULAR MEDICINE 217

INDEX 239

Case Studies and Case Study Answers are available online on Student Consult www.studentconsult.com
 x

Series Preface
How to Use This Book confident in the subject matter of many of the integration
The idea for Elsevier’s Integrated Series came about at a boxes, so they will serve as helpful reminders.
seminar on the USMLE Step 1 exam at an American At the back of the book we have included case study ques-
Medical Student Association (AMSA) meeting. We noticed tions relating to each chapter so that you can test yourself as
that the discussion between faculty and students focused you work your way through the book.
on how the exams were becoming increasingly integrated—
with case scenarios and questions often combining two or Online Version
three science disciplines. The students were clearly con- An online version of the book is available on our Student
cerned about how they could best integrate their basic Consult site. Use of this site is free to anyone who has
science knowledge. bought the printed book. Please see the inside front cover
One faculty member gave some interesting advice: “read for full details on the Student Consult and how to access
through your textbook in, say, biochemistry, and every time the electronic version of this book.
you come across a section that mentions a concept or piece In addition to containing USMLE test questions, fully
of information relating to another basic science—for example, searchable text, and an image bank, the Student Consult site
immunology—highlight that section in the book. Then go to offers additional integration links, both to the other books
your immunology textbook and look up this information, and in Elsevier’s Integrated Series and to other key Elsevier
make sure you have a good understanding of it. When you textbooks.
have, go back to your biochemistry textbook and carry on
reading.” Books in Elsevier’s Integrated Series
This was a great suggestion—if only students had the time, The nine books in the series cover all of the basic sciences.
and all of the books necessary at hand, to do it! At Elsevier The more books you buy in the series, the more links that
we thought long and hard about a way of simplifying this are made accessible across the series, both in print and
process, and eventually the idea for Elsevier’s Integrated online.
Series was born.
The series centers on the concept of the integration box. Anatomy and Embryology
These boxes occur throughout the text whenever a link to
another basic science is relevant. They’re easy to spot in the
text—with their color-coded headings and logos. Each box Histology
contains a title for the integration topic and then a brief
summary of the topic. The information is complete in itself—
you probably won’t have to go to any other sources—and you Neuroscience
have the basic knowledge to use as a foundation if you want
to expand your knowledge of the topic.
You can use this book in two ways. First, as a review book … Biochemistry
When you are using the book for review, the integration
boxes will jog your memory on topics you have already
covered. You’ll be able to reassure yourself that you can iden-
Physiology
tify the link, and you can quickly compare your knowledge
of the topic with the summary in the box. The integration
boxes might highlight gaps in your knowledge, and then you
Pathology
can use them to determine what topics you need to cover in
more detail.
Second, the book can be used as a short text to have at
hand while you are taking your course … Immunology and Microbiology
You may come across an integration box that deals with a
topic you haven’t covered yet, and this will ensure that you’re
one step ahead in identifying the links to other subjects Pharmacology
(especially useful if you’re working on a PBL exercise). On a
simpler level, the links in the boxes to other sciences and to
clinical medicine will help you see clearly the relevance of Genetics
the basic science topic you are studying. You may already be
 xi

Integration boxes:
Whenever the subject matter can be related to another
science discipline, we’ve put in an Integration Box.
Clearly labeled and color-coded, these boxes include
nuggets of information on topics that require an inte-
Artwork: grated knowledge of the sciences to be fully under-
The books are packed with 4-color illustrations stood. The material in these boxes is complete in itself,
and photographs. When a concept can be and you can use them as a way of reminding yourself
better explained with a picture, we’ve drawn of information you already know and reinforcing key
one. Where possible, the pictures tell a dynamic links between the sciences. Or the boxes may contain
story that will help you remember the informa- information you have not come across before, in which
tion far more effectively than a paragraph of text. case you can use them a springboard for further
research or simply to appreciate the relevance of the
subject matter of the book to the study of medicine.

Text:
Succinct, clearly written text, focusing on
the core information you need to know and
no more. It’s the same level as a carefully
prepared course syllabus or lecture notes.
This page intentionally left blank
Basic Mechanisms 1
CONTENTS approximately 150 nucleotide pairs wrapped around the
histone core. H1 histone anchors the DNA around the core.
This structure leads to a superhelix of turns upon turns upon
CHROMATIN
turns called a solenoid structure. In the solenoid structure,
CHROMOSOME ORGANIZATION each helical turn contains 6 nucleosomes and approximately
1200 nucleotide pairs. Additional turns form minibands that,
GENE ORGANIZATION
when tightly stacked upon each other, give the structure rec-
GENETIC CHANGE ognized as a chromosome. In each nucleus, chromatin is orga-
nized into 46 chromosomes. In a fully relaxed configuration,
ERRORS IN DNA AND DNA REPAIR
DNA is approximately 2 nm in diameter; chromatids are
approximately 840 nm in diameter. Twisting and knotting are
extremely effective at compacting DNA within the nucleus
(Fig. 1-2).
The essence of genetics is an understanding of the hereditary A DNA molecule comprises two long chains of nucleotides
material within a cell and the influence it has on survival of arranged in the form of a double helix. Its shape may be
the cell through every function and response the cell and its compared to a twisted ladder in which the two parallel sup-
organelles undertake. Without these fundamental concepts, ports of the ladder are made up of alternating deoxyribose
no aspect of human development and well-being can be sugars and phosphate molecules. Each rung of the ladder is
adequately explained. composed of one pair of nitrogenous bases, held together by
specific hydrogen bonds. Hydrogen bonds are weak bonds;
however, the total number of hydrogen bonds between the
●●● CHROMATIN strands assures that the strands of the double helix are firmly
One of the finest triumphs of modern science has been the associated with each other under conditions commonly found
elucidation of the chemical nature of chromatin and its role in living cells.
in the transfer of information from nucleic acids into proteins,
known as the central dogma. James Watson built on his earlier
work, which outlined the fundamental unit and chemical BIOCHEMISTRY
composition of the complex molecule composing chromatin
deoxyribonucleic acid (DNA). Briefly stated, the central DNA Configuration
dogma “oversimplifies” the mechanism whereby the chemical There are three basic three-dimensional configurations of
message held in DNA is transferred to ribonucleic acid (RNA) DNA. The most common is the B form in which DNA is
through transcription and this RNA blueprint is translated wound in a right-handed direction with 10 bp per turn.
into protein: DNA → RNA → protein. Other proteins associ- Within the turned structure are a major groove and a minor
ated with DNA contribute to its structure and many play roles groove, where proteins can bind. The A form also has a
right-handed turn and is composed of 11 bp per turn. This
in regulating functions. In its simplest form, chromatin is
form is seen in dehydrated DNA such as in oligonucleotide
composed of DNA and histone proteins.
fibers or crystals. The third form, Z-form DNA, was named
Histones are small, highly conserved, positively charged for its zigzag appearance and has a left-handed turn
proteins that bind to DNA and to other histones. The five composed of 12 bp per turn. This form occurs in regions of
major histones are H1, H2A, H2B, H3, and H4. The presence DNA with alternating pyrimidines-purines: CGCGCG.
of 20% to 30% lysine and arginine accounts for the positive
charge of histones and distinguishes these from most other
proteins. All histones except H1 are highly conserved among The molar concentration of adenine equals thymine and
eukaryotes. that of guanine equals cytosine. This information is best
DNA is packaged into the nucleus by winding the double accommodated in a stable structure if the double-ring purines
helix twice around an octamer of histones; this DNA-histone (adenine or guanine) lay opposite the smaller, single-ring
structure is called a nucleosome (Fig. 1-1). Each nucleosome pyrimidines (thymine or cytosine). The combination of one
is composed of two of each histone except H1 and purine and one pyrimidine to make up each cross-connection
 2 Basic Mechanisms

DNA double helix

DNA double helix

2 nm diameter

DNA Histone
Nucleosomes

6–8 Nucleosomes
per turn
200 bp of DNA
Solenoids

Solenoid
30 nm
diameter

Chromatin
300 nm
Chromatin diameter

Figure 1-1. Chromosome Chromatin

organization. The shortening, or loop contains
condensation, of chromatin results approximately
in a diminished volume of each 100,000 bp of DNA
chromosome and a reduction in the
exposed chromosome surface. This is a
dynamic process beginning with the
Chromatid
least condensed form, the DNA double 840 nm
helix, and proceeding to chromatin diameter
visible in interphase and prophase. The Chromatid
level of greatest condensation occurs at
metaphase.

is conveniently called a base pair (bp). In a DNA base pair, Because of the configuration of phosphodiester bonds
adenine (A) forms two hydrogen bonds with thymine (T), and between the 3′ and 5′ positions of adjacent deoxyribose
guanine (G) and cytosine (C) share three hydrogen bonds. molecules, every linear polynucleotide can have a free,
The sequence of one strand of DNA automatically implies the unbounded 3′ hydroxyl group at one pole of the poly-
sequence of the opposite strand because of the precise pairing nucleotide (3′ end) and a free 5′ hydroxyl at the other
rule A = T and C = G. pole (5′ end). There are theoretically two possible ways
for the two polynucleotides to be oriented in a double
helix. They could have the same polarity—that is, be paral-
BIOCHEMISTRY lel, with both strands having 3′ ends at one pole and 5′
ends at the other pole. Or, by rotating one strand 180
Nitrogenous Bases
degrees with respect to the other, they could have opposite
Purines are adenosine (A) and guanine (G). Pyrimidines are polarity—that is, be antiparallel—with a 3′ and a 5′ end
cytosine (C) and thymine (T). In the double helix structure, A at one pole of the double helix and a 5′ and a 3′ end at
binds to T with two hydrogen bonds; C binds to G with
the other pole of the double helix. Only the antiparallel
three hydrogen bonds.
orientation actually occurs. The antiparallel nature of the
Uracil (U) is found in RNA in place of T in DNA. The
structure of U is T without the methyl group at carbon 5. double helix dictates that a new DNA chain being repli-
Hypoxanthine is found in certain tRNAs. cated must be copied in the opposite direction from the
template (Fig. 1-3).
 Chromosome Organization 3

●●● CHROMOSOME ORGANIZATION

DNA of eukaryotes is repetitive—that is, there are many
DNA sequences of various lengths and compositions that do
not represent functional genes. Three subdivisions of DNA
are recognized: unique DNA, middle repetitive DNA, and
highly repetitive DNA. Unique DNA is present as a single
copy or as only a few copies. The proportion of the genome
taken up by repetitive sequences varies widely among taxa.
In mammals, up to 60% of the DNA is repetitive. The highly
repetitive fraction is made up of short sequences, from a few
to hundreds of nucleotides long, which are repeated on the
average of 500,000 times. The middle repetitive fraction con-
sists of hundreds or thousands of base pairs on the average,
which appear in the genome up to hundreds of times.

Figure 1-2. Generally, chromosomes are shown as in this

photograph—in a highly condensed stage known as
metaphase. This structure, however, represents one
chromosome that has been replicated and is composed of
two identical sister chromatids. At a later stage, the sister BIOCHEMISTRY
chromatids will separate at the centromere, and two
chromosomes will exist. Note: when in doubt about the Phosphodiester Bonds and
number of chromosomes present, count the number of Deoxyribose Molecules
centromeres present! A phosphodiester bond occurs between carbon 5 on one
deoxyribose and carbon 3 on an adjacent deoxyribose. The
sugar in DNA is deoxyribose: H+ replaces OH− at carbon 2.
The energy to form this bond is derived from the cleavage of
two phosphates from the ribonucleotide triphosphate.

5′ 3′

O O CH3
P H
O O H O H N N H
CH2 O T
N N H N A N
O N O H2C
H
O O HH O O
P P
P O O H N H O N H O O
S CH2 O C
T A P
N N H N G N
N
O H N O H2C
S C G S
H
P O O O O
P P H CH3 P

S A T O O H N
N H O H
O O
S
CH2 O T
P
G C
N A N H N N
S N O H 2C
H O
P
O O O O
P H P
O O H N H O O
O H N H
CH2 O
N G N H C Figure 1-3. DNA is organized in an
N N
N
N H O O H 2C antiparallel configuration: one strand is
OH
H O O
P
5′ to 3′ in one direction and the other
O O strand is 5′ to 3′ in the opposite
3′ 5′
direction. A purine is bound to a
pyrimidine by hydrogen bonds: A:T and
G:C. The helix occurs naturally because
of the bonds in the phosphate
backbone.
 4 Basic Mechanisms

Most unique-sequence genes code for proteins and are

essentially structural or enzyme genes. Human DNA encodes BIOCHEMISTRY
20,000 to 25,000 different gene products. The identification
of many genes is known, along with their sequence, but the Restriction Enzymes
number of variations that occur within these is harder to A restriction enzyme cleaves both strands of the DNA helix.
predict. A phenotype, or an observable feature of specific Sites of cleavage may produce blunt ends, 3′ overhanging
gene expression, is associated with a smaller proportion of ends, or 5′ overhanging ends. Many sequences recognized
these variations. (See the Online Mendelian Inheritance of are palindromes. Alu elements contain an AGCT site that
produces a blunt digestion site when exposed to the
Man, available at: http://www.ncbi.nlm.nih.gov/omim.) Much
restriction enzyme AluI, isolated from Arthrobacter luteus.
of the time variations in genes are discussed relative to abnor-
The name of the organism from which the enzyme is
mal gene expression and disease; however, many mutations isolated provides the abbreviation for the enzyme (Alu).
may have either no effect on gene expression or little effect
on the function of the protein in the individual. For example,
a protein may have less than 100% activity with little or no
●●● GENE ORGANIZATION
effect until the activity drops below a certain level.
Middle repetitive sequences represent redundant, tandemly Each cell has 23 pairs of chromosomes, or 46 separate DNA
arrayed copies of a given gene and may be transcribed just double helices, with one chromosome from each pair inher-
as unique-sequence genes. Specifically, these sequences refer ited maternally and the other paternally. Twenty-two pairs are
to genes coding for transfer RNA (tRNA) and ribosomal RNA called autosomes and one pair is called the sex chromosomes.
(rRNA). Because these RNAs are required in such large quan- Each pair of autosomes is identical in size and organization
tities for the translation process, several hundred copies of of genes. The genes on these homologous chromosomes are
RNA-specifying genes are expected. As a striking example, organized to produce the same proteins. However, slight
the 18S and 28S fractions of rRNA are coded by about 200 variations may occur, which changes the organization of the
copies of DNA sequences, localized in the tip regions of five base pairs and can lead to a change in a protein. These changes
acrocentric chromosomes in the human genome. It is esti- can be called polymorphisms (from Greek “having many
mated that human DNA is about 20% middle repetitive forms”) and result from mechanisms creating changes, or
DNA. mutations, within the DNA. Another name for variation in
Highly repetitive DNA is usually not transcribed, appar- the same gene on homologous chromosomes is allele. Stated
ently lacking promoter sites on which RNA polymerase can another way, an allele is an alternative form of a gene. Two
initiate RNA chains. These highly repeated sequences may alleles in an individual occur at the same place on two homol-
be clustered together in the vicinity of centromeres, or ogous chromosomes, and these may be exactly the same or
may be more evenly distributed throughout the genome. they may be different. The presence of few alleles indicates
Presumably, the clustered sequences are involved in binding the gene has been highly conserved over the years, whereas
particular proteins essential for centromere function. The genes with hundreds of alleles have been less stringently
most common class of dispersed sequences in mammals is conserved. An example of the latter is the gene responsible
the Alu elements. The name derives from the fact that for cystic fibrosis, which may have one or more of over 1500
many of these repetitious sequences in humans contain reported changes, or mutations. Different alleles, or combina-
recognition sites for the restriction enzyme AluI. The entire tions of alleles, may cause different presentations of a disease
group has been referred to as the Alu family. The Alu among individuals, although some alleles may not lead to any
sequences are 200 to 300 bp in length, of which there are appreciable change in the clinical presentation.
an estimated million copies in the human genome. They As noted above, the central dogma states that DNA is
constitute between 5% and 10% of the human genome. transcribed into RNA, which is then translated into protein.
Various debatable roles have been ascribed to the Alu ele- It is now known that a gene may express RNA that is not
ments, from “molecular parasites” to initiation sites of DNA translated into a protein; these genes represent less than 5%
synthesis. of the genome. More commonly, a gene is a coding sequence
that ultimately results in the expression of a protein. The
sequence of bases in unique DNA provides a code for the
sequence of the amino acids composing polypeptides. This
DNA code is found in triplets—that is, three bases taken
MICROBIOLOGY together code for one amino acid. Only one of the two strands
of the DNA molecule (called the transcribed, or template,
Restriction Endonucleases
strand) serves as the genetic code. More precisely, one strand
Restriction endonucleases, also called restriction enzymes, is consistent for a given gene, but the strand varies from one
are normal enzymes of bacteria that protect the bacteria
gene to another.
from viruses by degrading the viruses. Restriction enzymes
The eukaryotic gene contains unexpressed sequences that
also recognize and cleave specific short sequences of
human DNA, making them highly useful in gene interrupt the continuity of genetic information. The coding
characterization and clinical diagnostics. sequences are termed exons, whereas the noncoding inter-
vening sequences are called introns (Fig. 1-4). The coding
 Gene Organization 5

Promoter Exons
Template strand
DNA C T A G T C
3′
T A C G G A U C A C G T C
PPP
Introns A T G C RNA polymerase C A G
G
5′
G G T A C A G
Coding strand
A U
Figure 1-4. Organization of a gene showing the upstream 5′
promoter region, exons, and introns. Introns are removed by mRNA
splicing during the formation of mRNA. Recognized by tRNA

Figure 1-5. RNA is transcribed from the template strand

region of the gene begins downstream from the promoter at
and has a complementary sequence to the coding strand.
the initiation codon (ATG). It ends at a termination codon Therefore, the coding strand sequence more accurately
(UAG, UAA, or UGA). Sequences before the first exon and reflects the genetic code.
after the last exon are generally transcribed but not translated
in protein.
The 5′ region of the gene contains specific sites important The entire gene is transcribed as a long RNA precursor, com-
for the transcription of the gene. This region, called the pro- monly referred to as the primary RNA transcript, or premes-
moter, has binding sites for transcription factors that regulate senger RNA; this is sometimes called heterogeneous nuclear
transcription initiation. Many cells contain the well-known RNA (hnRNA). Through RNA processing, the introns of the
seven-base-pair sequence TATAAAA, also referred to as the primary RNA transcript are excised and the exons spliced
TATA box. The TATA binding protein binds to this site, which together to yield the shortened, intact coding sequence in the
assists in the formation of the RNA polymerase transcrip- mature messenger RNA (mRNA). Specific enzymes that rec-
tional complex. Other promoter elements include the initia- ognize precise signals at intron-exon junctions in the primary
tor (inr), CAAT box, and GC box. The latter is very important transcript assure accurate “cutting and pasting.” There is no
in regulating expression through methylation. More specific rule that governs the number of introns. The gene for the β
binding sites within the promoter vary from gene to gene. As chain of human hemoglobin contains two introns, whereas the
imagined, this is an extremely complex region. It is the unique variant gene that causes Duchenne-type muscular dystrophy
combination of different transcription factors binding that has more than 60 introns. Nearly all bacteria and viruses have
regulates differential expression of the gene in different cells streamlined their structural genes to contain no introns.
and tissues. Among human DNAs, genes with no introns are less common.
The concept, mentioned above, of only one strand being
transcribed for a gene can be confusing when trying to under-
BIOCHEMISTRY stand how the DNA code is transferred to RNA, which is, in
turn, the message used to translate the code into a precise
DNA Orientation: Basic Concepts amino acid sequence of a protein. As noted, the two DNA
DNA is arranged in a 5′-to-3′ orientation. By convention, the strands of the double helix are antiparallel, with a 5′ and 3′
5′ end is to the left and the 3′ end is to the right. Similarly, end at each end of the molecule. Transcription occurs in a
sequences to the left of a point are upstream and those to 5′-to-3′ direction from the transcribed, or template, strand
the right are downstream. For example, the promoter is (Fig. 1-5). The sequence of this hnRNA, and subsequently the
upstream of the initiation site. Although these sequences are mRNA, is complementary to the antiparallel strand that is
not transcribed, they are important for binding proteins to opposite the template strand. The antiparallel strand is also
allow proper binding of polymerase and initiation of
referred to as the coding strand. The anticodons of tRNA find
transcription. Similarly, sequences at the end of the gene are
the appropriate three-base-pair complementary mRNA codon
important for termination, and signaling sites are important
for the addition of polyadenosine (polyA) that is not to attach the amino acid specified.
specified in the DNA template.

BIOCHEMISTRY
Some gene expression may be facilitated by transcription
Transcription and RNA Processing
factors binding to special sequences known as enhancers.
Enhancers may be found hundreds to thousands of base pairs Transcription is the synthesis of RNA from a DNA template,
away from the promoter, upstream or downstream of the requiring RNA polymerase II. RNA is single stranded with an
untranslated 5′ cap and 3′ polyA tail.
gene, or even within the gene. Binding of these sites increases
Small nuclear ribonucleoproteins (snRNPs) stabilize intron
the rate of transcription. It is suggested that the factor binding
loops, in a complex called a spliceosome, for removal of
to the enhancer may cause DNA to loop back onto the pro- introns. snRNPs are rich in uracil and are identified as U and
moter region and interact with the proteins binding in this a number: U1, U2, U3, etc.
region to increase initiation.
 6 Basic Mechanisms

within the protein and the consequences would depend on

●●● GENETIC CHANGE
the importance of that particular amino acid. Other changes
Variability in genetic information occurs naturally through may alter a splice site recognition sequence or sites of post-
fertilization when two gametes containing 23 chromosomes transcriptional or posttranslational modification. It is also
join to make a unique individual. No two individuals except possible that a change in a nucleotide may have no conse-
identical twins have identical DNA patterns. DNA changes quence, owing to the redundancy of the genetic code or the
are more likely to occur within highly repetitive sequences importance of the amino acid in the protein, and thus it is a
than within genes transcribing nontranslated RNAs and func- silent mutation.
tional genes, in which change could lead to a failure to func-
tion and potentially threaten the existence of the cell and
ultimately the individual. Changes within the repetitive
regions usually have little consequence on the cell because
of the apparent lack of function. Repetitive sequences are
BIOCHEMISTRY
similar but not identical among individuals and represent a
great reservoir for mutational changes. These sequences rep- Genetic Code
resent the DNA “fingerprint” of an individual, most often Three nucleotides code for one amino acid. A change in
referred to in court proceedings, because these regions dem- the third nucleotide may have no effect on the code for
onstrate the same heritability observed with expressed regions a particular amino acid; this is the “wobble effect.” For
of the chromosomes. example, arginine is coded for by CGU, CGC, CGA, and
Aside from fertilization, which brings together chromo- CGG. A change in the first or second nucleotide will
somes that have undergone recombination during gamete change the amino acid inserted into the protein. There is
formation and chromosomes that have assorted randomly one codon for methionine and tryptophan. Other amino
into gametes, changes in genetic material are generally acids may be specified by two to six codons (none are
observed as numerical or structural. These changes are called specified by five). There are three stop, or “nonsense,”
codons.
mutations. Numerical changes generally occur as a result of
nondisjunction. This error in the separation of chromosomes
may occur in the division of somatic cells, called mitosis, or 1ST 3RD
in the formation of gametes, called meiosis. In meiosis, non- POSITION POSITION
(5′ END) 2ND POSITION (MIDDLE) (3′ END)
disjunction may occur in either the first or second stage of
meiosis, called meiosis I or meiosis II, respectively. The great-
U U C A G
est consequences of nondisjunction are those observed in
Phe F Ser S Tyr Y Cys C U
meiosis because the resulting embryo has too many or too Phe F Ser S Tyr Y Cys C C
few chromosomes. Humans do not tolerate either excess or Leu L Ser S STOP STOP A
insufficient DNA well. Except for a few situations, the absence Leu L Ser S STOP Trp W G
of an entire chromosome (monosomy) or the addition of an C Leu L Pro P His H Arg R U
Leu L Pro P His H Arg R C
entire chromosome (trisomy) is incompatible with life for
Leu L Pro P Gln Q Arg R A
more than a few weeks to perhaps as long as a few months Leu L Pro P Gln Q Arg R G
(see Chapter 2). A Ile I Thr T Asn N Ser S U
Changes in genetic material, less dramatic than in an entire Ile I Thr T Asn N Ser S C
chromosome, are generally tolerated inversely to the size of Ile I Thr T Lys K Arg R A
Met M Thr T Lys K Arg R G
the change: the smaller the change, the better the cell may
G Val V Ala A Asp D Gly G U
tolerate the change. Changes may occur at a single nucleotide Val V Ala A Asp D Gly G C
—a point mutation—or involve a large portion of a chromo- Val V Ala A Glu E Gly G A
some. At the nucleotide level, a purine may be replaced by Val V Ala A Glu E Gly G G
another purine, or a pyrimidine by another pyrimidine. This
substitution process is known as a transition. However, if a
purine replaces a pyrimidine, or vice versa, a transversion
occurs. Consequences of these changes depend on where the
change occurs. Obviously, there is a greater opportunity for
an effect within an exon rather than within noncoding More observable changes can occur when regions of a
sequences. Even within an exon, the location of the change chromosome are deleted or duplicated. Loss of genetic mate-
is important. If the change results in the creation of a stop rial may occur from within a chromosome or at the termini
codon, known as a nonsense mutation, the resulting protein and results in what may be called partial monosomy. Just as
may be truncated and hence either nonfunctional or with with base changes, a single nucleotide may be added or
reduced function. If the change results in a different codon deleted from a sequence, with the consequences depending
being presented for translation, the change may cause a dif- on its location. These changes, called frameshift mutations,
ferent amino acid at a certain position (missense mutation) within a coding sequence can alter the reading frame of the
 Genetic Change 7

mRNA during translation. Altered reading frames may create will receive extra copies of genetic material while others
a stop codon, or incorrect amino acids will be inserted into will be missing genetic material (see Chapter 2).
the protein, resulting in suboptimal function. A common rearrangement is the fusion of two long arms
Many deletions of larger regions of chromosomes have of acrocentric chromosomes leading to the formation of two
been described in which partial monosomies result in spe- new chromosomes. When this fusion occurs at the centro-
cific syndromes that are sometimes called microdeletion mere, it is called a robertsonian translocation. There are five
syndromes. As might be expected, a deletion that involves acrocentric chromosomes among the 23 pairs (chromosomes
more than one gene may have a worse effect than a muta- 13, 14, 15, 21, and 22), and all are commonly seen in trans-
tion in a single gene. Many of the described disorders locations. Robertsonian translocations are the most common
involve deletions of millions of base pairs and numerous chromosomal rearrangement. In a balanced arrangement, no
genes. Most of these are de novo mutations and have such problems are evident in the individual. However, the unbal-
significant presentations that the individuals do not pass anced form presents the same concerns as partial monosomy
the deletion on to another generation (Box 1-1). Duplica- or partial trisomy.
tion of genetic material results from errors in replication. As noted, a mutation is a heritable change in genetic mate-
These may occur when a segment of DNA is copied more rial. It may be spontaneous, as with some nondisjunctions,
than once or when unequal exchange of DNA occurs insertions, or deletions, or induced by an external factor. This
between homologous chromosome pairs. The results may external factor, a mutagen, is any physical or chemical agent
be a direct, or tandem, repeat or an inverted repeat of that increases the rate of mutation above the spontaneous
the DNA. Unequal exchange, or recombination, occurs in rate; the spontaneous rate of mutation for any gene is 1 ×
meiosis when homologous chromosomes do not align prop- 10−6 per generation. Therefore, determining whether a muta-
erly. The recombination results in a deletion for one chro- tion results from a spontaneous event within the cell or from
mosome and a duplication for the other. In either case, a mutagen requires evaluation and comparison of the rates of
DNA that has been gained or lost can result in unbalanced mutation.
gene expression. Mutagens are generally chemicals and irradiation (Box 1-2).
Genetic material may also be moved from one location to Chemical mutagens can be classified as (1) base analogs that
another without the loss of any material. Such movements mimic purines and pyrimidines; (2) intercalating agents that
may occur within a chromosome or between chromosomes. alter the structure of DNA, resulting in nucleotide insertions
Within a chromosome, movements are usually seen as inver- and frameshifts; (3) agents that alter bases, resulting in dif-
sions. Inversions either include the centromere (pericentric ferent base properties; and (4) agents that alter the structure
inversion) or are in one arm of the chromosome (paracentric of DNA, resulting in noncoding regions, cross-linking of
inversion) (Fig. 1-6). These changes provide significant chal- strands, or strand breaks.
lenges to the chromosome during meiosis. Proper alignment Ionizing radiation damages cells through the production of
of homologous chromosomes is impossible. If recombination free radicals of water. The free radicals interact with DNA and
is attempted, distribution of genetic material to gametes can protein, leading to cell damage and death. Obviously, those
become unbalanced; some gametes may receive duplicate cells most vulnerable to damage are rapidly dividing cells. The
copies of DNA segments while others lack these DNA extent of the damage is dose dependent. Cells that are not
segments. killed have damage—mutations—to the DNA at sublethal
The movement of genetic material between chromosomes doses. Such damage is demonstrated by base mutations, DNA
is called a translocation. Translocations that exchange
material between two chromosomes are called reciprocal
translocations. These translocations generally have little
consequence for the individual in whom they arise. However,
translocations become important during the formation of Pericentric inversion
gametes and segregation of the chromosomes. Some gametes
a b c d e f n m l k j i h g o p q r s t

a b c d e f g h i j k l m n o p q r s t

Box 1-1. EXAMPLES OF DELETION SYNDROMES

a b c d e f g h i j k l q p o n m r s t
Cri du chat syndrome (5p15)
Prader-Willi syndrome (15q11-13) Paracentric inversion
Angelman syndrome (15q11-13)
DiGeorge syndrome (22q11.2) Figure 1-6. Inversions of DNA on a chromosome are
Smith-Magenis syndrome (17p11.2) distinguished by the involvement of the centromere.
Wolf-Hirschhorn syndrome (4p16.3) Pericentric inversions include the centromere. Paracentric
inversions occur in either the p or q arm.
 8 Basic Mechanisms

Box 1-2. EXAMPLES OF MUTAGENS

Damaged DNA
5′ 3′
Chemicals
Base analogs
Aminopurine: resembles adenine and will pair with T or C
Bromouracil: resembles thymine
3′ 5′
Intercalating agents
Ethidium bromide DNA glycolase
Acridine orange removes base
Nitrous oxide: causes deamination 5′ 3′
Nitrosoguanidine
Methyl methanesulfonate: adds methyl or ethyl groups
Ethyl methanesulfonate
Psoralens: cause cross-linking
Peroxides: cause DNA strand breaks 3′ 5′

Irradiation AP endonuclease removes

several nucleotides
Ionizing radiation
5′ 3′
X-rays
Gamma rays
Ultraviolet radiation
UV-A: creates free radicals and some dimers
UV-B: forms pyrimidine dimers, blocking transcription and 3′ 5′
replication
UV-C: forms pyrimidine dimers, blocking transcription and DNA polymerase
replication and ligase
5′ 3′

3′ 5′
cross-linking, and breaks in DNA. Breaks in the DNA of
chromosomes may result in deletions, rearrangements, or Figure 1-7. Base excision repair is the mechanism most
even loss. commonly employed for incorrect or damaged bases.
Ultraviolet (UV) radiation is non-ionizing because it pro- Specificity of repair is conferred by specific DNA
duces less energy. UV-A (≥320 nm) is sometimes called N-glycosylases, such as uracil (or another base) DNA
“near-UV” because it is closer to visible light wavelength. N-glycosylase. These glycosylases hydrolyze the N-glycosidic
bond between the base and the deoxyribose. AP, apurinic/
UV-B (290–320 nm) and UV-C (190–290 nm) cause the
apyrimidinic.
greatest damage. The most damaging lesion is the formation
of pyrimidine dimers from covalent bonds formed between
adjacent pyrimidines. These dimers block transcription and
replication.
which no base is attached to the phosphate-sugar backbone.
Unlike other types of mutations, these examples cause little
●●● ERRORS IN DNA AND DNA REPAIR distortion of the DNA and are repaired by base excision (Fig.
DNA mutations can be significant if the expression of a gene, 1-7). DNA glycosylases release the base by cleaving the gly-
or its alleles, and its allelic products are altered and the cosidic bonds between the deoxyribose and the base. DNA
alteration cannot be repaired. Cells obviously have mecha- polymerase I replaces the base to restore the appropriate
nisms to repair DNA damage, since each individual encoun- pairing (A:T or G:C), followed by ligation to repair the ends.
ters many spontaneous mutations that do not progress to a Glycosylases are specific for the base being removed, and if
disease state. Three general steps are involved in DNA repair: there is a deficiency of a particular glycosylase, repair is
(1) mutated DNA is recognized and excised, (2) the original compromised.
DNA sequence is restored with DNA polymerase, and (3) More extensive damage to DNA than single base pairs
the ends of the replaced DNA are ligated to the existing may distort the DNA structure. Damage of this type requires
strand. The mechanisms employed by cells to accomplish the removal of several nucleotides to accomplish repair.
these steps include base excision, nucleotide excision, and Nucleotide excision repair (Fig. 1-8) differs from base exci-
mismatch repair. sion repair, which requires specific enzyme recognition of
Individual bases need replacing because of oxidative the base needing repair and of the size of the repair.
damage, alkylation, deamination, or a structural error in The general mechanism of nucleotide excision repair is
 Errors in DNA and DNA Repair 9

recognition of a bulky distortion, cleavage of the bonds matching the template. There are nine major proteins
on either side of the distortion with an endonuclease, involved in nucleotide excision repair. Any of these proteins
removal of the bases, replacement of the fragment with can be mutated and affect the repair process. This is exactly
DNA polymerase I, and ligation of the ends to the DNA what is seen in the inherited diseases xeroderma pigmento-
strand. sum and Cockayne syndrome. Mutations in different genes
Nucleotide excision repair requires a complex system of yield the same general clinical presentation (Table 1-1).
proteins to stabilize the bulky region of the DNA being Patients with xeroderma pigmentosum have flaking skin with
removed and then to resynthesize the correct segment abnormal pigmentation and numerous skin cancers, such as
basal and squamous cell carcinomas as well as melanomas.
Combinations of different mutated genes result in variations
in the severity and spectrum of disease presentation. In Cock-
ayne syndrome, another DNA repair disorder, affected indi-
Damaged DNA: dimer
viduals share several clinical features with xeroderma
5′ 3′ pigmentosum, such as sensitivity to sunlight. Two primary
genes have been identified as causing Cockayne syndrome:
CSA and CSB. However, not only have abnormal proteins
involved in the DNA repair process been identified in Cock-
ayne syndrome, but some are also responsible for xeroderma
3′ 5′
pigmentosum. Clinical features of these two distinct syn-
Proteins bind and dromes become less distinct when similar mutations are
endonuclease removes shared (Table 1-2).
several nucleotides
5′ 3′

PATHOLOGY

3′ 5′ Skin Tumors
Basal cell carcinoma is a slow-growing tumor that rarely
DNA polymerase I metastasizes. It presents as pearly papules with subepidermal
and ligase telangiectasias and basaloid cells in the dermis.
5′ 3′
Squamous cell carcinoma is the most common tumor
resulting from sun exposure. The in situ form does not
invade the basement membrane but has atypical cellular
and nuclear morphology. Invasive forms occur when the
3′ 5′ basement membrane is invaded.
Melanoma of the skin demonstrates a variation in
pigmentation with irregular borders. Some malignant
Figure 1-8. Nucleotide excision repair. Damaged DNA is melanomas may develop from dysplastic nevi, but the
recognized on the basis of its abnormal structure or abnormal association of multiple dysplastic nevi with malignant
chemistry. A multiprotein complex binds to the site to initiate melanoma is strongest for familial forms of melanoma.
excision and repair by a DNA polymerase and ligase.

TABLE 1-1. Specific Genes Associated with Xeroderma Pigmentosum*

LOCUS NAME GENE SYMBOL CHROMOSOME LOCUS PROTEIN

XPA XPA 9q22.3 DNA repair protein complementing XP-A cells

XPB ERCC3 2q21 TFIIH basal transcription factor complex helicase XPB
subunit
XPC XPC 3p25 DNA repair protein complementing XP-C cells
XPD ERCC2 19q13.2-q13.3 TFIIH basal transcription factor complex helicase subunit
XPE DDB2 11p12-p11 DNA damage binding protein 2
XPF ERCC4 16p13.3-p13.13 DNA repair protein complementing XP-F cells
XPG ERCC5 13q33 DNA repair protein complementing XP-G cells
XPV POLH 6p21.1-p12 Error-prone DNA photoproduct polymerase
XPH ERCC1 19q13.2-q13.3 Excision repair protein ERCC1

*Mutations in these genes, as well as in others, may cause clinical features of the disease. These represent locus heterogeneity, or a condition in which a mutation
in more than one gene can cause the same presentation.
 10 Basic Mechanisms

TABLE 1-2. Relationship Between Genes Involved in the Xeroderma Pigmentosum–Cockayne Syndrome–
Trichothiodystrophy Spectrum*

DISEASE PHENOTYPES

XP XP/CS COFS
GENE XP CS XP/TTD TTD
with Neurologic Abnormalities Complex Syndrome

XPC
DDB2 (XPE)
ERCC4 (XPF)
POLH (XPV)
XPA
ERCC2 (XPD)
ERCC3 (XPB)
ERCC5 (XPG)
CSA
CSB
TTD-A

CS, Cockayne syndrome; COFS, cerebro-oculo-facio-skeletal syndrome; TTD, trichothiodystrophy; XP, xeroderma pigmentosum.
*Trichothiodystrophy is a group of disorders in which half of those affected are photosensitive, which is correlated with a nucleotide excision repair defect.

The mechanism of mismatch repair (Fig. 1-9) does not

Mismatched DNA on
nontemplate strand recognize damage to DNA; it recognizes bases that do not
recognized by proteins match those of the template strand. Proteins of the mismatch
repair system recognize a mispairing and bind to the DNA.
Other proteins bind to the site, and several nucleotides are
excised by an exonuclease and replaced by DNA polymerase
III and ligase. The DNA template strand and the newly syn-
5′ 3′
thesized strand are distinguished early in the replication
process by methylation present on specific nucleotides of the
Methylation template strand, allowing the repair machinery to differenti-
ate between correct and incorrect nucleotides at the mis-
3′ 5′ match site. Hereditary nonpolyposis colon cancer (HNPCC)
is a hereditary cancer. Mutations in mismatch repair system
Proteins bind and
exonuclease removes proteins occur in most cases of HNPCC. Because repair is
several nucleotides defective, mutations accumulate in cells leading from normal
5′ 3′ to abnormal cancer cell progression (see Chapter 5).
Overall, the combination of DNA polymerase 3′→5′ proof-
reading and the above three postreplication DNA repair
mechanisms reduce the error rate of DNA replication to 10−9
to 10−12.
3′ 5′

DNA polymerase III

and ligase
5′ 3′

KEY CONCEPTS
■ DNA is a double-stranded, antiparallel molecule.

3′ 5′ ■ Organization of DNA provides instructions for RNAs that can be

processed and translated into proteins or remain as RNA.
■ Changes in DNA sequences are mutations with a range of effects
Figure 1-9. Mismatch repair. Mismatches most commonly from none to severe, depending on the type and location.
occur during replication; however, they may occur from other
mechanisms such as deamination of 5-methylcytosine to ■ Many mutations are repaired each day by repair mechanisms.
produce thymidine improperly paired to G.
 Questions 11

●●● QUESTIONS of these options are among the differentials for a patient
presenting with an XP-looking presentation. It is the fine
1. A 6-year-old male presents with multiple brownish points of observation that separate the options for a pre-
freckles on the cheeks, nose, and upper lip. Freckles sumptive diagnosis.
are scattered on both forearms and thighs. No telan-
giectasias (dilated capillaries causing red spots) or 2. A study of 600 families previously diagnosed with
malignant skin tumors were present. No physical or hereditary nonpolyposis colon cancer found 100 indi-
neurologic abnormalities were noted on physical viduals with no evidence of mutations in the MLH1
examination, and mental development was normal for gene as expected. Further analysis of these 100 indi-
age. Past medical history reveals the boy demon- viduals revealed that 25 had mutations in both alleles
strated severe photosensitivity at age 6 months. of the gene encoding adenine glycosylase. Which of
Which of the following is the most likely presumptive the following is most likely affected in these 25
diagnosis? individuals?
A. Acanthosis nigricans A. Base excision repair
B. Acute lupus erythematosus B. DNA proofreading repair
C. Bloom syndrome C. Mismatch repair
D. Cockayne syndrome D. Nucleotide excision repair
E. Xeroderma pigmentosum E. SOS repair

Answer. E Answer. A

Explanation: This patient demonstrates xeroderma pig- Explanation: Hereditary nonpolyposis colon cancer
mentosum (XP) caused by mutations in one of several (HNPCC) is caused by mutations in several genes produc-
genes involved in nucleotide excision repair. Both Bloom ing proteins for mismatch DNA repair. In this specific study
and Cockayne syndromes are related to XP in that they diagnoses were most likely not based upon gene mutation
have defects in DNA repair. Table 1-2 shows the genotype- confirmation but upon patient and family presentation.
phenotype overlap between XP and Cockayne syndrome. Further analysis revealed a subset of patients who surpris-
XP should be suspected in early onset of photosensitivity, ingly did not have the expected mutation, and among these
pigment changes, tumors, and skin aging. The defect a subset was found that had mutations in the gene for
results in the inability to correct DNA damage caused by adenine glycosylase. DNA glycosylases are required for
ultraviolet radiation. With Cockayne syndrome, patients base excision repair, and in particular, specific nucleotide
present with skin aging, psychomotor delay, progressive glycosylases are required to make specific corrections.
ophthalmic changes leading to cataracts, and photosensi- Mutations in the adenine glycosylase allow damaged bases
tive rashes. Bloom syndrome is also called congenital opposite an adenine in the template strand to go unre-
telangiectatic erythema. In this patient telangiectasias are paired. This can lead to possible transversions and a change
absent. The mutation responsible for Bloom syndrome in the gene sequence. DNA proofreading repair is a function
encodes a DNA helicase activity contributing to genome of DNA polymerase. Mismatch repair enzymes are a family
stability. Acanthosis nigricans are dark, thick velvety areas of enzymes that include best-studied HNPCC. These include
of skin associated with insulin resistance and several dis- seven genes of which two represent the majority of cases.
orders, including Bloom syndrome. Acute lupus erythe- Nucleotide excision repair requires many proteins to effec-
matosus is characterized by a typical butterfly eruption tively repair an area of DNA. The diseases most often asso-
pattern on the malar region of the face and generalized ciated with nucleotide excision repair are xeroderma
photosensitive dermatitis. pigmentosum and Cockayne syndrome. SOS repair is a
The significance of this question in Chapter 1 is to postreplication mechanism best associated with Escherichia
underscore several features of questions and answers. coli as a last resort for repair. No template is required and
Clinical presentation of commonly discussed disorders is it is very error-prone.
important. In this case XP is the most commonly studied
of the options presented. The answer options should all Additional Self-assessment Questions can be Accessed
be related even if they have not been presented. Four at www.StudentConsult.com
Chromosomes in the Cell 2
CONTENTS letters A through G. Each pair of autosomes is identical in
size, organization of genes, and position of the centromere
(Fig. 2-1). The genes on these homologous chromosomes are
CHROMOSOME STRUCTURE AND NOMENCLATURE
organized to produce the same product. In addition, there are
Identification of Chromosomes two sex chromosomes, which are unnumbered and of differ-
ent sizes. The male has one X chromosome and one Y chro-
CELL CYCLE AND MITOSIS
mosome. The female has two X chromosomes of equal size
MEIOSIS and no Y chromosome. Thus, the complement of 46 human
chromosomes comprises 22 pairs of autosomes plus the sex
Meiosis and Gamete Formation
chromosome pair—XX in normal females and XY in normal
ROLE OF CHROMOSOMAL ABNORMALITIES IN males—and the female is described as 46,XX and the male
MEDICAL GENETICS as 46,XY.
Cytogenetic analysis and preparation of a karyotype pro-
Chromosomal Numerical Abnormalities
Chromosomal Structural Abnormalities
vide physical identification of metaphase chromosomes. At
this stage of visualization, each chromosome is longitudinally
doubled, and the two strands (or chromatids) are held together
at a primary constriction, known as the centromere. A chro-
mosome with a medially located centromere is technically
called metacentric. When the centromere is located away
from the midline, one arm of the chromosome appears longer
than the other. Such a chromosome is termed submetacen-
Replication and segregation of chromosomes from progenitor tric. In acrocentric chromosomes, the centromere is nearly
cells to daughter cells is a fundamental requirement for the terminal in position (Fig. 2-2). Cytogeneticists betrayed their
viability of a multicellular organism. Defects in the replica- sense of humor by designating the short arm of the chromo-
tion and distribution of this chromosomal material during some as p (for petite) and the long arm as q (the next letter
cell division give rise to numerical (aneuploidy) or structural of the alphabet!).
(translocations, deletions, duplications, or inversions) chro-
mosomal defects. Down syndrome is a well-known example
of a disorder that can be caused by either a numerical
Identification of Chromosomes
error or a structural error and is discussed several times in Chromosomes are most easily identified in the metaphase
this chapter; other disorders are highlighted to a lesser stage of the cell cycle. Here, each homologous chromosome
extent. These and many other abnormalities have pleiotropic is doubled and has a sister chromatid; the sister chromatids
consequences, or multiple phenotypic effects from a single are held together by a single centromere. Beginning with a
event, and can result in severe clinical presentations that sample of blood, phytohemagglutinin, which stimulates cell
are readily recognizable. Cytogenetics, the study of chromo- division in human white blood cells, and colchicine, which
some abnormalities, enables techniques for the visualization arrests cell division at the metaphase stage, can be used to
of an individual’s chromosomal complement. provoke a large number of cells to the metaphase stage. At
this point, chromosomes are ordinarily stained for visualiza-
tion under the light microscope. Two of the more traditionally
employed techniques are Q-banding and G-banding.
●●● CHROMOSOME STRUCTURE
Quinacrine dye stains chromosomes and is detected with
AND NOMENCLATURE a fluorescent microscope. The banding pattern produced is
Genetic information in DNA is organized on chromosomes called Q-banding. Pretreating cells with the enzyme trypsin,
as genes. As noted in Chapter 1, each cell has 22 autosomal which partially digests the chromosomal proteins, and then
pairs and one pair of sex chromosomes. The autosome pairs staining the preparation with Giemsa dye, results in the
are numbered 1 to 22, in descending order of length, and formation of G-bands, which are visible under the ordinary
further classified into seven groups, designated by capital light microscope as demonstrated in Figure 2-1. The Giemsa
 Chromosome Structure and Nomenclature 13

1 2 3 4 5

6 7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 X Y

Diagrammatic Relative
Group Number Representation Length*
Large chromosomes
A 1 8.4
2 8.0
3 6.8

B 4 6.3
5 6.1
Medium chromosomes
C 6 5.9
7 5.4
8 4.9
9 4.8
10 4.6
11 4.6
12 4.7

D 13 3.7
14 3.6
15 3.5
Small chromosomes
E 16 3.4
17 3.3
Figure 2-1. A, Normal karyotype. 18 2.9
Chromosomes are arranged as
homologs in descending order by size. F 19 2.7
(Courtesy of Dr. Linda Pasztor, Sonora 20 2.6
Quest Laboratories.) B, Characteristics
of metaphase chromosomes showing G 21 1.9
22 2.0
groups with similar lengths and
Sex chromosomes
centromere positions. Groups D and
G chromosomes with acrocentric X 5.1 (group C)
Y 2.2 (group G)
centromeres are often involved in
translocations. (Data from the * Percentage of the total combined length of a haploid set of 22 autosomes.
International System for Chromosome B
Nomenclature, 2005.)
 14 Chromosomes in the Cell

for the detection of an abnormal chromosome number or

a rearrangement between chromosomes.

p arm ●●● CELL CYCLE AND MITOSIS

Cells can be described as existing within a cell cycle. The cell
Centromere cycle has two essential components: mitosis, the period of
cell division, and interphase, and the period between mitoses.
Interphase is defined by three stages: the first gap phase (G1),
q arm the synthesis (S) phase, and the second gap (G2) phase. Cells
in a state of quiescence are in G0 but can be stimulated to
reenter G1. Progression through the cell cycle occurs rapidly
Metacentric Submetacentric Acrocentric or quite slowly, and often this is controlled by the length of
time that the cell spends in the G1 phase. The S phase is the
Figure 2-2. Anatomy of a chromosome. A chromosome is period of DNA replication, and each G1 chromosome that had
divided by a centromere into a long arm (q) and a short arm been a single chromosome now comprises two identical
(p). By convention the p arm is always at the top. The (sister) chromatids. Thus, at the end of the G2 phase, each
centromere is designated by its location as metacentric,
chromosome is represented as a pair of homologous chromo-
submetacentric, or acrocentric.
somes and each member of the pair is composed of two sister
chromatids. The cell is now ready for mitosis (Fig. 2-4). Under-
standing the details of the cycle is important for recognizing
mechanisms that can cause normal cells to progress to cancer
cells. These are detailed and discussed in Chapter 5.
bands, those stained with the dye, are rich in adenine and
thymine (AT-rich), whereas the light bands are rich in
guanine and cytosine (GC-rich). Quinacrine and Giemsa
dyes produce identical banding patterns. The advantage of
Giemsa over quinacrine is that it does not necessitate
expensive fluorescent microscopy. These banding procedures
are the cornerstones of karyotypic analysis. BIOCHEMISTRY
The key point in karyotypic analysis is that each chromo-
some is visualized as consisting of a continuous series of dark Cell Cycle
and light bands. In each chromosome arm, the bands are Regulation of the cell cycle is very complex. Some important
numbered from the centromere to the terminus. In describing features include the following:
a particular site, the chromosome number is listed first, fol- ■ Cyclin-dependent kinases (CDKs), along with cyclins, are

lowed by the arm (p or q), then the region number within an major control switches regulating transitions from G1 to S
arm, and finally the specific band within that region. For and G2 to M.
■ CDKs and cyclins trigger progression through the cell
example, 1q32 refers to chromosome 1, long arm, region 3,
and band 2. Higher-resolution techniques have permitted the cycle.
portrayal of prophase chromosomes and, concomitantly, the M
subdivision of existing bands. To indicate a sub-band, a (mitosis)
decimal point is placed after the original band designation,
followed by the number assigned to the sub-band. In the G2 G1
(gap 2) (gap 1)
example used, the identification of two sub-bands would be
designated 1q32.1 and 1q32.2.
Today, technical advances allow researchers to identify a
given region or a particular gene-specific sequence on a
chromosome spread with a fluorescent DNA-specific probe
that hybridizes with its corresponding sequence on the chro-
mosome. The hybridized probe is revealed by fluorescence
under ultraviolet light. This nonradioactive technique is called
fluorescent in situ hybridization, or FISH. The technique is
useful in defining specific chromosome sequences in both
interphase and metaphase nuclei. It is favored for detecting S phase
(DNA synthesis)
many chromosomal aberrations prenatally. Each chromosome
can also be labeled by chromosome-specific fluorophores, a Cells that
cease
technique known as chromosome painting, and readily dis- division
tinguished (Fig. 2-3). This technique is particularly useful
 Cell Cycle and Mitosis 15

Figure 2-3. Spectral karyotyping

(SKY) and multiplex fluorescence in
situ hybridization (M-FISH) of human
chromosomes permit simultaneous
visualization of all chromosomes in
different colors. Chromosome-specific
probes are generated from flow-sorted
chromosomes that are amplified by
polymerase chain reaction and
fluorescently labeled. Each human
chromosome absorbs a unique
combination of fluorochromes. Both
spectral karyotyping and M-FISH
(multiplex-FISH) use spectrally
distinguishable fluorochromes, but they
employ different methods of detection.
(Courtesy of Evelin Schröck, Stan du
Manoir, and Thomas Ried, National
Institutes of Health.)

During mitosis, the cell undergoes fission and each daugh- and a nuclear membrane re-forms around the chromosomal
ter cell receives a complete genetic complement that is identi- material, thereby reconstituting the nucleus. Associated with
cal to the progenitor cell. This is a highly complex process of telophase is cytokinesis, or cytoplasm division, which ulti-
the cell cycle with five distinct phases. Prophase begins mately results in two complete, chromosomally identical
mitosis and is characterized by a condensation of the chro- daughter cells.
mosomes and the initial stages of the mitotic spindle forma-
tion. A pair of organelles called centrioles form microtubule/
mitotic spindle organization centers and migrate to opposite
ends of the cell. Prometaphase features the dissolution of the HISTOLOGY
nuclear membrane and attachment of each chromosome to a
Centrioles
spindle microtubule via its centromere. During metaphase,
chromosomes are maximally condensed, and thus most easily Centrioles occur as a pair of organelles in the cell, and they are
arranged perpendicular to each other. They are composed of
visualized by light microscopy, and align along the equatorial
microtubules—nine sets of triplets—and organize the spindle
plane of the cell. Anaphase is characterized by replication of
apparatus of spindle fibers and astral rays on which
chromosomal centromeres and the migration of sister chro- chromosomes move during mitosis and meiosis. Similar to
matids to opposite poles of the cell. Finally, in telophase, the mitochondria, centrioles replicate autonomously.
chromosomes begin to decondense, spindle fibers disappear,
 16 Chromosomes in the Cell

Mitosis Meiosis
Parent cell

Interphase Diploid
Prophase
Chromatin condenses into
chromosomes.
Nuclear envelope disappears.

Metaphase Meiosis I
Diploid Homologous
Chomosomes align chromosomes
at the equatorial plate.

Anaphase
Meiosis II
Sister chromatids separate. Haploid Haploid
Centromeres divide.

Telophase

Chromatin expands.
Cytoplasm divides.
Haploid Haploid Haploid Haploid

Two daughter cells Haploid gametes

Figure 2-4. Mitosis is the process of forming identical Figure 2-5. Meiosis occurs in gonads and results in the
daughter cells. There are four basic stages: prophase, formation of gametes. In the first stage, meiosis I,
metaphase, anaphase, and telophase. homologous pairs of chromosomes are separated, thereby
reducing the number of chromosomes to 23. In meiosis II,
sister chromatids are separated, resulting in gametes with 23
chromosomes.

●●● MEIOSIS
Meiosis is cell division that occurs only during gamete forma- Prophase of meiosis I is the signature event of the meiotic
tion. This variation from the mitosis observed in somatic cells process, since it is here that genetic recombination takes
is essential because human somatic cells—including gamete place. Prophase is complex and is subdivided into five stages.
progenitor cells—are diploid, containing two complete copies During leptotene, chromosomes begin to condense to the
of each chromosome. The genetic material must be reduced point where they are easily visible. The chromosomes are rep-
by 50%, to a haploid state, during gamete formation for a resented by pairs, each with a single centromere and two
newly formed zygote to have a complete chromosomal com- sister chromatids. In zygotene, homologous chromosomes
plement. Meiosis involves two separate cell divisions that are associate with each other and pair, via the synaptonemal
conceptually similar to the stages of mitosis (Fig. 2-5). The first complex, along the entire length of the chromosomes. Further
cell division, meiosis I, is referred to as a “reductive division” coiling and condensing of the chromosomes and completion
because the chromosomal number is reduced to a haploid of the synapsis process characterize pachytene. Synapsed,
number in the resulting daughter cells. Here, homologous paired homologous chromosomes are termed bivalent—
chromosomes, each comprising two sister chromatids, line up indicating two joined or synapsed chromosomes—or tetrad—
along the equatorial plate during metaphase I and separate representing the four separate chromatids in the bivalent
during anaphase I. Meiosis II directly follows meiosis I in the structure. Importantly, bivalent chromosomes in pachytene
absence of further DNA replication. During anaphase of undergo an exchange of chromatid material in a process called
meiosis II, centromeres are duplicated and sister chromatids recombination or crossing-over. In practice, genetic recombi-
segregate to opposite poles of the cell. Each gamete formed nation is vital to the chromosomal exchange of parental
contains a haploid genome consisting of 23 chromosomes. genetic material during gamete formation. This process is the
 Meiosis 17

major source of genetic variation, and it permits an extremely dictyotene until sexual maturity is reached and ovulation
high degree of variability among gametes produced by an occurs. At ovulation, the oocyte completes meiosis I, produc-
individual. Homologous chromosomes begin to pull apart in ing a secondary oocyte that contains most of the cytoplasm
diplotene, and chiasmata—points of attachment between from the primary oocyte; the cell with little cytoplasm is
paired chromosomes—are apparent. Chiasmata indicate posi- termed the first polar body and undergoes atresia. The second-
tions where crossing-over has occurred. In the next stage, ary oocyte initiates meiosis II, but this process is completed
diakinesis, homologous chromosomes continue to separate only at fertilization of a mature ovum, at which point a second
from each other and attain a maximally condensed state. polar body is formed. Hence, in females, only one mature,
haploid gamete is produced during gametogenesis, and the
process may take from 10 to 50 years. Spermatogenesis, on the
HISTOLOGY other hand, is a much more rapid and dynamic process, taking
roughly 60 days to complete. Here, puberty signals the mitotic
Synaptonemal Complex and Synapsis maturation of diploid spermatogonia to diploid primary sper-
Synapsis is the pairing of homologous chromosomes during matocytes. Primary spermatocytes undergo meiosis I to form
prophase I of meiosis. The synaptonemal complex is the haploid secondary spermatocytes, which, in turn, proceed
protein scaffolding structure present between homologous through meiosis II to form spermatids that differentiate further
chromosomes that facilitates genetic recombination. into mature sperm. In contrast to oogenesis, four mature,
haploid gametes are derived from one primary spermatocyte.

HISTOLOGY
Following diakinesis, the rest of meiosis I proceeds quite
similarly to mitosis. During metaphase I, a spindle apparatus Ovary
forms and the paired chromosomes align along the equatorial The ovary is attached by the mesovarium to the broad
pole of the cell. During anaphase I, the individual bivalents ligament. The ovary is covered by a simple squamous or
completely separate from each other; then homologous chro- simple cuboidal epithelium/germinal epithelium (a
mosomes, with their cognate centromere, are separated and misnomer) that forms the ovarian cortex. Deep beneath the
drawn to opposite poles of the cell. Finally, in telophase I, the germinal epithelium is the tunica albuginea, which is the
haploid chromosomal complement has segregated to both dense irregular collagenous connective tissue capsule of the
ovary that surrounds the ovarian cortex. Ovarian follicles,
poles of the cell and cytoplasmic cleavage yields two daughter
containing primary oocytes, are embedded in the stroma of
cells. Two critical features can be appreciated at this point.
the cortex. The medulla of the ovary consists of a looser
First, the number of chromosomes has been reduced from connective tissue and blood vessels.
diploid (46 chromosomes) in one cell to haploid (23 chro-
mosomes) in daughter cells. Second, genetic recombination
has generated a new arrangement of genetic material, which
originated from parental chromosomes, in each of the daugh- HISTOLOGY & PHYSIOLOGY
ter cells. Each chromosome in the daughter cell can be
thought of as hybrid, or recombinant, representing a unique Seminiferous Tubules
combination of the two parental chromosomes. Seminiferous tubules constitute the exocrine portion of the
Meiosis II proceeds just as in mitosis except the starting cell testes. There are two major cell types: Sertoli cells and
is haploid and no DNA replication (typically an interphase the spermatogenic cells that lie between the Sertoli cells.
event) occurs. Each of the 23 chromosomes is represented by The immature germ cells are located near the periphery of
the seminiferous tubules, and as they mature they move
two sister chromatids sharing a centromere. These chromo-
toward the lumen.
somes thicken and align along the equatorial plane of the cell.
Sertoli cells are a nonproliferative columnar epithelium
The centromere replicates and each chromatid is then pulled connected by tight junctions. These junctions form the
to opposite poles of the cell during anaphase II. Subsequent blood-testis barrier that subdivides the lumen of the
cytoplasmic division yields two haploid (23 single chromatid seminiferous tubule into a basal and an adluminal
chromosomes with one centromere) gametes. Overall, a single compartment to protect developing germ cells from an
gamete progenitor cell may yield four independent gametes. immunologic response. Sertoli cells provide support and
nutrients to sperm cells during spermatogenesis and also
regulate the release of spermatozoa. Sertoli cells are the
Meiosis and Gamete Formation primary testicular sites of follicle-stimulating hormone (FSH)
Meiosis is the signature event in gamete formation. However, action, and androgen-binding proteins (ABPs) are secreted
under the influence of FSH. During embryonic development,
marked sex-specific differences exist in the production of
these cells secrete antimüllerian hormone, a member of the
sperm and egg. By birth, germ cells in females have nearly
transforming growth factor-β (TGF-β) superfamily of
completed oogenesis as primary oocytes—derived from glycoproteins involved in regulation of growth and
oogonia via roughly 30 mitotic divisions—and have initiated differentiation that prevents feminization of the embryo.
prophase of meiosis I. Primary oocytes are suspended at
 18 Chromosomes in the Cell

●●● ROLE OF CHROMOSOMAL

ABNORMALITIES IN
MEDICAL GENETICS
Having considered chromosomal structure, nomenclature,
and behavior during gamete formation, it is now possible to 1 2 3 4 5
consider the impact of chromosomal defects on human health.
Chromosomal abnormalities generally fall into two catego-
ries: numerical or structural. Each category is considered
separately. 6 7 8 9 10 11 12

Chromosomal Numerical Abnormalities 13 14 15 16 17 18

Euploidy versus Aneuploidy

Cells with normal chromosome complements have euploid 19 20 21 22 X Y
karyotypes (Greek eu, “good”; ploid, “set”). The euploid
A
states in humans are the haploid (23 chromosomes) germ cells
(gametes) and the diploid (46 chromosomes) somatic cells.
Aneuploid cells have an incomplete or unbalanced chromo-
some complement owing to a deficiency or excess of indi-
vidual chromosomes. A cell lacking one chromosome of a
diploid complement is called monosomic (46 − 1). A trisomic
1 2 3 4 5
cell has a complete chromosome complement plus a single
extra chromosome (46 + 1). Tetrasomics (46 + 2) carry a
particular chromosome in quadruplicate; the remaining chro-
mosomes are present twice as homologous pairs. Polyploidy
6 7 8 9 10 11 12
describes the condition in which a complete extra chromo-
somal set is present (e.g., 69 or 92 chromosomes). Only
aneuploidy is relevant for live births, since polyploidy is 13 14 15 16 17 18
incompatible with life.
19 20 21 22 X Y
Cause and Incidence of Aneuploidy
Down syndrome, or trisomy 21, illustrates the principles of B
aneuploidy (Fig. 2-6). The additional extra chromosome 21 in
Figure 2-6. Down syndrome karyotypes. A, Trisomy Down
somatic cells of individuals with Down syndrome was initially
syndrome: 47,XX,+21. B, Translocation Down syndrome:
thought to be the next-to-smallest chromosome. When 46,XY,t(14q;21q). (Courtesy of Dr. Linda Pasztor, Sonora
improved karyotypic techniques revealed that chromosome Quest Laboratories.)
21 is actually smaller than chromosome 22, no reversal in
numbers occurred because of the firm association of number
21 with Down syndrome. Geneticists acknowledge the pre-
vailing inconsistency that chromosome 21 (not 22) is in
reality the smallest chromosome in the human complement.
Down syndrome is the most common congenital chromo-
somal disorder associated with severe mental retardation. The
clinical features of this syndrome are quite distinctive and
readily discernible at birth. Characteristic features include a Box 2-1. CLINICAL FEATURES ASSOCIATED WITH
prominent forehead, a flattened nasal bridge, a habitually DOWN SYNDROME
open mouth, a projecting lower lip, a protruding tongue,
slanting eyes, and epicanthic folds. Additional features are Hypotonia Flat, depressed nasal bridge
shown in Box 2-1. Mental retardation Palpebral fissures slant upward
Many of these features are variably expressed and not Short stature Brushfield spots
present in all affected individuals. Even the highly unusual Prematurity Single palmar crease
Lower birth weight Posterior-rotated ears
iris of a Down syndrome infant is not universal. White (or
Brachycephaly Upturned nose
light yellow) cloud-like specks may circumscribe the outer
Macroglossia Flattened occiput
layer of the iris (Fig. 2-7) and are known as Brushfield spots.
Small mandible and maxilla Short, broad hands
The specks are infrequently associated with brown irides and Excess nuchal skin Clinodactyly of the fifth finger
have not been found in black infants with Down syndrome. Epicanthal folds Diastasis
The ultimate confirmation of Down syndrome must come
 Role of Chromosomal Abnormalities in Medical Genetics 19

woman ages. By age 50, the production of functional eggs

is drastically diminished.
For numerous years, scientists were comfortable in the
belief that the eggs of the human female are subject to the
hazards of aging and that aging alone accounted for most
trisomy cases. However, the simple focus on older women
and aged eggs is inadequate. Since 1970, the mean maternal
age for all live births has declined substantially because of
the decreasing number of children born to women over 35
years of age. Women under age 35 are currently responsible
for more than 90% of all births. Presently, women under the
age of 35 have 75% of the children affected with Down
syndrome, demonstrating that some factors in its etiology are
still not understood. Data show that the egg is not always at
fault, as surmised earlier. In 5% to 15% of the cases of Down
syndrome, the extra chromosome is of paternal origin.

Figure 2-7. Brushfield spots. Brushfield spots are white or

yellow-colored spots seen on the anterior surface of the iris.
The spots may be arranged concentrically to the pupils or, as
PATHOLOGY
seen here, along the pupillary periphery. They are present in
85% of blue- or hazel-eyed patients with trisomy 21. Only Screening and Diagnostic Tests for
17% of Down syndrome patients with a brown iris have Down Syndrome
Brushfield spots, since they can be obscured by pigment.
(Courtesy of Dr. Usha Langan, New Delhi, India.) The triple screen is a noninvasive screening test to
determine whether there is an increased risk for Down
syndrome. It is only a screening test and not a diagnostic
test. Increased risk is associated with the following:
100 ■ Low maternal serum α-fetoprotein (MSAFP)
90 ■ Low unconjugated estriol (uE3)
Incidence per 1000 births

80 ■ Elevated human chorionic gonadotropin (hCG)

70 Diagnostic tests include amniocentesis, chorionic villus

60 sampling (CVS), and percutaneous umbilical blood sampling
50 (PUBS).
40
30
20
Origin of Trisomy 21: Nondisjunction in Meiosis
10
The process of meiosis is complex and subject to error. It does
0
not always proceed normally. Accidents occur that affect the
<30 30 35 40 45 50
normal functioning of the spindle fibers and impede the
Mother’s age
proper migration of one or more chromosomes. During
Figure 2-8. Maternal age versus Down syndrome. the first meiotic division, a given pair of homologous chro-
mosomes may fail to separate from each other. This failure of
separation, known as nondisjunction, can result in a gamete
from analysis of the chromosome complement. Individuals containing a pair of chromosomes from one parent rather
with Down syndrome have 47 chromosomes rather than 46. than a single chromosome homolog (Fig. 2-9). Stated another
The incidence of Down syndrome rises markedly with way, nondisjunction of chromosome 21 homologs during
maternal age—from about 1 in 2000 live births at maternal oogenesis results in an egg that possesses two copies of chro-
age 20 years to 1 in 100 at age 40 (Fig. 2-8). Among infants mosome 21 rather than the usual one copy. Fertilization by
born to women over age 45, Down syndrome is expected a normal sperm gives rise to an individual who is trisomic for
to affect 1 in 40 infants. It was immediately surmised that chromosome 21.
the extra chromosome in the affected infant is acquired It should be noted in Figure 2-9 that nondisjunction of one
during the production of the egg by the mother. As noted chromosome pair in meiosis I results in two types of cells in
above, all eggs a woman produces during her reproductive equal proportions at the end of meiosis I; one cell contains
life are present from the moment of birth. At birth, the both members of the chromosome pair and the other lacks
ovaries contain 1 to 2 million germ cells; by puberty this the particular chromosome. In normal meiosis, the gametes
number has declined to 300,000 to 400,000 germ cells formed at the end of meiosis II have a single homolog of each
through normal follicular atresia. There is a progressive chromosome and fertilization returns the zygote to a diploid
decline in the number of eggs that mature perfectly as the state with homologous pairs of chromosomes. If a normal
 20 Chromosomes in the Cell

Meiosis II

Nondisjunction at 23 chromosomes +
meiosis I 1 additional chromosome
Trisomy at fertilization

22 chromosomes
Monosomy at fertilization

Nondisjunction at
meiosis II
23 chromosomes +
1 additional chromosome
Trisomy at fertilization
Meiosis I

22 chromosomes
Monosomy at fertilization Figure 2-9. Nondisjunction. A,
Nondisjunction occurs in meiosis I
when homologous chromosome pairs
segregate to the same daughter cell.
B, Nondisjunction occurs in meiosis II
when sister chromatids segregate to
23 chromosomes the same daughter cell. When
Euploid at fertilization nondisjunction occurs in meiosis I, all
gametes are abnormal, whereas when it
occurs in meiosis II, there is a 50%
chance that a normal gamete will be
fertilized.
B

sperm fertilizes an egg cell lacking the particular chromo- complement. If nondisjunction occurs in a secondary sper-
some, the outcome is monosomy. Theoretically, autosomal matocyte undergoing meiosis II, only two of the four sperm
monosomies should be equally as common as autosomal will be abnormal and only two of the zygotes will be
trisomies. However, monosomy, when it occurs in the auto- chromosomally abnormal; the other two will be normal
somes, is largely incompatible with life. In fact, any rare euploids. There is also a difference in chromatids, and
viable newborn with one autosome completely missing is therefore in alleles present on chromatids, depending on
short lived. Ironically, a person can survive with one missing whether nondisjunction occurs during meiosis I or meiosis
X chromosome; 45,X is known as Turner syndrome. Indeed, II. If nondisjunction occurs during meiosis I, all three chro-
of all disorders involving missing or additional chromosomes, matids in a fertilized egg have unique parental (or really
those involving the sex chromosomes are the most likely to grandparental) origin. If nondisjunction occurs during meiosis
demonstrate survival beyond the early days and months II, the result is two chromatids that originated from the
of life. replication of the same DNA strand and one unique chro-
Meiotic nondisjunction may occur during either the first matid occurring in the fertilized egg. The potential for inher-
or the second meiotic division (see Fig. 2-9). If nondisjunc- iting similar alleles is more likely with nondisjunction in
tion occurs in a primary spermatocyte during meiosis I, then meiosis II than in meiosis I even if consideration is not
all sperm derived from the primary spermatocyte will be given to the recombination that may have occurred. In
abnormal and all zygotes will have an aberrant chromosome both cases—nondisjunction in meiosis I and in meiosis
 Role of Chromosomal Abnormalities in Medical Genetics 21

II—the result is aneuploidy, or an abnormal number of chro-

mosomes. The nomenclature is somewhat burdensome, since
the standard terminology differs for the gamete and the
zygote. For a gamete, nullisomic (23 − 1) signifies the absence
of one chromosome in the haploid complement and disomic
(23 + 1) signifies the addition of one chromosome in the
haploid complement. For a zygote, monosomic (46 − 1)
specifies the absence of one chromosome in the diploid set
and trisomic (46 + 1) specifies the presence of one additional
chromosome in the diploid set.

Other Trisomies
Several thorough investigations have revealed that 40% to
50% of first-trimester spontaneous abortuses are trisomic for
one of the autosomes. All human autosomal trisomic condi-
tions are associated with marked developmental disorders.
The frequencies of trisomies in different autosomal groups
vary widely. Trisomies for chromosomes 13, 16, 18, 21, and
22 occur most often, especially chromosome 16. For reasons
not well understood, chromosome 16 appears to be particu-
larly vulnerable to nondisjunction. Trisomy 16 is the most
common (one third) autosomal trisomy found in abortuses.
Interestingly, trisomy 16 in abortuses shows little association Figure 2-10. Features of trisomy 13 (Patau syndrome).
with increasing maternal age, suggesting that an unusual age- Trisomy 13 female infant with cleft palate and bilateral lip,
independent mechanism is responsible for this extraordinarily low-set malformed ears, hypotelorism, and postaxial
polydactyly of the left hand. This infant also has an
common trisomic condition.
omphalocele. (From Moore KL, Persaud TVN. The Developing
Other than in Down syndrome, the trisomic condition is Human: Clinically Oriented Embryology, 7th ed. Philadelphia,
rare in live-born infants. Two autosomal trisomies other WB Saunders, 2003, p 164.)
than trisomy 21 demonstrate survival to term and occur
with sufficiently significant frequency to be well-described
syndromes—namely, trisomy 13 (Patau syndrome, Fig. 2-10)
and trisomy 18 (Edwards syndrome, Fig. 2-11). Both disorders
are associated with severe mental retardation and a broad
spectrum of severe developmental anomalies (Table 2-1).
Prominent features of a trisomy 13 baby are bilateral clefts
of the lip and palate, a forehead that slopes backward, defec-
tive eye development, and an excess number of fingers and
toes (polydactyly). Common clinical features of trisomy 18
infants are recessed chin, elongated head, small eyes, “rocker-
bottom” feet, and tightly clenched hands and fingers with the
second and fifth fingers overlapping the third and fourth.
Estimates for trisomy 13 and trisomy 18 range widely from
1 in 4000 to 1 in 10,000 live births. Each leads to death in
early infancy, invariably within a year of birth.

Mitotic Nondisjunction and Mosaicism

A small percentage (1% to 3%) of infants with Down syn-
drome have two populations of cell types with respect to
chromosome 21. Such mosaic individuals, with both normal
(46 chromosomes) and trisomic (47 chromosomes) cells, typi-
cally have less severe features of Down syndrome. Actually,
there is appreciable phenotypic variability among mosaics,
depending on the proportion of trisomic cells.
One route to mosaicism is nondisjunction during the early Figure 2-11. Features of trisomy 18 (Edwards syndrome).
Trisomy 18 female infant with a characteristic clenched fist,
cleavage stages of the zygote. A mitotic nondisjunction at the short sternum, narrow pelvis, hypertelorism, short palpebral
first cleavage division leads to two dissimilar cell populations; fissures, and rocker-bottom feet. (From Moore KL, Persaud
one cell line will be trisomic and the other will be monoso- TVN. The Developing Human: Clinically Oriented Embryology,
mic. If the monosomic cell line perishes, then only trisomic 7th ed. Philadelphia, WB Saunders, 2003, p 164.)
 22 Chromosomes in the Cell

TABLE 2-1. Comparison of Trisomy 13 and Trisomy 18

FEATURE TRISOMY 13 TRISOMY 18

Life span 50% die by age 1 month 90% die by age 1 year
75% die by age 6 months
Clinical Seizures Clenched fist; second and fifth fingers overlap the third
presentation Microcephaly; micrognathia and fourth
Scalp defects (absent skin) Intrauterine growth retardation (IUGR)
Cleft lip, cleft palate Rocker-bottom feet
Hypotelorism Micrognathia, prominent occiput, micro-ophthalmia
Coloboma defects of the iris Low-set ears
Low-set, abnormally shaped ears Cardiac defects
Polydactyly Generalized muscle spasticity
Simian crease Renal anomalies
Hernias Mental retardation
Cryptorchidism
Hypotonia
Severe mental and motor retardation
Associated Congenital heart defects in 80% Congenital heart defects in 90%
disorders Dextrocardia in 20–50% (heart is on right side of Joint contractures
chest rather than left) Spina bifida in 6%
Omphalocele in 10% Eye abnormalities in 10%
Holoprosencephaly in 66% Hearing loss—high
Radial bone aplasia in 5–10%
Problems associated with survival past 1 month of age:
Feeding difficulties
Gastroesophageal reflux
Slow postnatal growth
Apnea
Seizures
Hypertension
Kidney defects
Developmental disability
Scoliosis

cells remain. Now, if nondisjunction occurs in one cell during the exception of chromosome 21 and some combinations of
the second cleavage division, three different cell types arise. sex chromosomes, are lethal. Therefore, the finding of two X
If the monosomic cell line perishes (as is usually the case), chromosomes in females but only one in males raised several
then the embryo will be a mosaic consisting of cells with a questions. Why do females survive with twice as much gene
normal chromosome number and cells with a trisomic number product as males? Or, why do males survive with only half
of chromosomes. When several thousand normal divisions as much gene product as females?
ensue before a mitotic error occurs, the mosaicism may be The answers to these questions became clear with a better
clinically inconsequential inasmuch as the number of normal understanding of the mechanism of compensation for this
cells far exceeds the abnormal cells. Mosaics may also arise apparent gene dosage discrepancy. In the 1940s, Murray Barr
by chromosome loss, better designated as anaphase lag. In and Ewart Bertram noted differences in the position of a
this situation, one chromatid may lag so far behind during darkly staining mass in the nuclei of interphase cells. They
anaphase that it fails to become incorporated in a daughter further noted that the darkly staining mass, which became
nucleus. know as a Barr body, was associated only with interphase
cells from females. This led to the speculation that the Barr
Sex Chromosome Numerical Abnormalities body was a tightly condensed X chromosome. Because of its
Unlike autosome pairs that are the same size and contain correlation with the X chromosome, Barr bodies are also
homologous alleles, sex chromosomes are strikingly different referred to as sex chromatin.
in size with little similarity in the genes found on each. This A common method to observe sex chromatin is on a buccal
discrepancy is critically important in the developing embryo smear of cells scraped from the inside of the cheek, spread
because the two X chromosomes found in cells of females on a glass slide, stained, and examined with a light micro-
represent twice as many coding genes, and potentially gene scope. The number of Barr bodies observed is the number of
products, as the one X chromosome found in cells of males. X chromosomes minus 1. In embryos, it is first observed
It is now understood that most conditions of aneuploidy, with around the sixteenth day of development. Although easy to
 Role of Chromosomal Abnormalities in Medical Genetics 23

detect, disadvantages of Barr body analysis are that structural X chromosomes in early development for normal female
abnormalities of the X chromosome are not detected and that development but not normal male development (see Chapter
mosaicism can be missed. Currently, FISH analysis followed 11).
by G-banding is preferred over buccal smears for X chromo-
some studies.
In 1959, the first male with Klinefelter syndrome and a
Chromosomal Structural Abnormalities
karyotype of 47,XXY was identified. As expected, these Certain chromosomal defects do not involve numerical
males possess a Barr body, because of the presence of an deficiency or excess. Rather, they feature morphologic or
extra X chromosome, whereas normal males do not. At about structural abnormalities such as translocations, deletions,
the same time, a female with gonadal dysgenesis was described duplications, or inversions.
with a karyotype of 45,X, and the disorder became known
as Turner syndrome. These two events, along with research Translocation Errors
data, led scientists to recognize the importance of the Y chro- One chromosomal aberration that does not involve nondis-
mosome in sexual development and underscored the impor- junction is translocation—the transfer of a part of one chro-
tance of two normal X chromosomes for female development mosome to another, generally a nonhomologous chromosome.
(see Chapter 11). An embryo develops as a male in the pres- This occurs when two chromosomes break and then rejoin in
ence of a Y chromosome and as a female in the absence of a another combination. The exchange of broken parts is often
Y chromosome. reciprocal and may not involve loss of chromosomal material.
The first translocation observed was reported in the bone
Lyonization and Dosage Compensation marrow cells of an infant with Down syndrome born to a
In 1961, Mary Lyon proposed the inactive-X hypothesis to mother only 21 years old. The researchers found 46 chromo-
explain what happens to genes on the Barr body. She hypoth- somes in the affected child instead of the expected 47.
esized that (1) the genes found on the condensed X chromo- However, detailed examination of the chromosomes revealed
some are genetically inactive, (2) inactivation occurs very that one of the chromosomes had an unusual configuration.
early in development during the blastocyst stage, and (3) It appeared to consist of two chromosomes fused together
inactivation occurs randomly in each blastocyst cell. The net (see Fig. 2-6B). The interpretation was that the affected child
effect of this inactivation equalizes the phenotypes in males had inherited an extra chromosome, but this extra chromo-
and females through a phenomenon known as dosage com- some had become integrally joined to another chromosome.
pensation. The process of X chromosome inactivation is called Stated another way, one chromosome was in fact represented
lyonization. Contrary to Lyon’s original hypothesis that X three times, but the third instance was concealed as part of
inactivation occurs randomly, it has now been demonstrated another chromosome.
that gene inactivation may not always be random and that Translocations that exchange material between two chro-
the inactive X chromosome has some genes that are indeed mosomes are called reciprocal translocations (Fig. 2-12).
expressed. This work also highlights the need for two active These translocations generally have little consequence for the

1 2 3 4 5

6 7 8 9 10 11 12

13 14 15 16 17 18

Figure 2-12. Reciprocal translocation: 19 20 21 22 X Y

46,XY,t(1p;10p). (Courtesy of Dr. Linda
Pasztor, Sonora Quest Laboratories.)
 24 Chromosomes in the Cell

14q21q 21

14
A. Synapsis

21 14q21q 14q21q 21 14q21q 21

14 14 14

Balanced Balanced Unbalanced Unbalanced Unbalanced Unbalanced

B. Gametes normal karyotype translocation

Normal Translocation
chromosomes carrier Trisomy 21 Monosomy 21 Trisomy 14 Monosomy 14
14, 21 14, 21 14, 21 14, 21 14, 21 14, 21
C. Fertilization 14, 21 14q21q 14q21q21 14 14q21q14 21
with normal Normal Normal Down Lethal Lethal Lethal
gamete phenotype phenotype syndrome

21q 21q

14 14 21 21 14 14q 21 14 14q 21 21
D. Karyotypes and
chromosome
numbers 46 45 46

Figure 2-13. Possible gametes produced by an individual with translocation Down syndrome and the consequences of these
gametes becoming fertilized. A, Synapsis of the translocation chromosome 14q21q and normal chromosomes 21 and 14; 14p
and 21p have been lost. B, Six types of gametes are possible; three of these are viable. C, Fertilization with a normal gamete
will produce one normal genotype, one translocation carrier with a normal phenotype, one translocation Down syndrome
zygote, and three lethal zygotes. D, Karyotypic results for chromosomes 14 and 21 after fertilization.

individual in whom they arise. However, reciprocal transloca- translocation chromosome; it is written more precisely as
tions become an important issue during the formation of t(14q;21q). This large translocation chromosome carries the
gametes and segregation of the chromosomes. Some gametes essential genes of chromosomes 14 and 21. The two small
will receive extra copies of genetic material while others will p arms, containing tandemly arrayed ribosomal RNA genes,
be missing genetic material. are lost.
A common rearrangement is the fusion of two long arms When all genetic material is present, chromosomes are said
of acrocentric chromosomes leading to the formation of a to be in a balanced rearrangement and the carrier is typically
new chromosome. This fusion occurs at the centromere and asymptomatic. The loss of 14p and 21p in the 14/21 translo-
is called a robertsonian translocation. There are five acrocen- cation is inconsequential; since ribosomal RNA genes repre-
tric chromosomes (see Fig. 2-1) among the 23 pairs (chromo- sent middle repetitive sequences repeated many times in the
somes 13, 14, 15, 21, and 22), and all are commonly seen in genome on several chromosomes (see Chapter 1).
translocations. Robertsonian translocations are the most Translocation is without clinical consequence to the
common chromosomal rearrangement. In Down syndrome, mother, inasmuch as redundant copies of ribosomal RNA
the translocation always involves chromosome 21, of course, genes occur in other acrocentric chromosomes. However, the
often fused to chromosome 14. Initially, breaks occur in the consequences for her children can be significant, since the
two chromosomes in the region of the centromere. Then, the woman carrying the t(14q;21q) translocation chromosome
two long arms of broken chromosomes 14 and 21 become can produce several kinds of eggs. Eggs with only three
joined together at the centromere. This newly formed, chromosomal complements are viable, or capable of being
relatively large chromosome is referred to as a 14/21 fertilized (Fig. 2-13). Specifically, the three types of viable
 Role of Chromosomal Abnormalities in Medical Genetics 25

eggs, when fertilized by normal sperm, result in three pos- new gene is created! The new fusion gene consists of a
sible outcomes: (1) a completely normal child with a normal sequence of DNA from the original chromosome 22 known
chromosome set; (2) a normal child with the 14/21 translo- as a breakpoint cluster region (BCR) plus a gene from
cation chromosome who potentially can transmit transloca- chromosome 9, called ABL, which becomes attached to
tion-type Down syndrome; and (3) a Down syndrome child BCR (see Fig. 5-7). The fusion gene, BCR-ABL, codes for
with three copies of chromosome 21, one of which is fused an abnormally large chimeric product of this composite
to chromosome 14. gene and is fundamental to the pathogenesis of chronic
myelogenous leukemia (CML). The ABL gene is an onco-
gene, which is fairly innocuous in its normal location on
chromosome 9. When associated with an unfamiliar DNA
sequence (in this case, BCR), the ABL gene product fosters
an uncontrolled proliferation of white blood cells. The ABL
BIOCHEMISTRY gene has potent tyrosine kinase activity. Cell proliferation
rRNAs is enhanced as activated tyrosine kinase results in auto-
phosphorylation of a number of sites on the fusion protein
Ribosomal RNAs are an integral part of ribosomes. There
and phosphorylation of other proteins (see Chapter 5).
are four rRNAs: 5.8S, 18S, 28S, and 5S. The 45S precursor
gives rise to 5.8S, 18S, and 28S. 5S is transcribed from
separate 200- to 300-gene clusters and requires RNA
polymerase III for transcription. Each acrocentric
chromosome (13, 14, 15, 21, and 22) has 30 to 40 tandem PATHOLOGY
repeats of 45S genes that are transcribed by RNA
polymerase I. Chronic Myelogenous Leukemia (CML)
The nucleolus forms around the tandemly repeated rRNA
CML is a myeloproliferative disease of bone marrow
genes, and this combination of a nucleolus and rRNA genes
characterized by an increased proliferation of the
is called the “nucleolar-organizing region” (NOR). These
granulocytic cell line that does not lose the capacity to
regions are responsible for rRNA transcription and for
differentiate. The blood profile has increased granulocytes
assembly of components for ribosome synthesis.
and immature precursors, including occasional blast cells.
CML is responsible for 20% of all adult leukemias. There are
three phases of disease: chronic, blast, and accelerated.
Splenomegaly is the most common physical finding.

The t(14q;21q) translocation event is not confined to the

mother. Cases are known in which the father has been the
carrier. Curiously, when the father carries the translocation, Deletion Errors
the empirical chance of having a Down syndrome child is Sometimes a piece of chromosome breaks off, resulting in a
only about 1 in 20. This may reflect a lack of viability of deletion of genetic material. The effects of the loss of a portion
chromosomally unbalanced sperm cells. of a chromosome depend on the particular genes lost. One
In trisomy 21 caused by nondisjunction, recurrence of of the earliest deletions noted with staining techniques was
Down syndrome in a given family is a rare event. Most the loss of a portion of the short arm of chromosome 5.
of the cases of nondisjunction Down syndrome are isolated Affected infants have a rounded, moonlike face and utter
occurrences in an otherwise normal family. In sharp con- feeble, plaintive cries described as similar to the mewing of
trast, translocation Down syndrome runs in families. When a cat, and the disorder is named cri du chat (French, “cat cry”)
a parent is a balanced carrier for a robertsonian transloca- syndrome.The cry disappears with time as the larynx improves
tion that involves chromosome 21, the risk of an affected, and is rarely heard after the first year of life. The facial features
translocation Down syndrome child among those developing also change with age, and the moon-shaped face becomes long
to term is 1 chance in 3. This is the theoretical expecta- and thin. Most patients survive beyond childhood, but they
tion. Empirically, from studies of actual family pedigrees, rank among the most profoundly retarded (IQ usually <20).
the chance is nearer to 1 in 10, the difference reflecting Examples of deletion syndromes are shown in Table 2-2.
decreased viability of the trisomic embryo. Nevertheless, Deletions of varied types, notably interstitial and terminal,
the situation is very different from nondisjunction trisomy, played a role in delineating the segment of chromosome 21
in which the risk of giving birth to an affected child is responsible for Down syndrome. Deletions of different seg-
about 1 in 700. About 5% of all Down syndrome children ments of one of the long arms of chromosome 21 in trisomy
have the translocation-type abnormality. 21 individuals (resulting in partial trisomy) have made it pos-
Another example of a balanced translocation occurs sible to identify the chromosome region responsible for the
between the terminal regions of chromosome 22 and chro- phenotypic features of Down syndrome. The “Down syn-
mosome 9. The new chromosome 22 is referred to as the drome critical region” has been identified as a 5- to 10-Mb
Philadelphia chromosome, reflecting where it was originally region of the chromosome and encompasses bands 21q22.2
described. The translocation event is dramatic because a to 21q22.3.
 26 Chromosomes in the Cell

TABLE 2-2. Some Deletion Syndromes Duplication and Inversion Errors

As noted in Chapter 1, genetic material may be duplicated
CLINICAL because of errors in replication and failure of repair mecha-
SYNDROME DELETION PRESENTATION nisms to function properly. The results of such errors can
occur in mitosis or meiosis but have greater consequences if
Angelman 15q11-13 Mental retardation, ataxia, they occur during gamete formation. Even sequences that are
uncontrolled laughter, outside coding sequences of expressed genes can have a pro-
seizures
Velocardiofacial 22q11 DiGeorge anomaly, found effect upon the function of genes. For example, fragile
characteristic facies, X disease has an amplification of triplet repeats within the
cleft palate, cardiac promoter region of the gene that can silence gene expression
defects if the number of repeats surpasses a threshold number.
Miller-Dieker 17p13.3 Lissencephaly, It has also been discussed that genetic material can be
characteristic facies
Prader-Willi 15q11-13 Mental retardation, moved from one location to another and that this movement
hypotonia, obesity, short may involve the centromere (Fig. 2-14). In most cases, these
stature, small hands and translocations result in inversions and the nucleotide sequence
feet is oriented in the opposite direction in its new location. Sig-
Smith-Magenis 17p11.2 Mental retardation,
nificant consequences can occur during meiosis when homol-
hyperactivity,
dysmorphic features, ogous chromosomes are misaligned, leading to unbalanced
self-destructive distribution of genes.
behaviors
Williams 7q1 Developmental disability,
characteristic facies,
supravalvular aortic
stenosis
Cri du chat 5p15.2 Mental retardation, KEY CONCEPTS
characteristic facies, ■ Mitosis occurs in somatic cells and meiosis occurs in germ cells.
characteristic high-
pitched cat-like cry ■ Recombination occurs in meiosis I prophase; meiosis II is similar
to mitosis.
■ Techniques for visualizing chromosomes generally rely on meta-
phase chromosomes.
■ Chromosome abnormalities are classified as structural or
11.32 numerical.

p 11.31 ■ Structural abnormalities are caused by translocations, deletions,

11.2 duplications, and insertions.
11.1 ■ Numerical abnormalities are caused by nondisjunction and
11.1
occasionally anaphase lag.
11.2
12.1
■ The larger the chromosome involved in an aneuploid presenta-
12.2 tion, the greater the phenotypic effect and the poorer the clinical
12.3 outcome.
q 21.1 ■ In mosaicism, the greater the number of cells with an abnormal
21.2
21.3
karyotype, the more severe the outcome.
■ Lyonization explains dosage compensation differences between
22 male and female embryos.

23

Normal Inverted
Chromosome 18
●●● QUESTIONS
Figure 2-14. Paracentric inversions of chromosome 18:
46,inv(18),(q11.2;q23). 1. A newborn female is the second child born to a
36-year-old mother and 47-year-old father. The infant
has a round face, low hairline, hypertelorism, epi-
canthal folds, up-slanting palpebral fissures, long
philtrum, high-arched palate, short and webbed
neck, small hands and feet, clinodactyly of the fifth
fingers, overlapping toes, and diastasis of the first
and second toes. There is no family history of a
 Questions 27

similar presentation. Which of the following proce- evokes an understanding of nondisjunction completely.
dures is recommended to establish a diagnosis? Penta-X requires three nondisjunction events to occur, two
A. Linkage analysis in the formation of one gamete and one in the formation
B. Expression analysis of the other gamete.
C. Gene analysis 3. The laboratory performed analysis of blood chemis-
D. Karyotype tries and enzyme levels on an infant with penta-X
E. Triple screen syndrome. What is the theoretical expectation for
Answer. D these results compared to normal newborn levels?
Explanation: This infant presents with characteristics similar A. Decreased
to Down syndrome. The most common presentation of B. Increased
Down syndrome is trisomy 21, and there is a correlation with C. Same
increasing maternal age and chromosome nondisjunction, D. Uninterpretable
which is the mechanism causing trisomy 21. The triple Answer. C
screen is a screening assay done during pregnancy that
Explanation: The results of this comparison are theoreti-
measures α-fetoprotein (AFP), human chorionic gonadotro-
cally the same. Recall that lyonization of additional X chro-
pin (hCG), and unconjugated estriol (E3). This test can be
mosomes occurs at the blastocyst stage. After this occurs,
informative for trisomy 21. Gene analysis is not a good
there is only one active X chromosome in cells. The detri-
choice because this child presents with many phenotypes
mental effects in the child are from expression of the extra
suggestive of a syndrome involving multiple genes and
chromosomes. In reality, there are some genes active only
systems. Likewise, expression assays look at what genes
from the inactive X and there are some genes that are acti-
are being expressed; many genes are expressed constitu-
vated and inactivated at differential times. The best answer,
tively, and sorting through what should be expressed versus
however, is that the expected expression of genes should
what is expressed in different syndromes would be tedious
be the same as in a normal newborn.
and time prohibitive. Linkage analysis with family informa-
tion is not likely to be informative because there is no family 4. Among gametes produced by an individual carrying
history of a similar presentation. Therefore, the karyotype is a chromosome 14/21 translocation, fewer infants are
the best option provided. Karyotyping, whether by Giemsa actually born with translocation Down syndrome
banding, fluorescent in situ hybridization, or spectral analy- than expected. Which of the following best explains
sis, is most effective in determining extra chromosomes or the discrepancy between observed and expected
rearranged chromosomes. findings?
A. In utero loss of fetuses with Down syndrome
2. A karyotype of a 1-week-old child revealed 49,XXXXX B. Increased viability of trisomy Down syndrome
in all cells and the child was diagnosed with penta-X C. Reduced fertilizing capacity of balanced gametes
syndrome with multiple congenital anomalies. Which D. Unbalanced gamete missing a chromosome 14
of the following is the most likely etiology of this E. Unbalanced gamete missing a chromosome 21
disease?
Answer. A
A. Chimera
B. Dispermy Explanation: Individuals who carry a translocation are gen-
C. Mosaicism erally not affected in any way because they have a balanced
D. Nondisjunction complement of chromosomes. However, in the formation of
E. Tetraploidy gametes, the translocation chromosome has trouble align-
ing at the metaphase plate in order to segregate into germ
Answer. D
cells. Figure 2-13 shows the gamete possibility for transloca-
Explanation: The extra chromosomes occur in this infant tion Down syndrome carriers. Shown is that the risk of having
because of nondisjunction during gamete formation. A an infant with Down syndrome is about 1 in 3 but the empiric
chimera is the fusion of two different cell lines and the risk is less about 1 in 10 to 1 in 20. Monosomies, other than
presence of two different karyotypes, which is not the the sex chromosome 45,X, generally do not survive. Option
case here since all cells have the same karyotype. In B is a true statement but it does not explain the discrepancy
humans, chimerism can occur in nonidentical twins when between what is expected (1 in 3) and what is observed
anastomoses of placental blood vessels occur. Likewise, when a parent is a translocation carrier. Option C is not
mosaicism is not a good choice because all cells were appropriate since the best capacity for fertilization is with
49,XXXXX. Dispermy occurs when two sperm fertilize an balance chromosomes. Options D and E are not good
ovum. When this occurs, there is an additional haploid options because these gametes yield a monosomy after
complement of chromosomes, not just extra sex chromo- fertilization, and neither alone fully explains the discrepancy
somes. Tetraploid cells have four chromosome sets, or in observed versus expected findings. Monosomies, other
96 chromosomes. Note that “ploidy” refers to sets of chro- than the sex chromosome 45,X, generally do not survive.
mosomes, whereas “somy” refers to chromosome, and
“aneuploid” means having extra or missing chromosomes. Additional Self-assessment Questions can be Accessed
In this particular case, penta-X syndrome is very rare but at www.StudentConsult.com
Mechanisms of 3
Inheritance
CONTENTS inheritance, mechanisms of unifactorial inheritance are often
called mendelian inheritance and the other mechanisms are
referred to as nonmendelian inheritance.
MENDELIAN INHERITANCE
Multifactorial inheritance is more complex because of the
Autosomal Dominant Inheritance variation of traits within families and populations. Individual
Autosomal Recessive Inheritance genes within a disease demonstrating multifactorial inheri-
X-Linked Recessive Inheritance tance may have a dominant or recessive inheritance pattern;
X-Linked Dominant Inheritance but when numerous nongenetic factors and genes interact to
Penetrance and Expressivity cause the disease, the mechanisms can be difficult to interpret
Late-Acting Genes
and explain.
NONMENDELIAN INHERITANCE

Triplet Repeats ●●● MENDELIAN INHERITANCE

Genomic Imprinting
Genes are found on autosomes and sex chromosomes, and
Mosaicism
Mitochondrial Inheritance
evidence for the existence of genes prior to the molecular
revolution was based on measurable changes in phenotype.
MULTIFACTORIAL INHERITANCE These changes resulted from allelic variation. Observing vari-
Phenotypic Distribution
ation depends on the relationship of one allele to another.
Liability and Risk The terms used to describe this relationship are dominant and
Risk and Severity recessive. If only one allele of a pair is required to manifest
Gender Differences a phenotype, the allele is dominant. If both alleles must be
Environmental and Epigenetic Factors the same for a particular phenotypic expression, the allele is
Characteristics of Multifactorial Inheritance recessive. This is described by the notations AA, Aa, and aa,
where “A” is dominant and “a” is recessive. The AA condition
is called homozygous dominant, Aa is called heterozygous,
and aa is called homozygous recessive.
One of the most remarkable characteristics of chromosomes Sex chromosomes also have alleles with dominant and
is the ability to sort precisely the genetic material represented recessive expression. However, this situation is different
in homologous pairs of chromosomes into daughter cells and because for males all X chromosome genes are expressed
gametes, as previously discussed. This assortment is recog- from the same single chromosome. Females have two X chro-
nized through the visible characteristics of individuals. This mosomes, but the scenario is different from that of autosomes
phenotype, or visible presentation of a person, is influenced because of lyonization.
by the expression of alleles at different times during develop- Variation in alleles results from mutations, and the effects
ment, at different efficiencies, and in different cells or tissues. of any mutation can influence the character and function
Observed differences are the result of a cell’s genotype, or of the protein formed. Many times a mutation will create
molecular variation in alleles. a protein with a recessive nature, but this is not always the
Mechanisms of inheritance generally refer to traits resulting case. Several mechanisms through which an allele can affect
from a single factor or gene, called unifactorial inheritance, or a function are shown in Table 3-1. These mechanisms are
from the interaction of multiple factors or genes, called multi- independent of mode of inheritance.
factorial inheritance. Because it is the simplest inheritance
pattern, unifactorial inheritance is the best understood. Gregor
Mendel first investigated this type of inheritance in his famous
Autosomal Dominant Inheritance
studies of garden peas in 1865. Because the underlying prin- Mendelian inheritance is classified as autosomal dominant,
ciples of Mendel’s work became hallmarks to understanding autosomal recessive, and X-linked (Box 3-1). A diagram
 Mendelian Inheritance 29

TABLE 3-1. Selected Mechanisms of Allele Action Box 3-1. EXAMPLES OF INHERITED DISORDERS

MECHANISM EXAMPLE Mendelian Nonmendelian

Autosomal dominant Triplet repeats
Loss of Gene product Waardenburg Achondroplasia Fragile X syndrome
function or activity is syndrome results Marfan syndrome Myotonic dystrophy
reduced from mutations in Neurofibromatosis Spinocerebellar ataxia
PAX3, a DNA Brachydactyly Friedreich ataxia
binding protein Noonan syndrome Synpolydactyly
important in Autosomal recessive Genomic imprinting
regulating Albinism Prader-Willi syndrome
embryonic Cystic fibrosis Angelman syndrome
development.
Phenylketonuria Mitochondrial
Gain of Gene product Charcot-Marie-Tooth
function is increased disease results Galactosemia LHON
from the Mucopolysaccharidoses MERRF
overexpression of X-linked dominant MELAS
PMP22 (peripheral Hypophosphatemic rickets
myelin protein) Orofaciodigital syndrome
caused by gene X-linked recessive
duplication. Duchenne/Becker type
Protein Normal protein Kennedy disease muscular dystrophies
alteration function is results from CAG Hemophilia A and B
disrupted (polyglutamine)
Glucose-6-phosphate
expansion at the 5′
end of the dehydrogenase deficiency
androgen receptor. Lesch-Nyhan syndrome
The mutant protein
misfolds,
aggregates, and
interacts abnormally
with other proteins,
hence their offspring and further descendants are not bur-
leading to toxic
gain of function and dened with the dominant trait.
alteration of normal There are numerous examples in humans of defective genes
function. that are transmitted in a dominant pattern. Achondroplasia,
Dominant Alleles are Retinoblastoma is a form of dwarfism, is inherited as an autosomal dominant
effects of recessive at inherited as a
trait. Achondroplasia is a congenital disorder, a defect present
recessive the molecular recessive allele. A
mutation level, but mutation in the at birth. Affected individuals are small and disproportionate,
show a second, normal with particularly short arms and legs. With an estimated fre-
dominant allele (also known quency of 1 in 15,000 to 40,000 live births, achondroplasia
mode of as the two-hit is one of the more common mendelian disorders. Most infants
inheritance hypothesis) results
affected by achondroplasia with two mutated alleles, repre-
in tumor formation.
senting a homozygous condition, are stillborn, or die in
infancy; heterozygous individuals surviving to adulthood
produce fewer offspring than normal. This observation under-
scores an important point for many autosomal dominant
representing family relationships is called a pedigree and can disorders—two mutated alleles often have severe clinical
be informative about inherited characteristics. Figure 3-1 consequences.
shows conventional symbols used in pedigree construction.
The family pedigree shown in Figure 3-2 has features sug- Characteristics of Autosomal
gesting autosomal dominant inheritance. Note that each Dominant Inheritance
affected person has at least one affected parent. Moreover, Guidelines for recognizing autosomal dominant inheritance
the normal children of an affected parent, when they in turn in humans may be summarized as follows:
marry normal persons, have only normal offspring. In this 1. The affected offspring has one affected parent, unless the
particular instance, the mutant allele is dominant and the gene for the abnormal effect was the result of a new
normal allele is recessive. In nearly all instances of dominant mutation.
inheritance, as exemplified by the pedigree, one parent carries 2. Unaffected persons do not transmit the trait to their
the detrimental allele and shows the anomaly, whereas the children.
other parent is normal. The affected parent will pass on the 3. Males and females are equally likely to transmit the trait
defective dominant allele, on average, to 50% of the children. to males and females.
Normal children do not carry the harmful dominant allele; 4. The trait is expected in every generation.
 30 Mechanisms of Inheritance

Normal female Mating

Normal male Consanguineous mating

Unknown sex, normal

Dizygotic twins (two eggs)

Affected female

Affected male
Monozygotic twins (one egg)

Affected child of unknown sex

Male heterozygote (carrier of recessive allele) Proband or propositus/proposita

Female heterozygote (carrier of recessive allele) I, II Roman numerals designate generation number

Spontaneous abortion or stillbirth

1, 2 Arabic numerals designate individuals
within generations
Deceased

Figure 3-1. Conventional symbols used in pedigrees.

I
1 2

II
1 2 3 4 5 6 7

III
1 2 3 4 5 6 7 8 9 10 11 12 13 14

Figure 3-2. Pedigree of a family with an autosomal dominant trait.

5. The presence of two mutant alleles generally presents with In other words, recessive disorders in family histories tend
a more severe phenotype. Detrimental dominant traits are to appear only among siblings and not in their parents.
rarely observed in the homozygous state. This is demonstrated by the family pedigree in Figure 3-3.
This pedigree shows that a normal male marries a normal
woman. Apparently, both were heterozygous carriers, since
Autosomal Recessive Inheritance
one of the four children (the first child, designated II-1)
A gene can exist in at least two allelic forms. For the sake exhibited the recessive trait. This son, although affected,
of simplicity, two will be considered—A and its alternative had two normal offspring (III-1 and III-2). These two chil-
(mutant) allele, a. From these two alleles, there are three dren must be carriers (Aa), having received the a allele
different genotypes, AA, Aa, and aa, that can be arranged from their father (II-1) and the A allele from their unaf-
in six types of marriages. These genotypes and their offspring fected mother (II-2). The genetic constitution of the mother
are listed in Table 3-2. The outcome of each type of mar- (II-2) cannot be ascertained; she may be either homozygous
riage follows the mendelian principles of segregation and dominant (AA) or a heterozygous carrier (Aa). The mar-
recombination. riage of first cousins (III-3 and III-4) increases the risk that
In the vast majority of cases of recessive inheritance, both parents of IV-1 and IV-3 have received the same
affected persons derive from marriages of two heterozygous detrimental recessive gene through a common ancestor.
carriers; affected individuals receive a mutant allele from In this case, the common ancestors are the parents in
each parent and represent homozygous recessive expression. generation I.
 Mendelian Inheritance 31

TABLE 3-2. Possible Combinations of Genotypes and Phenotypes in Parents and the Possible
Resulting Offspring

GAMETES

MATING TYPE FIRST PARENT SECOND PARENT OFFSPRING

Genotype Phenotype 50% 50% 50% 50% Genotype Phenotype

AA × AA Normal × normal A A A A 100% AA 100% Normal

AA × Aa Normal × normal A A A a 50% AA 100% Normal
50% Aa
Aa × Aa Normal × normal A a A a 25% AA 75% Normal
50% Aa 25% Abnormal
25% aa
AA × aa Normal × abnormal A A a a 100% Aa 100% Normal
Aa × aa Normal × abnormal A a a a 50% Aa 50% Normal
50% aa 50% Abnormal
aa × aa Abnormal × abnormal a a a a 100% aa 100% Abnormal

I
1 2

II
1 2 3 4 5 6 7

III
1 2 3 4 5 6

IV
Figure 3-3. Pedigree of a family with 1 2 3 4 5
an autosomal recessive trait.

It can be deduced from this pedigree that the daughter happen to mate, as seen in Figure 3-3. This explains cases in
(II-6) of the first marriage was a carrier (Aa). Her two children which a trait, absent for many generations, can suddenly
were normal, but it is noted that her first child (III-4) married appear without warning.
a first cousin (III-3), and from this marriage affected children Often only one member in a family is afflicted with a par-
(IV-1 and IV-3) were born. Accordingly, the daughter of the ticular disorder. In such an event, it would be an error to jump
third generation (III-4) must have been heterozygous, and in to the conclusion that the abnormality is not genetic solely
turn, her mother (II-6) was most likely heterozygous (or else because there are no other cases in the family. Without a
she married a heterozygous man). Similarly, the male involved positive family history, and sometimes the corroboration of
in the cousin marriage (III-3) must have been heterozygous, diagnoses, the occurrence of a single afflicted individual may
as was his father (II-3). represent a new, sporadic mutation.
Pedigrees of the above kind typify the inheritance of such
recessively determined traits as albinism, cystic fibrosis, and Characteristics of Autosomal
phenylketonuria. Special significance is attached to the het- Recessive Inheritance
erozygous carrier—the individual who unknowingly carries Guidelines for recognizing autosomal recessive inheritance
the recessive allele. It is usually difficult to tell, prior to mar- may be summarized as follows:
riage, whether the individual bears a detrimental recessive 1. Most affected individuals are children of phenotypically
allele. Thus, a recessive allele may be transmitted without any normal parents.
outward manifestation for several generations, continually 2. Often more than one child in a large sibship is affected.
being sheltered by the dominant normal allele. The recessive On average, one fourth of siblings are affected.
allele, however, becomes exposed when two carrier parents 3. Males and females are equally likely to be affected.
 32 Mechanisms of Inheritance

4. Affected persons who marry normal persons tend to

have phenotypically normal children. (The probability
BIOCHEMISTRY & PHYSIOLOGY
is greater of marrying a normal homozygote than a Hemoglobin
heterozygote.)
Hemoglobin is composed of heme, which mediates oxygen
5. When a trait is exceedingly rare, the responsible allele is
binding, and globin, which surrounds and protects the
most likely recessive if there is an undue proportion of heme. Hemoglobin is a tetramer of globin chains (two α
marriages of close relatives among the parents of the chains and two β chains in adults), each associated with a
affected offspring. heme. There are many variants of hemoglobin. In sickle cell
anemia, the β-globin chain is mutated and is known as
hemoglobin S (Hb S). A missense mutation causes valine to
Consanguinity and Recessive Inheritance be placed in the protein in place of glutamic acid.
Offspring affected with a recessive disorder tend to arise The mutation that causes Hb S produces oxygenated
more often from consanguineous unions than from marriages hemoglobin that has normal solubility; however,
of unrelated persons (see Chapter 12). Close relatives share deoxygenated hemoglobin is only about half as soluble as
normal Hb A. In this low-oxygen environment, Hb S
more of the same alleles than persons from the at-large popu-
molecules crystallize into long fibers, causing the
lation. If a recessive trait is extremely rare, the chance is very
characteristic sickling deformation of the cell. The deformed
small that unrelated marriage partners would harbor the same cells, which can disrupt blood flow, are responsible for the
defective allele. The marriage of close relatives, however, symptoms associated with sickling crises such as pain, renal
increases the risk that both partners have received the same dysfunction, retinal bleeding, and aseptic necrosis of bone,
defective allele through some common ancestor. Not all and patients are at an increased risk for anemia owing to
alleles are equally detrimental. Stated in another way, identi- hemolysis of the sickled cells.
cal alleles may produce an extreme phenotype, whereas two
different alleles of the same gene may appear mild or even
normal.
With increasing rarity of a recessive allele, it becomes
increasingly unlikely that unrelated parents will carry the
IMMUNOLOGY
same recessive allele. With an exceedingly rare recessive dis-
order, the expectation is that most affected children will come ABO Blood Groups
from cousin marriages. Thus, the finding that the parents of There are over 25 blood group systems that account for
Toulouse-Lautrec, a postimpressionist artist who documented more than 400 antigens on the surface of red blood cells.
bohemian nightlife, particularly at the Moulin Rouge in Paris, The ABO blood group is one of the most important, and the
were first cousins is the basis for the current view that the antigens expressed are produced from alleles of one gene.
French painter was afflicted with pycnodysostosis, character- There are three major alleles—A, B, and O—but more than
ized by short stature and a narrow lower jaw. This condition 80 have been described.
is governed by a rare recessive allele unlike achondroplasia, The ABO gene encodes glycosyltransferases, which
another form of short stature that is determined by a domi- transfer specific sugars to fucosyltransferase-1, also known
nant allele. Thus, it was more likely that Toulouse-Lautrec as the H antigen. The H antigen is a glycosphingolipid
consisting of galactose, N-acetylglucosamine, galactose,
suffered a rare disorder expressed as a result of his parents’
and fructose attached to a ceramide. Types A, B, and O
relatedness rather than a common disorder that could only
blood are produced from the same glycosyltransferase
be explained by a new mutation. gene; amino acid changes distinguish the types. The A
allele encodes α1,3-N-acetylgalactosamyl transferase, which
Codominant Expression adds N-acetylgalactosamine to the H antigen to form the A
In some heterozygous conditions, both the dominant and antigen. The B allele produces α1,3-galactosyltransferase,
recessive allele phenotypes are expressed. From a molecular which transfers galactose to the H antigen, thus forming the
viewpoint, the relationship between the normal allele and the B antigen. The O allele has no enzyme activity, but the H
mutant allele is best described as codominant. This means antigen is present on the cell surface.
that, at the molecular level, neither allele masks the expres-
sion of the other. An example of codominance is sickle cell
anemia. In this example, two types of hemoglobin are pro-
duced: normal hemoglobin A and a mutant form, called
X-Linked Recessive Inheritance
hemoglobin S. Another example is the expression of both A
and B antigens on the surface of red blood cells in individuals No special characteristics of the X chromosome distinguish it
with type AB blood. from an autosome other than size and the genes found on the
The terms dominant and recessive have little, if any, utility chromosome, but these features distinguish all chromosomes
when both gene products affect the phenotype. Dominance from each other. X chromosome inheritance, often called
and recessiveness are attributes of the trait, or phenotype, not X-linked or sex-linked, is remarkable because there is only
of the gene. An allele is not intrinsically dominant or reces- one X chromosome in males. Most of these alleles are there-
sive—only normal or mutant. fore hemizygous, or present in only one copy, in the male
 Mendelian Inheritance 33

I
1 2

II
1 2 3 4 5 6 7 8 9 10

III
1 2 3 4 5 6 7 8 9 10

IV

1 2 3 4 5 6 7 8 9 10

Figure 3-4. X-linked inheritance of V

hemophilia A among descendants of 1 2
Queen Victoria (I-2) of England.

because there are no corresponding homologous alleles on Characteristics of X-Linked

the Y chromosome, with the exception of those in the pseu- Recessive Inheritance
doautosomal pairing region (see Chapter 11, Fig. 11-1). Pres- Guidelines for recognizing X-linked recessive inheritance
ence of a mutant allele on the X chromosome in a male is may be summarized as follows:
expressed, whereas in the female a single mutant allele may 1. Unaffected males do not transmit the disorder.
have a corresponding normal allele to mask its effects, as 2. All the daughters of an affected male are heterozygous
expected in the situation of dominance versus recessiveness. carriers.
The special features of X-linked recessive inheritance are 3. Heterozygous women transmit the mutant allele to 50%
seen in the transmission of hemophilia A (Fig. 3-4). This is of the sons (who are affected) and to 50% of the daughters
a blood disorder in which a vital clotting factor (factor (who are heterozygous carriers).
VIII) is lacking, causing abnormally delayed clotting. Hemo- 4. If an affected male marries a heterozygous woman, half
philia exists almost exclusively in males, who receive the their sons will be affected, giving the erroneous impression
detrimental mutant allele from their unaffected mothers. of male-to-male transmission.
Figure 3-4 shows part of the pedigree of Queen Victoria
of England. Queen Victoria (I-2) was a carrier of the mutant X-Linked Inheritance and Gender
allele that either occurred as a spontaneous mutation in As noted, X-linked inheritance is distinguished by the pres-
her germline or was a mutation in the sperm of her father, ence of one chromosome in males but two in females. To
Edward Augustus, Duke of Kent. Queen Victoria had one explain the appearance of a condensed body in female
son (II-9) with hemophilia and two daughters (II-3 and cells, known as a Barr body, and to justify the possibility
II-10) who were carriers. The result of these children mar- of twice as many X chromosome gene products in females
rying into royal families in other countries was the spread as in males, the Lyon hypothesis was proposed. This hypoth-
of the mutant factor VIII allele to Spain, Russia, and esis, which has been become well established, recognizes
Germany. The children of II-3 have hemophilia in two more the Barr body in female cells as an inactivated X chro-
generations (III-7, IV-3, IV-5, and IV-10). The families of mosome. Through inactivation, dosage compensation occurs
II-9 and II-10 also revealed hemophilia through two more that generally equalizes the expression between males and
generations (not shown). Though the grandson of III-2 females.
married V-1, no hemophilia allele was introduced back into To review, lyonization suggests that (1) alleles found on the
the family of the first son of Queen Victoria, Edward VII, condensed X chromosome are inactive, (2) inactivation occurs
and the royal family of England has remained free of hemo- very early in development during the blastocyst stage, and (3)
philia. Generation V is represented by Queen Elizabeth and inactivation occurs randomly in each blastocyst cell. Lyoniza-
Prince Philip. tion is more complicated than this simplistic presentation
For alleles on the X chromosome, each son of a carrier because some alleles are expressed only from the inactive X
mother has a 50% chance of being affected by hemophilia, chromosome, other alleles escape inactivation and are
and each daughter has a 50% chance of being a carrier. expressed from both X chromosomes, and still other alleles
Hemophilic females are exceedingly rare, since they can only are variably expressed. It is easiest to understand X inactiva-
derive from an extremely remote mating between a hemo- tion as a random event or that about 50% of cells have the
philic man and a carrier woman. A few hemophilic women maternal X chromosome inactivated and about 50% of cells
have been recorded in the medical literature; some have have the paternal X chromosome inactivated; however, this
married and given birth to hemophilic sons. situation does not always occur. It is possible to have skewed
 34 Mechanisms of Inheritance

Figure 3-5. Inheritance of an X-linked dominant trait. Note that daughters always inherit the trait from an affected father,
whereas sons of an affected father never inherit the trait.

disease occurs in both males and females, and only one

I
mutant allele is required. As might be expected, heterozygous
1 2 females may be less affected than males because of the pres-
ence of a normal, nonmutated allele. The distinguishing
II feature between an X-linked dominant and an autosomal
1 2 3 4 5 6 disorder is that an autosomal mutation is transmitted from
males and females to male and female offspring. When a
III mutation is located on the X chromosome and expressed in
1 2 3 4 a dominant manner, females transmit the mutant allele to
both male and female offspring; however, males can only
Figure 3-6. Nonpenetrance in a family with an autosomal
dominant disorder. The light-colored boxes indicate transmit it to females (Fig. 3-5). In addition, affected females
individuals who do not express the phenotype for the may only transmit the mutant allele to 50% of offspring;
disorder, but who have the genotype for the disorder. males will transmit the mutant allele to 100% of females.

inactivation, whereby the X chromosome from one parent is

Penetrance and Expressivity
more or less likely to become inactivated. Depending on the Not every person with the same mutant allele necessarily
degree of skewing, the clinical presentation will be affected. manifests the disorder. When the trait in question does not
The more extreme the occurrence of skewing in favor of appear in some individuals with the same genotype, the term
keeping the mutant X active, the poorer the prognosis for the penetrance is applied. Penetrance has a precise meaning—
individual. namely, the percentage of individuals of a specific genotype
The onset of X inactivation is controlled by the XIST gene. showing the expected phenotype. If the phenotype is always
This gene is expressed only from the inactive X chromosome expressed whenever the responsible allele is present, the trait
and is a key component of the X inactivation center (XIC) is fully penetrant. If the phenotype is present only in some
found at the proximal end of Xq. The cell recognizes the individuals having the requisite genotype, the allele express-
number of X chromosomes by the number of XICs in the cell. ing the trait is incompletely penetrant. For a given individual,
In the presence of two X chromosomes, XIST is activated and penetrance is an all-or-none phenomenon; that is, the pheno-
RNA molecules are produced that bind to regions of the X type is present (penetrant) or not (nonpenetrant) in that one
chromosome, rendering it inactive. It is not known how some individual. In penetrant individuals, there may be marked
genes escape the influence of the RNA molecules and remain variability in the clinical manifestations of the disorder. When
active. more than one individual is considered, such as a population
of individuals, a percentage is usually applied to the propor-
tion of individuals likely to express a phenotype. To illustrate
X-Linked Dominant Inheritance this point, if a trait occurs with 80% penetrance, expression
Disorders resulting from X-linked dominant inheritance occur is expected in 80% of individuals with the trait.
far less frequently than other forms of inheritance. As noted, Nonpenetrance is a cul-de-sac for clinicians and genetic
X-linked recessive inheritance can occur, and males are counselors. Figure 3-6 demonstrates a pedigree with an auto-
almost always the affected gender, although in very rare cases somal dominant trait in which nonpenetrance is pervasive.
it is possible for females to acquire two mutant alleles or Individual II-2 most likely carries the disease allele, unless
express milder phenotypes as carriers. With X-linked domi- offspring III-2 arose from a new dominant mutation. The
nant inheritance, there are no carriers; expression of the future offspring III-4 is at risk for the dominant disease. The
 Nonmendelian Inheritance 35

calculated mathematical risk would take into consideration (Table 3-3). The effects of location may result in a loss of
the empirical penetrance percentage for the trait (say, 60%) function, as seen with fragile X syndrome. A gain of function
and the probability that a person from the general population is seen with amplification of CAG, resulting in polyglutamine
(spouse II-6) would harbor the disease allele. tracts that cause neurotoxicity in several other neurodegen-
Expressivity is the term used to refer to the range of phe- erative diseases. Finally, RNA can be detrimentally affected
notypes expressed by a specific genotype. This is much more if the expansion occurs within a noncoding region. In myo-
frequent than nonpenetrance. A good example of expressivity tonic dystrophy, the expanded transcript is unable to bind
is seen in neurofibromatosis (NF). NF consists of two disor- RNA proteins correctly for splicing and remains localized in
ders, NF1 and NF2, caused by mutations in different genes. NF the nucleus (see Chapter 8).
is an autosomal dominant disorder, and in both forms over During normal replication, when the double helix separates
95% of affected individuals have café-au-lait spots. Café-au- into small, single-stranded regions, secondary structures form
lait spots are flat, coffee-colored macules. The expressivity of with complementary and repeated sequences. These struc-
these spots, which resemble birthmarks, is variable and differs tures, represented as loops and hairpins, hinder the progres-
in number, shape, size, and position among individuals. sion of replication by DNA polymerase. An example is
(GAA)n/(TTC)n expansions that bind to each other. As a
result, the polymerase may dissociate either slightly or com-
Late-Acting Genes pletely. If its realignment or reassociation does not occur at
Proper interpretation of penetrance and expressivity may be the exact nucleotide where it should, DNA has slipped. Con-
complicated when the genes involved are expressed in the sequently, synthesis continues, but it may “resynthesize” a
adult rather than the child. These late-acting genes include short region, resulting in amplification. This amplified region
many genes involved with aging but may also include certain distorts the helical structure of DNA—a distortion under the
disease genes. Huntington disease is an inherited disorder surveillance of mismatch repair proteins. Ordinarily, proteins
characterized by uncontrollable swaying movements of the stabilize the DNA not matching the template strand into a
body and the progressive loss of mental function. The muta- loop that can be excised followed by repair and ligation of
tion in the gene is present at birth in all cells of the individual, any correct nucleotides inserted with the DNA strand. Mis-
but the effect of the protein is not evident until much later. match repair is the mechanism responsible for slippage repair.
The symptoms usually develop in an affected person between Failure of the mismatch repair mechanism to remove the
the ages of 30 and 45 years. Penetrance is 100%, there is no extra DNA does not imply a mutation of any of the repair
cure, and the progress of the disease is relentless, leading to proteins but rather an inability to adequately repair all regions
a terminal state of helplessness. No therapy can significantly involved in slippage. This suggests that triplet repeat amplifi-
alter the natural progression of the disease, and there are no cation may occur through events of large slippage that over-
states of remission. Death occurs typically 12 to 15 years after whelm the repair system, through unequal recombination, or
the onset of the involuntary, jerky movements. both. The mechanism by which DNA avoids repair during
amplification is unknown.
●●● NONMENDELIAN INHERITANCE
Some clinical presentations do not fit the classical patterns of BIOCHEMISTRY
mendelian inheritance and represent examples of nontradi-
tional or nonmendelian inheritance (see Box 3-1). These Hairpin Structure
include triplet repeats, genomic imprinting, mosaicism, and Hairpins are fundamental structural units of DNA. They are
mitochondrial inheritance. formed in a single-stranded molecule and consist of a
base-paired stem structure and a loop sequence with
unpaired or mismatched nucleotides. Hairpin structures are
Triplet Repeats often formed in RNA from certain sequences, and they may
The expansion of short tandem arrays of di- and trinucleo- have consequences in DNA transcription, such as causing a
pause in transcription or translation, that result in termination.
tides from a few copies to thousands of copies demonstrates
a type of mutation with the potential of having profound
effects on the phenotype of offspring through an unusual G
Loop
mode of inheritance. First demonstrated with fragile X syn-
drome, the expansion of triplet repeats is found in several
J J J J J

J J J J J

neurologic disorders. The expansion probably occurs as a G C

result of faulty mismatch repair or unequal recombination in
C G
a region of instability. The proximity of the region of instabil- Stem
ity to an allele is of paramount importance. Trinucleotide G C
repeats can be found in any region of gene anatomy: the
G C
5′-untranslated promoter region, an exon, an intron, or the
3′-untranslated region of the gene. Interestingly, trinucleotide C G
expansions in any of these regions can also result in disease
 36 Mechanisms of Inheritance

TABLE 3-3. Neurologic Disease Due to Triplet Repeat Amplification

CHROMOSOME NORMAL RANGE DISEASE RANGE

LOCATION/DISORDER LOCUS REPEAT (REPEATS) (REPEATS)

In the 5′ Untranslated Region

Fragile X-A Xq27.3 CGG in FMR1 gene 6–54 50–1500

Fragile X-E Xq28 CGG/CCG in FMR2 gene 6–25 200+

Within the Translated Region of the Gene

Spinobulbar muscular atrophy Xq21.3 CAG in androgen 13–30 30–62

(Kennedy disease) receptor gene
Huntington disease 4p16.3 CAG in HD gene 9–37 37–121
Spinocerebellar ataxia type 1 6p24 CAG in ataxin-1 gene 25–36 43–81
Spinocerebellar ataxia type 3 14q CAG in undescribed gene 13–36 68–79
(Machado-Joseph disease)
Dentatorubropallidoluysian 12p13.31 CAG in atrophin gene 7–23 49–88
atrophy (DRPLA)

In the 3′ Untranslated Region

Myotonic dystrophy 19q13.3 CTG in cAMP-dependent 5–37 44–3000

muscle protein kinase

In an Intron

Friedreich ataxia 9q13 GAA in the first intron of 7–20 200–900

the FRDA gene

A process known as unequal crossing-over, or recombina- expected. Neither males nor females show any outward signs
tion, may further amplify duplications. In this process, there is of fragile X syndrome. However, male carriers of the fragile
physical exchange of genetic material between chromosomes. X premutation are at a high risk for fragile X–associated
During meiosis, homologous chromosomes may mispair with tremor/ataxia syndrome (FXTAS), an adult-onset neurologic
each synapsis. Should a crossover event occur, the DNA disorder characterized by ataxia, intention tremor, short-term
breaks, an exchange occurs, and the DNA ends are ligated. If memory loss, atypical Parkinson disease, loss of vibration and
the exchange of chromosome material (i.e., DNA) is unequal, tactile sensation and reflexes, and lower limb weakness. Pen-
chromatids either lose or gain DNA (Fig. 3-7). For amplifica- etrance of this disorder increases with age. With the appear-
tions, the result is a gain of triplet repeats for one chromatid. ance of these features in this group of males (premutation
The presence of triplet repeats is not an abnormal condi- males occur at a frequency of 1 in 813), the premutation
tion. It is when the number of repeats reaches a threshold presentation is a more common cause of tremor and ataxia
number that disease is expressed (see Table 3-3). When the in men over age 50 (1 in 3000) than are other ataxia/tremor-
number of repeats remains stable in the absence of amplifica- associated disorders.
tion, or with limited amplification below a threshold number, Females with premutations are also reported with FXTAS,
a normal condition exists. Once amplification begins to occur, although the incidence is lower. Two additional effects seen
a premutation may exist in which some individuals, but not in these females are premature ovarian failure occurring
all, may express some symptoms. At this stage, amplification before age 40 and an increased incidence of dizygotic twins.
can proceed in the gametes of a premutation individual to a Women with full mutations do not experience these features,
full mutation in which all individuals are affected. Depending just as men with full mutations have a different constellation
on the gene affected and its chromosomal location, a triplet of physical features. Approximately 22% to 28% of women
repeat disease may demonstrate autosomal dominant, auto- in this group experience premature ovarian failure. Some
somal recessive, or X-linked expression. studies suggest the increase in twinning may be linked more
Unlike most X-linked or recessive disorders, the premuta- closely to premature ovarian failure than to the premutation
tion phenotype presents a different clinical image than itself.
 Nonmendelian Inheritance 37

Centromere

Sister CGG CGG CGG CGG CGGn

chromatids
CGG CGG CGG CGG CGGn
Homologous
Recombination chromosomes

CGG CGG CGG CGG CGGn

CGG CGG CGG CGG CGGn

Centromere

CGG CGG CGG CGG CGGn

Figure 3-7. Unequal crossover and CGG CGG CGG CGG CGG CGGn
sister chromatid exchange. A, One
chromatid of sister chromatids CGG CGG CGG CGGn
incorrectly pairs with its corresponding
sister chromatid. B, The outcome
CGG CGG CGG CGG CGGn
shows one chromosome gained DNA,
one lost DNA, and two remained the
same. B

A particularly interesting feature of triplet repeat amplifica- explanation dispelled previous beliefs that the disease was
tion is that, in many disease presentations, the amplification occurring earlier and with greater severity in successive
is parent specific during gametogenesis. This is the underlying generations because the mothers were worrying during
cause of confusion about its mode of inheritance. For fragile pregnancy and beyond, and somehow contributing to the
X syndrome, two elements contribute to the expression of disease etiology.
trinucleotide repeats and disease expression. First, expansions
tend to occur through female meiosis I gamete formation.
Second, males are more often affected than carrier females
Genomic Imprinting
due to X chromosome inactivation. This explains why in For most autosome genes, one copy is inherited from each
fragile X syndrome the sons of carrier females are more parent and generally both copies are functionally active.
affected than daughters and why offspring of carrier males There are some genes, however, whose function is dependent
do not express the disorder. The risk of mental retardation on the parent from whom they originated. Stated another
and other physical features depends on the position of an way, allelic expression is parent-of-origin specific for some
individual in a pedigree relative to a transmitting male. The alleles. This phenomenon is known as genomic imprinting.
daughters of normal transmitting males inherit the same Genomic imprinting differs from X chromosome inactivation
regions of amplification as are present in the transmitting in that the latter has a somewhat random nature and involves
father. most of the chromosome. Genomic imprinting involves
During oogenesis in the daughter of a normal transmitting specific alleles on particular chromosomes.
male, further amplification occurs that is inherited by sons DNA is imprinted through methylation, though the signal
and daughters. Because males carry only a single X chromo- for initiating this process is unknown. It is a reversible form
some, the effect is more pronounced than in females carrying of allele inactivation. During gametogenesis, most DNA is
two X chromosomes, one of which presumably is normal. demethylated to remove parent-specific imprints in germ
Females are therefore obligate carriers. The reverse occurs in cells. Remethylation then occurs on alleles specific to the
Huntington disease, in which amplification occurs preferen- sex of the parent (Fig. 3-8); some alleles are methylated
tially in meiotic transfer from the father. In either situation, specifically in the copy inherited from the father, inactivating
a molecular explanation now exists for the observation in that copy of the gene, while others are methylated specifi-
some neurologic disorders of an increase in disease severity cally in the maternally inherited copy. In females, methylation
through successive generations. Referred to as genetic antici- occurs prior to ovulation when oocyte development resumes.
pation, amplification of triplet repeats provides a scientific In males, imprinting in spermatogonia is less clear but prob-
explanation to allay fears in an affected family. This ably occurs at birth when spermatogonia resume mitosis.
 38 Mechanisms of Inheritance

A number of clinically important genetic diseases are asso-

ciated with imprinting errors. The first recognized genomic
imprinting disorder was Prader-Willi syndrome. It is also one
of the most common microdeletion syndromes and involves
at least 12 genes at the chromosome 15q11.2-q13 locus. At
least two of these are imprinted genes depending on the
parent of origin and hold special importance for Prader-Willi
A and Angelman syndromes: SNRPN and UBE3A, respectively.
Maternal somatic cells Paternal somatic cells The SNRPN gene, producing small nuclear ribonucleoprotein
N, is methylated during oogenesis but not spermatogenesis.
The UBE3A gene, producing ubiquitin ligase, is methylated
during spermatogenesis but not oogenesis (Fig. 3-9). As a
common microdeletion, or contiguous gene, syndrome,
B
deletion of a region of the paternal chromosome 15 results
Maternally imprinted gametes Paternally imprinted gametes in Prader-Willi syndrome because no SNRPN protein is
expressed from the imprinted maternal chromosome 15
SNRPN allele. Likewise, deletion of the same region from the
maternal chromosome 15 yields Angelman syndrome and not
Prader-Willi syndrome. SNRPN protein is produced in Angel-
man syndrome, but UBE3A protein is not expressed from the
imprinted paternal chromosome.
C
Zygote

BIOCHEMISTRY
Figure 3-8. Genomic imprinting. A, Somatic cells have
methylated alleles from a specific parent. B, At gamete Ubiquitin
formation, the imprint is removed and all alleles are Ubiquitin is a highly conserved, small protein of 76 amino
re-imprinted for the sex of the parent. C, When gametes form acids involved in protein degradation and found in all cells.
a zygote, parent-specific alleles are present. Blue is a It attaches to proteins targeted for degradation by
paternal imprint and pink is a maternal imprint.
proteasomes or occasionally lysosomes.
■ UBE1: ubiquitin-activating enzyme, which converts
However, it is clear that DNA methyltransferase expression
ubiquitin to a thiol ester
in the nucleus correlates with maternal and paternal imprint- ■ UBE2: family of carrier proteins
ing. Methylation remains throughout embryogenesis and ■ UBE3: protein ligase that binds ubiquitin to proteins
postnatally. The consequence of imprinting is that there is
only one functional allele for these imprinted genes. This
has significant clinical implications if the functionally active
allele is inactivated by mutation. Prader-Willi and Angelman syndromes occur from micro-
deletions in 75% to 80% of cases and can be detected by
FISH analysis. However, as seen in Figure 3-9, other mecha-
BIOCHEMISTRY nisms exist, including the possibility of mutations within the
DNA Methylation individual genes. These represent the major mutation mecha-
nisms. Gross deletion of the promoter and exon 1 of SNRPN
DNA methylation occurs by the addition of a methyl group
has been reported; most mutations reported in the UBE3A
to cytosine. With the presence of “CpG islands,” or regions
of adjacent cytosines and guanines in promoter regions, gene are nonsense mutations resulting in a nonfunctional
methylation of these cytosines is an important aspect of protein. Molecular analysis with restriction enzymes can
gene regulation. Promoter regions that are highly methylated reveal changes in methylation sites.
provide fewer readily available target sites for transcription Not all chromosomes have imprinted genes. In fact, only
factors to bind. Therefore, methylation is associated with nine chromosomes with imprinted alleles have been reported.
down-regulation of gene expression and demethylation is Most of the genes that are imprinted occur in clusters and
associated with up-regulation of gene regulation. Methylation probably number only a few hundred.
occurs in the presence of DNA methyltransferase, which Uniparental disomy (UPD) is responsible for approximately
transfers a –CH3 group donated by S-adenosylmethionine. 20% of Prader-Willi and Angelman syndromes and occurs
The –CH3 group is added to carbon 5 of cytosine and
when two copies of one chromosome originated from one
becomes 5-methylcytosine (m5C).
parent by nondisjunction. This differs from a complete hyda-
Barr bodies, the physical presentation of inactive X
chromosomes, are heavily methylated. Aberrant DNA tidiform mole, which receives an entire complement of chro-
methylation can lead to disease. mosomes from one parent and is incompatible with life. When
a homologous pair of chromosomes is inherited from a single
 Nonmendelian Inheritance 39

Normal

Chromosome 15

SNRPN UBE3A

Prader-Willi syndrome Angelman syndrome

Figure 3-9. Differences between Deletion

Prader-Willi and Angelman syndromes. (~75%–80%)
The genes SNRPN and UBE3A are
SNRPN UBE3A SNRPN UBE3A
shown to demonstrate the effect of
parent-specific methylation. Prader-Willi
and Angelman syndromes may occur
from a microdeletion of chromosome UPD
(~20%)
15q11.2-q13, uniparental disomy, or an
imprinting error. Deletion areas contain
SNRPN UBE3A SNRPN UBE3A
several genes (e.g., contiguous gene
sign/microdeletion). No transcription
occurs from a gene if an unmethylated Imprinting
allele is deleted and a methylated allele error
(~2%)
remains. Similarly, no transcription
occurs when both alleles are SNRPN UBE3A SNRPN UBE3A
methylated. Paternally derived
chromosomes are blue; maternally = Methylation
derived chromosomes are pink.

parent, consequences arise if some genes on the chromosome required. Between the ages of 1 and 6 years, the child develops
are imprinted and thus not expressed (see Fig. 3-9). As seen hyperphagia, leading to morbid obesity. Individuals have
in Prader-Willi and Angelman syndromes, UPD is a factor in short stature. Children have cognitive learning disabilities but
a significant number of cases. are generally only mildly mentally retarded. Their behaviors
Uniparental disomy occurs in Prader-Willi and Angelman are distinctive and characterized by tantrums, stubbornness,
syndromes when a gamete has two of the same chromosome manipulative behaviors, and obsessive compulsiveness, such
from nondisjunction of chromosome 15. Upon fertilization, as picking at sores. Both males and females demonstrate
trisomy 15 occurs but fetal demise is avoided through “rescue” hypogonadism and incomplete pubertal development with a
and loss of one of the three copies. Most of the time, normal high incidence of infertility. Other features include small
disomy is restored. However, about a third of the time UPD hands and feet, almond-shaped eyes, myopia, hypopigmenta-
occurs. Most nondisjunction occurs in maternal meiosis I. tion, and a high threshold for pain. Obesity can be managed
Therefore, the resulting UPD is a heterodisomy, or the pres- by diet and exercise to yield a more normal appearance.
ence of two different homologous chromosomes from a
parent, rather than an isodisomy, or the presence of two
chromosomes with identical alleles. If genomic imprinting
Mosaicism
exists on these chromosomes, genetic disease occurs. The The presence of cells with different karyotypes in the same
fetus may have escaped the consequences of trisomy but not individual is mosaicism. It arises from a mutation occurring
the necessity of fine regulation of gene expression. during early development that persists in all future daughter
Clinically, Prader-Willi and Angelman syndromes present cells of the mutated cell. If the mutation occurs early in devel-
quite differently. Angelman syndrome is characterized by opment, more cells as well as tissues will be affected; thus,
microcephaly, severe developmental delay and mental retar- clinical presentations are generally more pronounced the
dation, severe speech impairment with minimal or no use of earlier a mutation occurs.
words, ataxia, and flapping of the hands. Symptoms become Mosaicism may either be chromosomal mosaicism or germ-
apparent beginning around age 6 months and are fully evident line mosaicism. With chromosomal mosaicism, the presence
by age 1. Because affected individuals often have a laughing, of an additional chromosome or the absence of a chromo-
smiling facies, the term “happy puppet” was used in the past some from nondisjunction will create some trisomic or mono-
to describe them. somic cells. Monosomic cells are likely to die, but trisomic
Prader-Willi syndrome may first be apparent in utero, where cells may persist, yielding a clinical presentation less severe
the fetus is hypotonic and displays reduced movements. This than complete trisomy, in which all cells have an extra chro-
hypotonia is apparent at birth; feeding may be difficult owing mosome. This underscores an important concept about chro-
to a poor sucking reflex, and nasogastric feeding may be mosomal mosaicism: the more cells with an extra chromosome,
 40 Mechanisms of Inheritance

II

III

Figure 3-10. Pedigree suggesting a

IV
germline mutation in individual I-1 or
I-2.

II

III

IV

Figure 3-11. Mitochondrial inheritance. mtDNA is inherited from females only.

the more severe the clinical presentation. Mosaicism may also nucleus. These genes are contributed to offspring through
result from a less dramatic event than nondisjunction. A new gametes from each parent. Mitochondria also contain DNA
mutation may occur on a particular chromosome in some (mtDNA) that contribute genes to the process of cellular
cells that persists in some tissues but not necessarily all. If the energy production. Mitochondria, however, are contributed
expression of the mutated gene or region of chromosome to the zygote only from the maternal gamete and thus rep-
adversely affects the cells or tissues in which it is located, a resent a maternal inheritance pattern. Females always pass
more discrete effect will occur. If germ cells are not affected mitochondrial mutations to both sons and daughters, but
by chromosomal mosaicism, gametes will be normal and off- males never pass these mutations to their offspring (Fig.
spring will be unaffected. A minority of Down syndrome 3-11).
cases as well as many types of cancers are examples of mtDNA is a circular molecule that encodes 37 gene prod-
somatic mosaicism affecting chromosomes. ucts on 16.5 kb of DNA. There may be a few to thousands of
In germline mosaicism, the mutation is not in somatic cells mitochondria per cell. If all copies within a cell are the same,
and an individual is unaware of the mutation until an affected the cell is homoplasmic. In part owing to a very high sequence
offspring is born. All cells of the affected offspring will carry evolution rate, some mtDNAs may become mutated while
the mutation. Parental testing will not reveal the mutation others remain normal within the same cell. This situation, in
unless germ cells are tested. With one affected child, the which normal and mutated mtDNAs exist in the same cell, is
occurrence of a de novo mutation in the child cannot be termed heteroplasmy. Segregation of mtDNA during cell divi-
distinguished from a germline mosaicism. De novo mutations sion is not as precise as chromosomal segregation, and daugh-
are also called spontaneous mutations. However, the occur- ter cells may accumulate different proportions of mutated and
rence of the same mutation or condition in more than one normal mtDNA. The random segregation of mtDNA during
offspring is highly suggestive of a parental germline mutation mitosis may yield some cells that are homoplasmic or cells
(Fig. 3-10). Germline mosaicism is suspected in about one with variable percentage of heteroplasmy. For this reason,
third of young males developing Duchenne type muscular many members of the same family may have different propor-
dystrophy (see Chapter 7). tions of mutated mtDNAs. Unlike nuclear chromosomal
allele mutations demonstrating autosomal dominant, autoso-
mal recessive, or X-linked inheritance, a threshold of mutated
Mitochondrial Inheritance mtDNAs is generally required before a disease results. Typi-
All inheritance models, with the exception of mitochondrial cally, clinical manifestations result when the proportion of
inheritance, involve genes found on chromosomes in the mutant mtDNA within a tissue exceeds 80%. This threshold
 Multifactorial Inheritance 41

is tissue and mutation dependent. As a result, there is vari-

ability in symptoms, severity, and age of onset for most mito- Box 3-2. EXAMPLES OF MULTIFACTORIAL
chondrial diseases. Stated another way, both penetrance and INHERITANCE
expressivity are dependent on the degree of heteroplasmy
within an individual with a mitochondrial disease. Congenital Malformations Adult-Onset Diseases
Mitochondria are extremely important in producing ATP Cleft lip/palate Diabetes mellitus
through oxidative phosphorylation. It may then be intuitive Congenital dislocation of the hip Epilepsy
that those tissues with the highest energy requirements might Congenital heart defects Hypertension
be the most highly affected by mtDNA mutations. This also Neural tube defects Manic depression
Pyloric stenosis Schizophrenia
suggests that those tissues with the greatest energy demands
may also have a lower threshold for mtDNA mutations (i.e., a
lower proportion of heteroplasmy will result in disease). Mito-
chondrial diseases often involve muscle, heart, and nervous
tissues and present with central nervous system (CNS) abnor-
malities with or without neuromuscular degeneration. Exam-
ples of mitochondrial disease are Leber hereditary optic
neuropathy (LHON), mitochondrial encephalomyopathy with
lactic acidosis and stroke-like episodes (MELAS), and myo-
clonic epilepsy and ragged red fibers (MERRF) (see Chapter 7).
It is important to point out that mitochondrial diseases
have two different origins. Mutations within mtDNA lead to 66.7%
mitochondrial disease dependent on the degree of hetero-
plasmy in cells containing the mutation and exhibiting a
maternal inheritance pattern. A second type of mitochondrial
disease results from mutations in nuclear genes affecting the
expression and function of proteins required in mitochondria.
There are approximately 3000 of these proteins, and not all 62 64 66 68 70 72 74
have been identified. The criterion for distinguishing between Height in inches
the two forms of mitochondrial disease is that one is mater-
Figure 3-12. Height in adult males demonstrates a bell-
nally inherited and the other demonstrates mendelian pat- shaped curve as expected for multifactorial, polygenic traits.
terns of inheritance, the latter reflecting nuclear chromosome
expression. Risk to families with mitochondrial disease is dif-
ferent with the two modes of inheritance. The average, or mean, is 68 inches, with a standard deviation
of 2.6 inches. Standard deviation (SD) is a measure of the
variability of a population. Briefly, if a given population is
●●● MULTIFACTORIAL INHERITANCE normally distributed, then approximately two thirds of the
Many conditions are represented by a complex interaction of population lies within 1 SD on either side of the mean—in
several to many genes, and environmental factors may also this case, 68 − 2.6 and 68 + 2.6, or between 65.4 and 70.6
influence their expression. Individual alleles in this complex inches. Ninety-five percent of the individuals, or 19 in 20,
interaction may individually demonstrate any of the mende- may be expected to fall within the limits set by 2 SD on either
lian or nonmendelian inheritance patterns previously dis- side of the mean. Exceptionally short people (<62.8 inches)
cussed. However, the expression of these individual alleles is and exceptionally tall people (>73.2 inches) occupy the
dependent on other alleles and factors. Therefore, the under- extreme limits of the curve.
standing of these types of interactions and the diseases dem- The bell-shaped distribution characterizes traits such as
onstrating multifactorial inheritance is quite complex (Box height and weight in which there is continuous variation
3-2). Several examples will be discussed briefly to demon- between one extreme and the other. In regard to height,
strate the principles of multifactorial inheritance. A more those at the extremes of the curve—the exceedingly short and
detailed discussion of diabetes will ensue to illustrate a the exceptionally tall—are not generally recognized as having
disease with genetic and nongenetic influences that affects a disorder. An exceptionally tall person is not judged as
millions of individuals each year. having a clinical condition! In certain other situations,
however, those individuals at the tail of the distribution curve
are potential candidates for a congenital disorder such as
Phenotypic Distribution Marfan syndrome. The point in the distribution curve beyond
Many genes influence phenotypes such as height and weight. which there is a risk that a particular disorder will emerge is
As a result, the distribution of the many phenotypes demon- called the threshold level (Fig. 3-13). All individuals to the
strated by multifactorial inheritance is expected to form a left of the threshold level are not likely to have the disorder
bell-shaped curve. For example, the normal curve of distribu- and those to the right of the threshold value are predisposed
tion of heights of fully grown males is shown in Figure 3-12. to the disorder.
 42 Mechanisms of Inheritance

Frequency distribution in the population TABLE 3-4. Family Relationships and

Threshold
Distribution in those affected Shared Genes

PROPORTION OF
GENES IN COMMON
RELATIONSHIP TO A (COEFFICIENT OF
GIVEN SUBJECT RELATIONSHIP, R)
Frequency

Identical twin 1
Fraternal twin 1/2
First-degree relatives 1/2
Parent-child
Siblings
Second-degree relatives 1/4
Grandparent-grandchild
Uncle-nephew
Total liability (genetic and environmental)
Aunt-niece
Third-degree relatives 1/8
Figure 3-13. The threshold level is shown for the First cousins
continuous variation of a multifactorial, polygenic trait.

Liability and Risk

and the incidence of the disorder among first-degree relatives
The term liability expresses an individual’s genetic predispo- can be expected to be higher than in the general population.
sition toward a disorder and also the environmental circum- The distribution of second-degree relatives is also shifted to the
stances that may precipitate the disorder. As an analogy, in right, but in a direction less than that of first-degree relatives.
the case of an infectious disease, an individual’s susceptibility Third-degree relatives exhibit a distribution curve that tends to
to a virus or bacterium depends on inherent immunologic approximate that of the general population. Although first
defenses, but the liability also includes the degree of exposure cousins do not share as many genes as first-degree relatives, the
to the infective agent. In the absence of exposure to an infec- risk of a polygenically determined disorder is higher when the
tious virus or bacterium, the genetically vulnerable person parents are first cousins than when they are unrelated.
does not become ill. Likewise, in spina bifida, a strong genetic
predisposition renders the fetus susceptible or at a risk, but
the intrauterine environment may turn the risk into the
Risk and Severity
reality of the disorder. Environmental influences are thus The risk to relatives varies directly with the severity of the
superimposed on the multiple genetic determinants for high condition in the proband. Individuals with the more severe
risk. A condition such as spina bifida or cleft palate is often cases possess a higher number of predisposing genes and
referred to as a multifactorial trait, since it results from the accordingly tend to transmit greater numbers of risk genes.
interaction of both genetic factors involving multiple genes For example, for cleft lip, if the child has unilateral cleft, the
and environmental agents. risk to subsequent siblings is 2.5%. If the child has bilateral
The greater the number of risk genes possessed by the cleft lip and palate, the sibling risk rises to 6%. In the most
parents, the greater the probability that they will have an severe cases, the individual is at the extreme tip of the tail of
affected child. It also follows that the greater the number of the curve, having inherited a vast number of predisposing
risk genes in an affected child, the higher the probability that genes.
a sib will be affected. As a general rule, the closer the relation-
ship between two individuals, the greater the number of
genes that are shared by these individuals. Table 3-4 shows
Gender Differences
the proportion of genes that relatives have in common. A Both anencephaly and spina bifida occur more frequently in
parent and child share 50% of their genes, since the child females than in males. Anencephaly has a male-to-female
receives half of his or her genes from each parent. ratio of 1 to 2, while spina bifida approximates a male-to-
Figure 3-14 illustrates the liabilities of a disorder determined female ratio of 3 to 4. This suggests that there are sex-specific
by many genes, with a population incidence of 0.005, for rela- thresholds.
tives; the risk factors for relatives are 1, 5, and 10 times the Children of affected females with pyloric stenosis are more
general incidence for third, second, and first degree relatives, likely to be born with the pyloric stenosis than are children
respectively. On average, 50% of the genes of first-degree rela- of affected males. The threshold value for the female who is
tives (parents, children, and siblings) are shared with the affected is shifted to the left, with the consequence that the
affected individuals. The mean of the distribution for first- affected female possesses a large quantity of predisposing
degree relatives is shifted to the right. Thus, first-degree rela- genes required for the expression of the disorder. The affected
tives have more risk genes than does the general population, female imposes a greater risk to relatives, particularly to the
 Multifactorial Inheritance 43

male child or sibling, because of the larger number of predis-

Risk threshold
posing genes. The threshold level of the male is closer to the
population mean than that of the female. Strange as it may
seem, the less frequently affected sex, or the female, in the
case of pyloric stenosis, transmits the condition more often
General
population to the more frequently affected sex, or the male in this
example.

Environmental and Epigenetic Factors

Neural tube defects are multifactorial traits, reflecting a
A genetic predisposition that is polygenic, with a threshold
beyond which individuals are at risk of developing the
malformation if environmental factors also predispose. The
dietary intake of folic acid by women tends to protect their
First-degree fetuses against neural tube defects. Many other examples
relatives exist that correlate exposure to environmental factors during
critical periods of development with adverse outcomes. Reti-
noic acid embryopathy and gestational diabetes are two addi-
tional examples. Others are included in later discussions.

B
BIOCHEMISTRY
Folic Acid
Folic acid is a vitamin, a water-soluble precursor to
Second-degree
relatives tetrahydrofolate. It plays a key role in one-carbon
metabolism and the transfer of one-carbon groups. This
makes it essential for purine and pyrimidine biosynthesis as
well as for the metabolism of several amino acids. It is also
important for the regeneration of S-adenosylmethionine,
known as the “universal methyl donor.”
Folate deficiency is the most common vitamin deficiency
in the United States, presenting clinically as megaloblastic
C anemia. However, the group most likely to be deficient in
folate is women of childbearing age, whose deficiency
should be treated. Folic acid prevents neural tube defects
and is recommended for all women prior to conception
Third-degree and throughout pregnancy in doses ranging from 0.4 to
relatives 4.0 mg/day.

Epigenetic factors are those heritable changes that occur

without a change in the DNA sequence. Imprinting is one
of the major mechanisms contributing to epigenetics; the
D
other is histone acetylation. In the cases of Prader-Willi and
Figure 3-14. Risk factors, and therefore the risk threshold Angelman syndromes, described above, the sequence of an
for relatives, increase with degree of relatedness.
allele may be normal but the presence of methylation in the
form of parent-specific patterns determines whether the gene
is expressed or not. The presence of two alleles from a parent
with imprinted regions of chromosome 15, as in uniparental
disomy, has clinical consequences even though there is no
deletion or mutation.
Other studies of epigenetic factors, such as diet, stress, and
environmental toxins, are demonstrating that such factors
can have profound effects on offspring even two generations
 44 Mechanisms of Inheritance

after the event. Diet, stress, and environmental toxins have

long been considered environmental factors, but only more BIOCHEMISTRY
recently have effects been recognized beyond the generation
in which they are identified. For example, studies of families Insulin
who survived winters of famine demonstrate that their chil- Insulin is produced by the β-cells of the pancreatic islets of
dren and grandchildren live significantly longer than children Langerhans, which are found predominantly in the tail of the
and grandchildren of individuals who had experienced an pancreas. Insulin is translated as preproinsulin and cleaved
overabundance of food in the winter and developed gluttony to proinsulin in the endoplasmic reticulum. During Golgi
packaging, proteases cleave the proinsulin protein, yielding
in a single season. Other studies show that men who smoke
C peptide and two other peptides that become linked by
as prepubertal boys have sons with significantly higher body
disulfide bonds. This latter structure is mature insulin. C
mass indices, suggesting they will be at higher risk for obesity peptide has no function but is a useful marker for insulin
and other health problems as adults, which can lead to secretion, since these should be present in a 1 : 1 ratio.
shorter life spans. Because the liver removes most insulin, measurements of C
peptide reflect insulin measurements.
Insulin secretion is initiated when glucose binds to
Characteristics of GLUT2 glucose transporter receptors on the surface of
Multifactorial Inheritance β-cells and the glucose is transported into the cell, thereby
stimulating glycolysis. The increase in ATP or ATP/ADP
The unique characteristics of multifactorial inheritance as inhibits the ATP-sensitive membrane K+ channels, causing
depolarization and leading to the activation of voltage-
they pertain to certain congenital conditions are as follows:
gated Ca++ membrane channels. Calcium influx leads to
1. The greater the number of predisposing risk genes pos-
exocytosis and release of insulin from secretory granules
sessed by the parents, the greater the probability that they into the blood.
will have an affected child. In addition to this primary pathway, the phospholipase C
2. Risk to relatives declines with increasingly remote degrees and adenylyl cyclase pathways can also modulate insulin
of relationship. secretion. For example, glucagon stimulates insulin via the
3. Recurrence risk is higher when more than one family adenylyl cyclase pathway by elevating cyclic AMP (cAMP)
member is affected. levels and activating protein kinase A. Somatostatin,
4. Risk increases with severity of the malformation. however, inhibits insulin release by inhibiting adenylyl
5. Where a multifactorial condition exhibits a marked differ- cyclase.
ence in incidence with sex, the less frequently affected sex
has a higher risk threshold and transmits the condition
more often to the more frequently affected sex.

Diabetes
Diabetes mellitus (DM) is an example of a complex disease
that is not a single pathophysiologic entity but rather several
distinct conditions with different genetic and environmental
etiologies. Individuals with two fasting glucose levels of PHARMACOLOGY
126 mg/dL or greater are considered to have diabetes melli-
Insulin Therapy
tus. Two major forms of DM have been distinguished: type 1
and type 2. Note that, unlike other diseases that typically use First-line therapies for type 2 diabetes mellitus are “insulin
Roman numerals to designate disease types, Arabic numerals sensitizers” such as the thiazolidinediones and metformin.
Insulin is used when this first approach fails to completely
are used for diabetes.
resolve the situation. Exogenous insulin, used for type 1 and
Type 1 has been referred to by obsolete expressions
type 2, can be administered intravenously or intramuscularly.
such as “juvenile-onset diabetes,” “ketosis-prone diabetes,” For long-term treatment, subcutaneous injection is the
“insulin-dependent diabetes mellitus,” and “brittle diabetes.” predominant method of administration.
Type 2 has been called “maturity-onset diabetes,” “ketosis- Several aspects of subcutaneous injection of insulin differ
resistant diabetes,” “non–insulin-dependent diabetes mel- from its physiologic secretion. The kinetics of the injected
litus,” and “stable diabetes.” Type 1 DM is predominantly form of insulin does not parallel the normal response to
a disease of whites or populations with an appreciable nutrients. Insulin from injection also diffuses into the
white genetic admixture and is characterized by β-cell peripheral circulation instead of being released into the
destruction in the pancreas. Type 2 DM is the more preva- portal circulation.
lent type, comprising 80% of the cases; it is characterized Preparations are classified by duration of action: short,
intermediate, or long-acting.
by insulin resistance and an insulin secretory defect in
■ Short: lasts 4 to 10 hours (insulin lispro/insulin aspart,
β-cells. In the United States, the prevalence of type 1 is
regular insulin)
about 1 in 400 by age 20. Over 95% of affected individu- ■ Intermediate: lasts 10 to 20 hours (NPH insulin)
als develop the disease by age 25 years. The mean age of ■ Long-acting: lasts 20 to 24 hours (insulin glargine)
onset is approximately 12 years.
 Multifactorial Inheritance 45

The two broad categories of DM are separable on the basis Children of a diabetic father have a greater liability to type
of several observations, such as mean age of onset, the asso- 1 DM than children of a diabetic mother. By the age of 20,
ciation with certain genes within the major histocompatibility 6.1% of the offspring of diabetic fathers had diabetes, whereas
complex (MHC), the presence of circulating islet cell antibod- only 1.3% of the offspring of diabetic mothers had the
ies, and the predisposition of β-cells to destruction by certain disease. Hence, type 1 is transmitted less frequently to the
viruses and chemicals. Affected individuals with type 1 DM offspring of diabetic mothers than to those of diabetic fathers.
may have some family history of type 1 DM, gluten enter- The mechanism responsible for the preferential transmission
opathy, or other endocrine disease. Most of these patients is not clear.
have the “immune-mediated form” of type 1 diabetes mel- In essence, the low incidence of hereditary transmission of
litus with islet cell antibodies and often have other autoim- type 1 DM suggests the intervention of one or more critical
mune disorders such as Hashimoto thyroiditis, Addison environmental insults. One hypothesis suggests that type 1
disease, vitiligo, or pernicious anemia. Evidence supports the requires two hits, analogous to the two hits required in the
view that early-onset type 1 is an autoimmune disease in development of some cancers. The first hit is an infection, and
which insulin-producing β-cells of the pancreas are ultimately the second hit is the selection of self-reactive T cells, which
and irreversibly self-destroyed by autoreactive T lympho- is influenced genetically through the MHC. The incisive ques-
cytes. Individuals with type 2 DM are more often women, tions are: What are the nongenetic (environmental) factors
older than age 40, and experience obesity. Type 1 and type 2 that trigger type 1, and how do they interact with the genetic
DM are genetically distinct, inasmuch as type 2 is not known factors?
to be associated with any particular human leukocyte antigen
(HLA) haplotype. Monozygotic Twin Studies
There is another form of diabetes termed type 1B, also To elucidate the role of genetic and environmental factors in
called idiopathic or type 1.5 diabetes. These individuals are the etiology of diabetes, pairs of identical (monozygotic)
initially responsive to medication because they have adequate twins have been studied. Theoretically, if diabetes is influ-
insulin production but gradually develop insulin resistance. enced strongly by inherited factors and one identical twin
Antibodies develop and more slowly destroy pancreatic β- manifests the disease, the other would be expected to display
cells. This explains those individuals who are diagnosed with the disease. The extent of genetic involvement is estimated
type 2 DM who gradually become insulin resistant and may from the degree of concordance (both twins developing dia-
become ketosis prone. Individuals of African, Hispanic, or betes) as opposed to discordance (only one twin developing
Asian descent are more likely to develop type 1B DM. diabetes).

ANATOMY ANATOMY & EMBRYOLOGY

Pancreas Twins and Fetal Membranes

The pancreas is a retroperitoneal organ except for the tail, Monozygotic (MZ) twins are identical twins that originate
which projects into the splenorenal ligament. It is an from one zygote, a process that usually begins during the
exocrine gland and produces digestive enzymes. It is also blastomere stage. Dizygotic (DZ) twins are fraternal twins
an endocrine gland and produces insulin and glucagon. The that originate from two zygotes.
main pancreatic duct joins the bile duct, which runs through The type of placenta depends on when twinning occurs.
the head of the pancreas, to form the hepatopancreatic Most MZ twins have monochorionic-diamniotic placentas
ampulla that enters the duodenum. (65% to 70%). If twinning occurs later (9–12 days after
fertilization), then monochorionic-monoamniotic placentation
may occur, but this is rare (1%). In this latter case, twin-to-
twin transfusion syndrome can occur. If twinning occurs after
Family Studies day 12, separation is incomplete and conjoined twins result.
Type 2 DM is more highly associated with familial diabetes DZ twins have dichorionic-diamniotic placentas, most of
which are separate (60%). If implantation sites are close,
than type 1 DM. Most studies show that at least one third of
placentas may fuse (40%). Since DZ twins occur more
the offspring of type 2 parents will exhibit diabetes or abnor-
frequently than MZ twins, the most prevalent placentation is
malities in glucose tolerance in late life. Specifically, the prev- dichorionic-diamniotic.
alence of type 2 among children of type 2 parents is 38%,
compared with only 11% among normal controls. In sharp
contrast, familial aggregation of type 1 is less common. The
usual finding in family studies is that 2% to 3% of the parents In a study of 100 pairs of identical twins for type 2 DM, it
and 7% of the siblings of a proband with type 1 have diabetes was found that, when one twin of a pair developed diabetes
(Table 3-5). Stated another way, the likelihood that a parent after age 50, the other twin developed the disease within
with type 1 DM will have a child with type 1 is only 2% to several years in 90% of cases. Thus, older (i.e., >50 years)
3%. If one child has type 1, the average risk that a second identical twins are usually concordant for type 2 DM. The
child will have type 1 is only 7%. very high concordance rate for late-onset type 2 is impressive
 46 Mechanisms of Inheritance

The DQ locus consists of two tightly linked genes:

TABLE 3-5. Lifetime Risk for Type 1 Diabetes DQA1 and DQB1. These encode α and β chains. Both
Mellitus in First-degree Relatives* loci are highly polymorphic. There are 8 and 15 major
allelic variations in DQA1 and DQB1, respectively. Alleles
RELATIVE RISK (%) at both loci demonstrate susceptibility to type 1 DM.
Certain DQ alleles that are usually inherited in conjunc-
Parent 2.2 ± 0.6 tion with DR3 and DR4 are recognized as prime sus-
Children 5.6 ± 2.8 ceptibility alleles. In white patients, DR3 and DR4 are
Siblings 6.9 ± 1.3
HLA nonidentical sib 1.2 almost universally associated with the DQB1*0302 and
HLA haploidentical sib 4.9 DQB1*0201 antigens.
HLA identical sib 15.9 It is clear that both HLA-DQA1 and HLA-DQB1 alleles
Identical twin 30–40 are important in establishing a susceptibility to diabetes.
General population 0.3 DQA1*0501-DQB1*0201 and DQA1*0301-DQB1*0302
Data from Harrison LC. Risk assessment, prediction and prevention of type 1 haplotypes, representing closely linked markers that are
diabetes. Pediatr Diabetes 2001;2:71–82. inherited together, confer the highest risk for type 1 DM.
*When diagnosed in the proband before age 20 years.
In combination, their effect is even stronger than that
observed for individuals homozygous for DQA1*0501-
DQB1*0201 or DQA1*0301-DQB1*0302, suggesting that
in that the diabetic condition arises at a time when twins heterodimers formed from gene products in trans confor-
usually live apart and ostensibly share fewer environmental mation (i.e., DQA1*0501 and DQB1*0302) may be par-
factors than during early childhood. The twin studies support ticularly diabetogenic. Other DQ haplotypes conferring a
the hypothesis that type 2 is determined primarily by genetic high risk for type 1 DM include DQA1*0301-DQB1*0201
factors. among blacks, DQA1*0301-DQB1*0303 in the Japanese,
On the other hand, when one twin developed the disease and DQA1*0301-DQB1*0401 in the Chinese. The DQA1*
before age 40, the other twin developed the disease in only 0102-DQB1*0602 haplotype is protective and is asso-
half the cases. Accordingly, younger (i.e., <40 years) identical ciated with a reduced risk for type 1 DM in most
twins are 50% discordant for type 1 DM—that is, if one has populations.
type 1, the other does not and shows no signs of developing
it in half the cases. These findings demonstrate that genetic
factors are predominant in type 2, and additional factors,
presumably environmental, are required to trigger type 1 DM. Autoimmunity
Type 1 DM is an autoimmune disease. Sera from newly diag-
HLA Studies nosed type 1 patients contain antibodies that react with the
Studies in several laboratories have revealed a strong associa- β-cells in the islets of Langerhans taken from normal, nondia-
tion between type 1 DM and HLA antigens at the DR locus betic individuals. Type 1 DM represents the culmination of a
of the MHC. The major antigens conferring enhanced risk to slow process of immune destruction of insulin-producing β-
type 1 are DR3 and DR4. Indeed, 95% of white patients with cells (Fig. 3-15) and is also classified as an HLA-associated
type 1 express either DR3 or DR4, or both. Individuals who autoimmune disease.
express both DR3 and DR4 antigens are at the highest risk, What triggers the production of antibodies against the
whereas DR2 and DR5 expression is uncommon in type 1. pancreatic β-cells? A promising hypothesis is that the
The DR3 and DR4 alleles are not in themselves diabetogenic antibody is the remnant of an immune response to com-
but, rather, are markers for the true susceptibility allele in ponents of the islet cells that were altered or damaged
the HLA region. by viruses. An intriguing association suggests a viral trig-
gering event from the observation that 20% of all chil-
dren with congenital rubella—primarily those who are
DR3 positive or DR4 positive—become diabetic later in
life. This form of diabetes may be a consequence of the
IMMUNOLOGY widespread effects of congenital rubella on the immune
system.
Human Leukocyte Antigens
Whatever triggering event may be operative, it is clear that
Human leukocyte antigens (HLAs) are alloantigens important destruction of insulin-producing cells is a slowly developing
for maintaining tolerance, and they serve as antigen- process, not an acute one. There is definitive evidence that T
presenting receptors for T lymphocytes. HLA genes are lymphocytes are the major determinants of this process.
clustered on chromosome 6p. Class I proteins such as
Essentially, then, the current popular theory of the pathogen-
HLA-A, HLA-B, and HLA-C are each independent allele
esis of type 1 DM encompasses β-cell damage by a foreign
products. Class II proteins such as HLA-D (DP, DQ, DR) are
formed from admixing maternal and paternal allele products. viral antigen, activation of the immune system, and the sub-
Each person has one haplotype from each parent. sequent induction of autoimmunity directed against the
β-cells.
 Multifactorial Inheritance 47

autoimmunity, preventing nonlymphoid cells from presenting

IMMUNOLOGY their own proteins as antigens. If pancreatic cells were to
express class II molecules inadvertently, they could cause an
Autoimmunity autoimmune response via T cells.
Autoimmunity is loss of self-tolerance in humoral or cellular What triggers the expression of class II antigens in the
immune function. Helper T (TH) cells are the key regulators pancreatic cells? A promising hypothesis is that the produc-
of immune responses to proteins and are MHC restricted. tion of class II molecules is the consequence of an immune
Major factors contributing to autoimmunity are genetic response to pancreatic cells, specifically to islet β-cells, that
susceptibility and environmental triggers. Autoimmune
have been altered or damaged by viruses. A viral infection
diseases may be systemic, as seen in systemic lupus
insult activates, in some manner vaguely understood, the
erythematosus, or organ specific, as demonstrated by type
1 diabetes mellitus.
pancreatic cells to express class II molecules (see Fig. 3-15).
A plausible scenario is that a viral protein shares appreciable
amino acid sequences with a pancreatic islet protein—an
instance of molecular mimicry.
When the pancreatic cells are abnormally triggered to
IMMUNOLOGY express class II molecules, they can then present their anti-
gens to helper T cells, just like macrophages. Stated another
Lymphocytes
way, the pancreatic cell protein receptor alongside the class
Lymphocytes are responsible for antigen recognition. B II molecule forms a functional unit capable of interacting with
lymphocytes—antibody-producing cells—make up 10% to
helper T cells. The outcome is a large-scale activation of T cells
15% of circulating lymphocytes. Antigen recognition is
and a cascade of effects that include the production of circu-
accomplished by antibodies.
T lymphocytes recognize antigens on antigen-presenting lating antibodies by plasma cells specifically directed against
cells and make up 70% to 80% of circulating lymphocytes. the surface receptors on the pancreatic B cells and other
Most T cells are distinguished by the presence of CD4 or components.
CD8 glycoproteins on their surface that determine function. Viruses may be only one of many triggering agents of type
CD8+ molecules, expressed on most cells, bind class I 1 DM. Other environmental insults such as drugs and toxic
histocompatibility molecules. CD4+ molecules bind class II chemicals might similarly damage β-cells and give rise to
histocompatibility molecules and are present on antigen- diabetes. In experimental animals, drugs such as alloxan and
presenting cells such as B cells, macrophages, and streptozotocin can induce diabetes by destroying β-cells. In
dendritic cells. CD8+ T lymphocytes are cytotoxic killer cells, 1975, a rodent poison known as Vacor, which has a molecular
while other lymphocytes produce interferons, tumor necrosis
structure resembling that of streptozotocin, was introduced in
factor, and interleukins. CD4+ T lymphocytes, also known as
the United States. It was accidentally ingested by a number
T helper cells, produce cytokines and are important in
cell-mediated and antigen-mediated immunity. of people, several of whom developed acute diabetes with
clear evidence of β-cell destruction. Not all of these people
developed diabetes, indicating that the environmental insult
interacts with a complex genetic background, which can be
Several studies have identified susceptibility genes for dia- protective.
betes. As noted, type 1 DM is associated with the HLA region
of chromosome 6. For type 2 DM, which is the most prevalent Type 2 Diabetes Mellitus
form of diabetes, several susceptibility genes have been iden- As stated earlier, type 2 DM has a greater genetic component
tified in different groups, including Mexican Americans, an than does type 1 in that concordance for type 2 among
isolated Swedish population living in Bosnia, Pima Indians in monozygotic twins approaches 100%, depending on the twin
the southwest United States, and Utah families of European cohort. Yet environmental factors also play a role; ironically,
descent. Each study identified different genes specific to that environmental factors are better known in type 1 than in
population. These data suggest that different combinations of type 2.
susceptibility genes have different effects within populations Type 2 most often occurs in individuals who are over age
and increase the incidence of disease within individuals and 40 and overweight. Obesity facilitates expression of the
populations. genetic predisposition to type 2. The changes in lifestyle that
result in both obesity and type 2 are vividly exemplified by
Molecular Mimicry the urbanization of the Pima Native Americans of Arizona.
There is evidence that a defect in the expression of HLA- The exceptionally high prevalence of type 2 among the Pima
directed class II molecules may establish the conditions for (affecting 50% of the adult population) reflects a modern
autoimmune disease. Class II molecules, which enable T cells change in dietary pattern from low caloric intake, in which
to perceive antigen, are normally expressed on antigen- both obesity and diabetes were rare, to caloric abundance, in
presenting cells that interact with helper T cells—namely, which both clinical conditions are common.
dendritic cells, macrophages, and B cells. The usual inability A susceptibility gene among the Pima Indians is that for
of nonlymphoid cells, such as pancreatic cells, to express class calpain-10, a protease that regulates the function of other
II surface markers apparently serves as protection against proteins. It is composed of 15 exons and undergoes
 48 Mechanisms of Inheritance

1. Virus infects b-cells in the

pancreatic islets

2. T-lymphocyte infiltration
and recognition of foreign
antigens on infected cells Infected islets
and local APC
Figure 3-15. Process depicting
destruction of insulin-producing β-cells
in a hypothetical model of viral-induced
3. Up-regulation of HLA class II
islet cell autoimmunity. Infection of the
T on surviving b-cells by IFNg
CD4 pancreatic islet by a virus (e.g.,
T coxsackie B4 or cytomegalovirus) may
T lead to a robust intra-islet
Periphery T-lymphocyte–mediated response. As a
T T 4. Selection and expansion
result of T-lymphocyte infiltration, local
T CD8 inflammation, and/or interferon (IFN)
of autoreactive THelper
clones secretion, induction of HLA class II
T
expression on the β-cell is enhanced,
Drain to leading to the selection of T-lymphocyte
local node clones. Through mimicry, reactivation of
these T-lymphocyte clones occurs when
5. Autoreactive B lymphocyte
acquires T-cell help
antigen-presenting, autoreactive B
lymphocytes capture and present
B specific β-cell antigens released from
T the damaged islet. The specific B/T-
lymphocyte interaction provides
Clonal selection co-stimulation and avoids anergic
Cytokines
Affinity maturation deactivation of autoreactive B cells. As
these clones survive and expand,
b-Cell–specific islet-specific autoantibodies accumulate
6. Autoantibody binds B in the circulating immunoglobulin pool.
to surviving b-cells This view is supported by studies of
and insulin High-affinity B-lymphocyte
differentiation to a plasma high-risk subjects showing that
cell that secretes antibodies antibodies to candidate autoantigens
may exist long before disease
develops. The presence of islet
immunity, however, does not
T-cell receptor Insulin
HLA class II necessarily imply loss of β-cell function.
(Courtesy of Dr. Ronald Garner, Mercer
University School of Medicine.)

differential splicing to form at least 8 different proteins These same individuals have a lower metabolism and increased
expressed in a tissue-specific manner. Calpain-10 is found insulin resistance suggestive of mild diabetes, characteristics
only in pancreatic islet cells. A specific A-to-G mutation in that also increase obesity. Calpain-10 itself does not cause
intron 3, referred to as UCSNP-43 (for University of Chicago diabetes, but it does interact with other factors such as diet
single nucleotide polymorphism 43), increases the risk for and exercise to cause diabetes. These mutations have also
diabetes. Two other mutations, UCSNP-19 in intron 6 and been found in other populations and when present increase
UCSNP-63 in intron 13, also affect risk. Two mutated UCSNP- the risk for diabetes.
43 alleles and two different alleles at the other two sites are Molecular analyses of the insulin gene and an adjacent
associated with the greatest risk for developing diabetes. The large, “hypervariable” region proximal (5′) to the gene itself
presence of two different DNA sequences at three sites in have revealed an array of mutational events, but thus far it
the same gene allows for eight different combinations of has been difficult to associate most known nucleotide changes
sequences. It is hypothesized that these alterations affect with specific physiologic mechanisms. It can be asserted that
expression in different tissues: the UCSNP-43 alleles alter the risk for transmission of type 2 DM is greater than that for
calpain-10 expression in the pancreas and the other alleles type 1 because of the need of an environmental stress or
affect expression in muscle or fat cells. insult to the β-cells. For first-degree relatives, the risk is 10%
Pima Indians with two UCSNP-43 mutations but without to 15%; the risk of impaired glucose tolerance, which is the
diabetes produced 53% less calpain-10 mRNA in muscle. usual precursor of type 2, is 20% to 30%. A good case can
 Multifactorial Inheritance 49

TABLE 3-6. Comparison of MODY Types

MUTATION
MODY TYPE GENE PROTEIN PROTEIN FUNCTION* EFFECT PREVALENCE

1 HNF4A Hepatocyte nuclear factor–4α Transcription factor

2 GCK Glucokinase Phosphorylates glucose Common
3 HNF1A Hepatic nuclear factor–1α Transcription factor Most common
4 IPF1 Insulin promoter factor-1 Transcription factor Loss of function
5 HNF1B Hepatic nuclear factor–1β Transcription factor
6 NEUROD1 Neurogenic differentiation Transcription factor
factor–1

*Each of these transcription factors is involved in the regulation of the insulin gene through a complex process affecting the gene directly or through regulation of
each other. Thus, mutations decrease transcription, leading to increased blood glucose. Ultimately, complete β-cell failure occurs.

be made for periodic screening of first-degree relatives with Glucose Levels in Two Subtypes of MODY
oral glucose tolerance tests: those with impaired tolerance Glucokinase and Transcription Factor Deficiencies
should be advised to maintain ideal body weight. In a minor-
Transcription
ity, but a significant percentage, of families, type 2 occurs 20 factor (HNF-1a) mutation
without the precondition of obesity. In those families, type 2
is probably caused by a different mechanism. 16
Glucose (mmol/L)

Maturity-onset Diabetes of the Young 12

A small subset, representing about 2% to 5% of individuals
Glucokinase
with diabetes, have maturity-onset diabetes of the young 8 mutation
(MODY). As the oxymoronic name suggests, this form of
Normal
disease resembles “normal” type 2 DM but can be present in 4
young adulthood, usually occurring before age 25 as opposed
to after age 40. Unlike types 1 and 2 diabetes, which represent 0
complex inheritance along with environmental factors, MODY 0 20 40 60 80 100
is transmitted as an autosomal dominant disease with high Age (yr)
penetrance; 50% of the offspring of an affected parent exhibit
at the least impaired glucose tolerance, which usually pro- Figure 3-16. MODY3 (HNF-1α) and MODY2 (glucokinase)
gresses to frank, but often mild, diabetes. The symptoms of cause more than 80% of maturity-onset diabetes of the
young. Glucose is elevated in both types but may be
MODY are quite variable, reflecting its genetic heterogeneity.
dramatically increased in MODY3. (Redrawn with permission
MODY, characterized by defects in pancreatic β-cell func- of The American Diabetes Association from Pearson ER,
tion, is caused by mutations in at least six genes representing Velho G, Clark P, et al. β-Cell genes and diabetes: quantitative
six MODY types (Table 3-6). Five of these are transcription and qualitative difference in the pathophysiology of hepatic
factors, and mutations in all six genes are loss-of-function nuclear factor-1α and glucokinase mutations. Diabetes
mutations. Seventy-five percent of cases of MODY are caused 2001;50:S101–S107.)
by transcription factor mutations. The most common form,
MODY3, representing 70% of cases, is caused by mutations
in a transcription factor (HNF1A) gene that regulates expres-
sion of several liver genes, including the hepatic nuclear functions in hepatocytes to produce a “liver” isoform. The
factor-1α (HNF-1α) protein. The second most common pre- liver form of GCK acts to process glucose from digestion. The
sentation is MODY2, caused by mutations in the glucokinase neuroendocrine form acts as a sensor to trigger cell responses
(GCK) gene. For these individuals, glucose levels may be affecting carbohydrate metabolism. One can hypothesize that
elevated to twice normal, whereas patients with mutations in patients with MODY2 have lower levels of glucose than those
HNF-1α may have glucose levels increased up to five times with MODY3 because of the tissue-specific nature of the
normal (Fig. 3-16). glucokinase with two promoters.
Glucokinase is a distinctive protein in that it has two pro-
moters with little homology that are functional in a tissue- Gestational Diabetes
specific manner. The first, most 5′ promoter functions in Finally, diabetes may also develop during pregnancy from an
pancreatic islet cells, neural tissue, and enterocytes to produce unknown cause. Gestational diabetes occurs in approximately
a “neuroendocrine” isoform. The more downstream promoter 4% of all pregnancies and usually resolves after pregnancy.
 50 Mechanisms of Inheritance

Insulin resistance is thought to occur as a result of hormone

levels during pregnancy. Symptoms generally occur in the KEY CONCEPTS
second half of pregnancy and are characterized by fatigue ■ Mendelian patterns of inheritance depend on expression from
resulting from a lack of glucose in tissues. autosomes or sex chromosomes.
If untreated, maternal hyperglycemia is harmful to the ■ Less straightforward mechanisms and factors of inheritance to
developing fetus. Since insulin does not cross the placenta interpret include triplet repeats, mitochondrial inheritance, mosa-
and glucose does, the fetal pancreas responds by increasing icism, penetrance, expressivity, and the presence of late-acting
insulin secretion. Extra glucose is stored and is responsible genes and imprinting.
for the large size of newborns, a condition known as macro- ■ Diseases caused by single genes are more easily interpreted
somia. Newborns subsequently suffer from hypoglycemia than those caused by multiple factors, including environmental
because of elevated insulin and have an increased risk of influences.
perinatal mortality and morbidity. This must be corrected to ■ Environmental factors may be epigenetic if their consequences
prevent mental retardation and other signs of failure to thrive. are observed in offspring of the affected individual.
These infants are at an increased risk for breathing problems. ■ Multifactorial inheritance interpretations involve population data
They also have an increased risk of later developing obesity where those individuals at risk (to the right of the risk threshold)
and type 2 DM. Similarly, up to 50% of the mothers of these are more susceptible to developing disease but may not actually
infants will develop type 2 DM. In addition, the risk of a develop disease.
mother experiencing gestational diabetes in future pregnan- ■ Risk, severity, and gender differences are explained by the pres-
cies is 67%. Clearly, there are many aspects to diabetes that ence of greater or lesser numbers of susceptibility genes.
result from a complex interaction between genetic factors and
■ Diabetes is an example of a complex disease with genetic and
nongenetic factors.
environmental influences.
■ MODY is a collection of disease caused by individual genes.

●●● QUESTIONS Explanation: Familial incontinentia pigmenti is usually lethal

prenatally in males. Females present with a variety of skin,
1. The pedigree shows a large extended family that hair, nails, teeth, and mental abnormalities. This case pres-
demonstrated mild symptoms similar to incontinen- ents a mild form suggesting a less severe mutation in the
tia pigmenti. Which of the following mechanisms of affected gene. This is the most difficult mechanism of inheri-
inheritance is demonstrated for this family? tance for students to quickly recognize because they often
A. Autosomal dominant opt for autosomal dominant inheritance. Affected sons pass
B. Autosomal recessive the trait to all daughters but to no sons. Affected daughters
C. Multifactorial pass the trait to 50% of sons and daughters.
D. X-linked dominant
E. X-linked recessive Additional Self-assessment Questions can be Accessed
Answer. D at www.StudentConsult.com
Genetics of 4
Metabolic Disorders
CONTENTS most metabolic diseases are rare, this chapter presents selec-
tive disorders that are most familiar to students and practi-
tioners because they represent classic studies of disease or
HISTORICAL PERSPECTIVE
disorders commonly tested for in newborn screening. They
MODEL OF BLOCKED METABOLIC REACTIONS include selected aminoacidopathies, a carbohydrate meta-
bolic disorder, and several organic acidemias. Additional dis-
NEWBORN SCREENING
orders, such as lysosomal storage disorders and cystinuria, are
AMINOACIDOPATHIES presented in other chapters more appropriate to the defect or
clinical presentation.
Phenylalanine Metabolism and Hyperphenylalaninemia
Tyrosine Metabolism
Homocystinuria and Hyperhomocystinuria ●●● HISTORICAL PERSPECTIVE
A CARBOHYDRATE METABOLISM DISORDER The first example of recessive inheritance occurred in patients
Galactosemia
excreting “black urine.” From an analysis of family histories,
Archibald Garrod concluded that this anomaly, known as
ORGANIC ACIDEMIAS alkaptonuria, appeared when affected children inherited one
Maple Syrup Urine Disease
defective gene from each parent. He later theorized that the
Biotinidase Deficiency defective gene prevented the formation of a particular
enzyme. As seen in Figure 4-1, the normal allele specifies the
TREATMENT OF GENETIC DISEASE

Normal

It is an accepted tenet that an inheritable change of an allele OH

J

can reveal itself as a modification in the structure and func-

tion of that specific allele product. Often these products are O
K
J

proteins, but modifications can also affect mRNAs in ways CH2COOH CH3C CH2COOH
J

that are not reflected in the protein; they may also occur in OH
tRNAs, rRNAs, and other less well recognized RNAs. Modi- Homogentisic acid Enzyme Acetoacetic acid
fications in protein structure result from any of the mecha-
Normal gene
nisms presented in Chapter 1, with effects ranging from
benign to an inactive or absent protein. Even a single amino Alkaptonuria
acid replacement in a protein may have important clinical
OH
consequences, as evidenced by sickle cell hemoglobin (see
J

Chapter 6). Both structural proteins and enzymes are subject

to these alterations; however, many of the earliest described Reaction blocked
J

inherited anomalies were biochemical defects, or inborn CH2COOH

J

errors of metabolism, resulting from enzymatic deficiencies. OH

The consequence of an enzyme deficiency is the direct impair- Homogentisic acid
ment of a specific metabolic pathway. Many of these have Accumulates and is excreted
in urine; urine turns black
been characterized and are responsible for the appearance of
metabolic disease. Figure 4-1. Alkaptonuria. The inability of homogentisic acid
Metabolic disorders represent an expansive literature. There to form acetoacetic acid because of deficient or defective
are several logical ways to group these disorders. Although enzyme results in alkaptonuria.
 52 Genetics of Metabolic Disorders

synthesis of the enzyme that converts homogentisic acid to

acetoacetic acid. In an alkaptonuric individual, the enzyme
is absent or deficient and homogentisic acid accumulates,
since its conversion to acetoacetic acid is blocked or reduced.
The excess homogentisic acid is then eliminated in urine. Genes
Urine containing homogentisic acid, on exposure to air, grad- No
enzyme c
ually darkens.
Subsequently, it became clear that each gene controls the
Enzyme a Enzyme b
formation of a single enzyme and therefore a single metabolic accumulates
reaction. This one gene–one enzyme thesis was reinforced A B C D
and expanded. It was extended to include all cellular proteins
and was, at the time, more precisely defined as the one gene– Substrate C
one polypeptide principle. However, as the mechanisms of accumulates
genetic variability have become further clarified, it has X Y Z
become even more amazing to find that the mechanisms of Increased utilization
alternative splicing may provide different protein products of accessory pathway
per gene and allele.
Figure 4-2. Model of metabolic disease. Genes altered by
different forms of mutations may cause altered enzymes that
prevent the conversion of a substrate to a product. In some
cases, substrate may be converted to alternative metabolites
by a different pathway.

BIOCHEMISTRY
Alternative Splicing
Alternative splicing is the differential joining of exons to form
more than one variant from the same gene. The significance
of this mechanism is that it increases the diversity of gene homogentisic acid along with a blue-black discoloration of
products. A prominent example at the DNA level is connective tissue, including bone, cartilage, and skin, caused
immunoglobulin class switching. At the RNA level, this is by deposits of ochre-colored pigment—a condition known as
demonstrated at dystrophin, the gene mutated in Duchenne ochronosis. The block between C and D may also cause
and Becker muscular dystrophies. Dystrophin has seven increased use of other pathways, resulting in greater produc-
promoters that are used to generate specific, cell-type tion of alternative products. For example, in phenylketonuria
expression. (PKU), a single enzymatic block results not only in the accu-
mulation of phenylalanine (product C) but also in gross over-
production of phenylpyruvic acid (product Z). This provides
a general orientation to inborn errors of metabolism also
called metabolic disorders.
An important consideration in the development of disease
●●● MODEL OF BLOCKED is the period of gene expression. Those genes important for
early development will have earlier consequences if mutated
METABOLIC REACTIONS
than will genes not expressed until puberty or after. These
The consequences of an alteration of a single enzyme are genes are differentially expressed; they are expressed at some
shown in Figure 4-2, which depicts the model reaction periods but not at other periods. Some genes are expressed
sequence A → D and the relevant genes for each step. In this continually and are called constitutively expressed genes. A
model, it is assumed that each gene acts independently and different group of genes may be expressed only in a particular
each is responsible for the formation of a specific enzyme. tissue. These tissue-specific genes may be either differentially
Substrate A is converted to end product D through a series or constitutively expressed (Table 4-1).
of metabolic steps that are each catalyzed by a separate A different consideration is the type of mutation and the
enzyme. As illustrated, a number of consequences may arise location of the mutation within the affected allele. As recog-
if enzyme “c” is not formed because gene “c” is mutated nized in Chapter 1, there are several types of mutations. In
and transcription does not occur. One effect would be the addition to mutations altering structure and function, other
lack of formation of product D, which could lead to a clinical mutations may cause a modification in the rate of synthesis,
disorder. In addition, the immediate precursor of the blocked posttranslational mechanisms, or transporting of proteins out
reaction could accumulate in abnormal amounts. For example, of the cell. Even if these changes produce products at normal
it has already been shown that homogentisic acid accumulates or near-normal levels, a modification may alter the function,
in alkaptonuria because its conversion to acetoacetic acid is efficiency, or half-life of the protein and result in a disease
blocked. The outcome is an increase in urinary excretion of condition.
 Newborn Screening 53

TABLE 4-1. Types of Gene Expression

TYPE OF GENE EXPRESSION EXPRESSION PRODUCT NATURE OF EXPRESSION PRODUCT

}
Constitutive tRNAs
rRNAs
Non-tissue-specific
Cell membrane enzymes
Some transcription factors
Insulin
Cytochrome P-450
G protein–coupled receptor
} Tissue-specific

Differential
Receptors
Transcription factors } Non-tissue-specific
Cortisol
Globins
Dystrophins
} Tissue-specific

BIOCHEMISTRY continuum of symptoms may be seen in individuals from mild

to very severe because of the differences between alleles. In
Posttranslational Modification reality, variable expressivity and clinical variability are not a
Posttranslational modification leads to the formation of 100 peculiarity of a few disorders. They should be expected and
or more different amino acid derivatives from the basic 20. are seen in the vast majority of genetic diseases. This variabil-
Modifications may be permanent or reversible. Examples ity is not only the effect of specific mutations but also a con-
include converting a protein to a functional form, directing sequence of the temporal interaction of gene products with
a protein to a specific site, aiding in protein secretion, or the environment of the cell. This is readily seen in individuals
altering protein activity or stability.
with cystic fibrosis, a condition caused by a mutation in the
Proteins are frequently modified at the ends. At the amino
cystic fibrosis transmembrane conductance regulator gene
terminus, modifications may include formylation, acetylation,
aminoacylation, and glycosylation. At the carboxyl terminus, (CFTR). Over 1500 mutations have been reported in this gene,
modifications may include methylation, glycosyl- which is composed of over 300,000 bp spanning 27 exons. The
phosphatidylinositol anchor formation, and ADP ribosylation. severity of cystic fibrosis is associated with specific mutations.
An example is insulin that is modified by cleavage in the The deletion of three base pairs coding for phenylalanine
Golgi complex, where disulfide bonds link two of the (referred to as the ΔF508 mutation) results in a severe presen-
fragments. Another example occurs with the addition of tation when both alleles have the deletion. An insertion of
oligosaccharides to proteins that form the glycoproteins. seven polythymidines is asymptomatic when present on both
Most proteins that are secreted or bound to the cell alleles but may present as a mild or classic phenotype when
membrane are modified by the attachment of various present with a different mutation (see also Cystic Fibrosis in
carbohydrates.
Chapter 9).

●●● NEWBORN SCREENING

Heterogeneity of disease among different individuals can Newborn screening programs in the United States and other
occur if any of these situations occurs; that is, mutations occur- countries have proved invaluable in the detection of serious
ring in different locations may affect expression, structure, or metabolic disorders. The goal is to identify infants with
function. For example, a particular mutation within the pro- genetic conditions who can be helped by early intervention
moter region may abolish transcription of an allele critical for programs. Mass screening of metabolic disorders became
a cellular function. A different mutation within an exon may feasible as rapid, cost-effective assays, such as the Guthrie
result in the incorporation of a different amino acid in the test, became available. All states screen for PKU, congenital
protein that will reduce, but not abolish, its function. Another hypothyroidism, galactosemia, and sickle cell disease (only
mutation may have no consequence for expression of the selected populations in some states). Most countries that
protein. These differences result from allelic variation, or reported tests had performed tests for PKU and hypothy-
allelic heterogeneity. Alleles differ in their expression because roidism. Screening tests for other disorders may include
they are defined by different mutations and thus result in dif- maple syrup urine disease, homocystinuria, biotinidase defi-
ferent versions of the same protein. Some alleles may present ciency, congenital adrenal hyperplasia, cystic fibrosis, and
the same clinical phenotype within a population (see human immunodeficiency virus. With the advent of newer
Achondroplasia in Chapter 7). However, for many diseases, a technologies, such as tandem mass spectrophotometry and
 54 Genetics of Metabolic Disorders

leucine, isoleucine, and valine, also known as branched-chain

TABLE 4-2. Considerations for Newborn amino acids. Most of the resulting conditions are rare but
Screening Tests clinically important because of the debilitating consequences
to the homozygous individual with two defective alleles and
GUIDELINE JUSTIFICATION the extreme financial and psychological burdens on their
family. Disorders are named for the amino acid that accumu-
Incidence The incidence of the disorder must lates in the blood or urine.
warrant screening
Benefit The test must provide a potential benefit
to the infant
Treatment A treatment must be available for
newborns BIOCHEMISTRY
Cost The cost-benefit ratio should favor
screening over not screening for the Amino Acids
infant
Feasibility The test must be practical for population Amino acids are classified as essential (those that must be
screening with minimal false-negative derived from the diet) and nonessential (those synthesized
results by the body). The essential amino acids include histidine,
isoleucine, leucine, lysine, methionine, phenylalanine,
threonine, tryptophan, and valine. The nonessential amino
acids include alanine, arginine, asparagine, aspartic acid,
glutamine, glycine, proline, serine, and tyrosine.
DNA-based technologies, newborn screening tests have
In extended periods of stress, the biosynthetic pathways
expanded to better identify infants at risk for metabolic cannot fulfill the requirement for certain nonessential amino
disorders. acids. These amino acids then become conditionally
Inborn errors of metabolism should be considered in all essential, and dietary supplementation is required. Cysteine
critically ill infants. Frequently, these infants are normal at and glutamic acid are examples of two amino acids that
birth. Good health deteriorates over a period of hours to become essential during periods of stress.
weeks with symptoms such as poor feeding, failure to thrive,
developmental delay, or delayed milestones. Older undiag-
nosed children may have recurrent problems such as vomit-
ing, lethargy, ataxia, or seizures. Failure to diagnose these Phenylalanine Metabolism
conditions may often lead to dysmorphologies, mental retar-
and Hyperphenylalaninemia
dation, or even death. Sometimes older children with mild or
subtle disorders may actually have undiagnosed metabolic Phenylalanine is the starting point for a series of reactions
disorders that went undetected at birth. essential in metabolism. Reflected in this pathway of reactions
There are several considerations for determining which dis- are several inherited defects: phenylketonuria, albinism, tyro-
orders should be screened routinely in newborns. Although sinemia, and alkaptonuria (Fig. 4-3). Each of the four meta-
many of the disorders currently tested for are not frequently bolic disorders results from a block at a different point. All
seen, the clinical value of screening tests must be established are recessive, and the heterozygote typically produces suffi-
(Table 4-2). Enough individuals should be affected that cient enzyme and is considered “normal.”
screening is valued. The infants should benefit from the test Classic phenylketonuria is recognized as a deficiency of
by having an effective treatment available, and early treat- phenylalanine hydroxylase. Shortly after birth, the affected
ment should be more beneficial than delayed treatment. infant has an unusually high concentration of phenylalanine,
Treatment should also be significantly less expensive than an “essential” dietary amino acid, in the blood. Some phe-
treating an affected individual with the disorder. Finally, it nylalanine is incorporated into various proteins. Most,
should be feasible to screen large numbers of individuals, and however, is normally converted to tyrosine, which is involved
the number of false negatives should be minimal (see Table in several metabolic pathways, including the formation of
13-3). pigment and neurotransmitters. The deficiency of the enzyme
phenylalanine hydroxylase (PAH) results in the accumulation
of phenylalanine in fluids and tissues. The increased concen-
●●● AMINOACIDOPATHIES tration of phenylalanine leads, in turn, to an increased rate
Inborn errors of amino acid metabolism generally are the of formation of phenylpyruvic acid, which is excreted in the
outcome of enzyme deficiencies that result in the accumula- urine (Fig. 4-4). Excess phenylpyruvic acid in sweat accounts
tion of a substrate. Most of these are autosomal recessive for the unusual “mousy” body odor of PKU patients. Phe-
single-gene defects in amino acid synthesis or catabolism. nylpyruvic acid is a competitive inhibitor of the pyruvate
Some amino acids are synthesized within the cell, whereas dehydrogenase complex, which is important in glucose
others require dietary intake. However, having amino acids metabolism and subsequently in synthesis of fatty acids and
in the diet does not guarantee a healthy state unless levels cholesterol. This may contribute to the lack of myelin forma-
are carefully monitored. The dietary amino acid may become tion and mental retardation in these individuals. Phenylala-
a substrate for another important product, as seen with nine will also interfere with hydrophobic amino acid transport
 Aminoacidopathies 55

Phenylalanine Tyrosine
Phenylketonuria Tyrosinemia type II
hydroxylase aminotransferase

Phenylalanine Tyrosine p-Hydroxyphenylpyruvic acid

Albinism Neonatal
p-Hydroxyphenylpyruvic
type IA and IB, Tyrosinase acid oxidase tyrosinemia
type II

4-Hydroxyphenylpyruvate
3,4-Dihydroxyphenylalanine
(dopa)
Tyrosine Homogentisate
hydroxylase

Catecholamines Dopa quinone Homogentisic

Alkaptonuria
acid oxidase

4-Maleylacetoacete
Melanins

Fumarylacetoacetate

Fumarylacetoacetate Tyrosinemia
hydrolase type I

Acetoacetate

Figure 4-3. Disorders of the phenylalanine-tyrosine pathways.

GTP

GTP cyclohydrolase

Dihydroneopterin

Dihydrobiopterin synthetase +Mg;;

type III
Sepiapterin

Dihydrobiopterin
Alternative
degradation
pathway
Tetrahydrobiopterin NAD;
Phenylalanine BH4

Phenylpyruvic acid Phenylalanine Dihydropteridine

hydroxylase reductase
type I type II
Phenylacetic acid Dihydrobiopterin
Tyrosine BH2 NADH;+H;

Phenylacetyl-
Phenyllactic acid
glutamine

Figure 4-4. Different forms of hyperphenylalaninemia. Defects in the ability to produce tetrahydrobiopterin efficiently will lead
to PKU types II and III. PAH may be normal, but without tetrahydrobiopterin (BH4) synthesis there is excess phenylalanine and a
decrease in tyrosine.
 56 Genetics of Metabolic Disorders

across the blood-brain barrier. It competes for the same active at a higher frequency than expected and account for approxi-
transport system as does leucine, an important component of mately 60% of mutations in Europeans. These “hot spots” for
myelin. Postmortem studies of PKU infants with microceph- mutations provide a better understanding of allelic heteroge-
aly suggest that the decreased brain size is due to a decreased neity. Stated another way, if 60% of PAH mutations are
amount of myelin and a reduction in other protein compo- represented by only 5 of 500 mutations, the phenotypical
nents of the brain. expectation among individuals with PKU and hyperphenyl-
alaninemia is more likely to be based on these 60% rather
than phenotypes that may be represented by other
PATHOLOGY mutations.
Both the excessive amount of phenylalanine and the lack
Changes in Urinary Amino Acids Associated with of tyrosine produce the abnormal consequences of PKU (see
Clinical Presentations Fig. 4-3). High levels of phenylalanine are damaging to rapidly
developing brain tissues in the developing infant. Brain
CONDITION EXAMPLES
damage in the untreated patient occurs after birth rather than
before because the placenta produces phenylalanine hydrox-
ylase. Therefore, the placenta converts excess phenylalanine
Inherited metabolic PKU, tyrosinemia,
disorders homocystinuria, cystinuria, to tyrosine for the developing fetus. Production of this
other hyperaminoacidurias enzyme in the infant’s liver begins after birth.
Malnutrition Dietary protein malabsorption, Figure 4-4 also demonstrates that a deficiency of tyrosine
extreme metabolic crises can occur in other ways that result in elevated levels of
such as fasting, starvation, phenylalanine and decreased levels of tyrosine. These forms
or muscle wasting
Hydroxyproline excretion is of hyperphenylalaninemia are often referred to as type II
associated with celiac disease and type III PKU to distinguish them from classic, or type
and malabsorption disease I, PKU. Each form can be detected with the Guthrie test or
Decreased protein Recent dietary insufficiency with amino acid profiling of urine or plasma. However, the
ingestion demonstrates decreased choice, and ultimately the success, of treatment depend on
amino acid excretion
Alcoholism Nutritional anemia and alcoholic which enzyme is affected. Individuals with classic PKU are
gastritis demonstrate altered treated with a phenylalanine-restricted diet; however, in type
amino acid excretion II and type III hyperphenylalaninemia, phenylalanine is
Cushing disease Increased cystine elevated but these individuals are not spared the effects of
Chronic fatigue Strongly associated with progressive neurologic deterioration by a phenylalanine-
syndrome β-alanine; increased ratio of
tryptophan to branched-chain restricted diet (Table 4-3). Defects in dihydrobiopterin syn-
amino acids thetase and dihydropteridine reductase resulting in deficiencies
Muscle catabolism 3-Methylhistidine indicates of tetrahydrobiopterin and dihydrobiopterin block or reduce
excessive muscle catabolism the conversion of phenylalanine to tyrosine, leading to
Osteoporosis Elevated hydroxyproline with inadequate neurotransmitter formation.
increased osteocalcin
secretion indicates high bone When relying on the Guthrie screening test, the physician
turnover must be cautious about positive results. Elevated tyrosine
associated with low birth weight, seen in infants on high-
protein diets, and with vitamin C deficiency may also cause
a positive Guthrie test. Liver disease and galactosemia can
also cause a positive test. Two additional tests can be used to
More than 500 mutations have been described in the PAH detect elevated levels of phenylalanine and other amino
gene. Most mutations result in hyperphenylalaninemia owing acids. A fluorometric assay is an automated test that produces
to reduced enzyme activity. Mutations have been described fewer false positives than does the Guthrie test. Tandem mass
in each of the 13 exons; however, 5 mutations are present spectrometry is used to identify numerous metabolic defects.

TABLE 4-3. Comparison of Hyperphenylalaninemias

PERCENTAGE OF ALL
TYPE ENZYME AFFECTED HYPERPHENYLALANINEMIAS

I Phenylalanine hydroxylase, <1% activity 60%

I (mild, persistent)* Phenylalanine hydroxylase, ≤35% activity 35%
II Dihydropteridine reductase 3%
III Dihydrobiopterin synthetase 1–3%

*Variation in severe versus mild, persistent form results from different PAH allele combinations.
 Aminoacidopathies 57

This test is extremely sensitive to trace metabolites. Its biggest

drawback for routine screening is cost-effectiveness. In spite TABLE 4-4. Clinical Features of Classic
of the cost factor, the use of tandem mass spectrometry Phenylketonuria at Different Periods
screening is increasing in newborn screening programs.
APPEARANCE SYMPTOMS

Newborn Usually no symptoms immediately

MICROBIOLOGY Lethargy
Feeding difficulty
Guthrie Test Reduced pigmentation of hair, skin,
eyes
The Guthrie test is a bacterial inhibition assay. β2-
Eczema
Thienylalanine is placed in the medium and normally causes
the inhibition of Bacillus subtilis growth. However, in the Child Irreversible after about 6 months
presence of an excess of phenylalanine, this inhibition is Seizures
overridden and bacterial growth occurs. This test is the least Nausea
Vomiting
expensive screening method available for determining
Hyperactivity
excess phenylalanine in the blood, but other tests are used Aggressive behavior
to confirm findings. Poor coordination
Ataxia
Abnormal posturing
Self-injurious behavior
“Mousy” odor of body and urine
An excessively high phenylalanine level during pregnancy
Mental retardation
is of particular concern because of its toxicity to the develop-
ing fetus. Maternal PKU results from the lack of PAH in the Pregnant female Spontaneous miscarriage
with untreated
liver and placenta that ordinarily would convert excess phe- PKU
nylalanine to tyrosine. This situation may occur when the
Infant of mother IUGR
mother fails to inform the physician that she has PKU or she
with untreated Microcephaly
forgets (often repressing the memories) the special diet she maternal PKU Psychomotor retardation
had as a child. When the phenylalanine-restricted diet is no Congenital heart defects
longer maintained, phenylalanine levels rise but may have Postnatal growth retardation
less dramatic effects than seen during early infant and child- Abnormal neurologic findings
Mild craniofacial dysmorphology
hood development. The mother’s mutated enzyme fails to
convert excess phenylalanine to tyrosine and subsequently
leads to fetal malformations; the infant may be genetically
normal but is adversely affected by the mother’s abnormal
PAH. Malformations include intrauterine growth retardation or age and is commonly characterized by nystagmus, photo-
(IUGR), microcephaly, heart abnormalities, and mental retar- phobia, and reduced visual acuity. Oculocutaneous albinism
dation (Table 4-4). It is important that the mother’s phenyl- type II is tyrosinase positive and is the most common form
alanine levels be monitored, since the earlier phenylalanine of albinism throughout the world. This albinism also results
levels are maintained within normal limits, the less prevalent from mutations in the tyrosinase gene. Type II differs from
malformations will be in the infant. A corollary is that tyro- type IB clinically. In type IB, there is a general reduction in
sine levels must not be too low, since this will affect the level pigmentation. Type II is also distinguished by pigmented nevi,
of neurotransmitters formed in the fetus. suggesting that it is more difficult to identify in blacks, who
have pigmented spots but also yellow hair. In some regions,
such as South Africa, type II is the most common recessive
Tyrosine Metabolism disorder.
Many genes are involved in the production of pigment of skin Tyrosine is essential for protein synthesis, catecholamine
and hair. The best-described condition affecting pigment pro- production, melanin production, and thyroid hormone syn-
duction is classic albinism, which results from a lack of tyrosi- thesis. Though tyrosine is available from the diet, a large
nase (see Fig. 4-3). Individuals with classic PKU may percentage is formed during phenylalanine metabolism.
demonstrate pale skin and hair coloration that might suggest Accumulation of tyrosine due to a metabolic disorder is rare,
albinism. However, once diagnosed, these individuals receive with the exception of neonatal tyrosinemia, which results
tyrosine in their diet, which bypasses the metabolic deficiency from delayed synthesis or stability of p-hydroxyphenylpyru-
of PKU and allows pigment formation. vic acid oxidase. Normal newborns, especially those of low
Classic albinism is also known as oculocutaneous albinism birth weight, tend to have unusually high levels of tyrosine
type I. Type IA is tyrosinase negative, whereas type IB has in their blood. This transient form of tyrosinemia is benign
reduced tyrosinase activity. In patients with albinism, the and rarely presents with symptoms. Blood tyrosine levels
body is incapable of synthesizing the pigment melanin, which usually become normal (<4 mg/100 mL) within a few weeks.
is a metabolite of tyrosine. Albinism does not vary with race Infants with neonatal tyrosinemia have a positive Guthrie
 58 Genetics of Metabolic Disorders

HISTOLOGY PHYSIOLOGY
Melanocytes Hyperaminoaciduria
Melanocytes are neural crest cell derivatives interspersed Hyperaminoacidurias, or the increased plasma concentration
among the cells of the stratum basale. They are of an amino acid, are explained by several mechanisms:
distinguished by pale, halo-like cytoplasm. Within (1) a defect may occur before the kidney, as seen in
melanocytes are melanosomes that produce melanin. The hyperargininemia, where the defect causes increased plasma
melanin-containing melanosomes are transported to and arginine; (2) competition for the same transporter may occur
take up locations covering the nuclei in keratinocytes, when the same transporter is used for more than one amino
protecting them from ultraviolet radiation. Hence, skin acid and the high filtering load of one amino acid inhibits the
color depends on activity of melanocytes rather than their others; and (3) a defective transporter may occur and result in
number. decreased reabsorption by the kidney. Finally, a generalized
proximal tubule dysfunction may occur from inherited or
acquired conditions affecting the proximal tubule cells. In this
type of hyperaminoaciduria, also called Fanconi syndrome,
the kidney fails to appropriately reabsorb the particular amino
NEUROSCIENCE & PHARMACOLOGY acids as well as Na+, Cl−, HCO3−, glucose, and water.

Catecholamines
Catecholamines serve as neurotransmitters and circulating
hormones. They are derived from tyrosine and include Homocystinuria and
dopamine, epinephrine, and norepinephrine. The rate- Hyperhomocystinuria
limiting step in this pathway, which begins with the essential
amino acid phenylalanine, is tyrosine hydroxylase. Methionine is an essential amino acid that is converted to
After release from chromaffin cells of the adrenal gland cysteine. These amino acids are the only two sulfur-containing
and sympathetic axons, catecholamine reuptake primarily amino acids in protein synthesis, and therefore, the roles they
occurs at synaptic clefts, although non-neuronal uptake also play in synthetic pathways are critical. For example, an
occurs. This mechanism is sodium dependent but not ATP important methionine derivative is S-adenosylmethionine
dependent; transporters are therefore referred to as (SAM), which serves as an endogenous methyl donor. Methi-
Na+-dependent transporters. An example is the Na+- onine is also important in the synthesis of carnitine, taurine,
dependent norepinephrine transporter (NET). After uptake, phosphatidylcholine, and phospholipids. Cysteine is critical
catecholamines are either stored or catabolized by
in the production of heparan, coenzyme A (CoA), biotin, and
monoamine oxidase (MAO) and catechol
glutathione.
O-methyltransferase (COMT).
Catecholamine receptors are specific for ligands.
Dopamine effects are mediated by D1 and D2 receptor
subfamilies. Norepinephrine and epinephrine act via two BIOCHEMISTRY
broad families of receptors (α and β) or five classes (α1, α2,
β1, β2, and β3). S-Adenosylmethionine (SAM)
SAM is an enzymatic cofactor involved in irreversible methyl
group transfers for methylation and required by most
methyltransferase reactions. Many targets of methylation are
screening test; hence, care must be exercised when diagnosing in the brain. In particular, catecholamines are degraded by
PKU in a newborn. Positive Guthrie tests are typically fol- the actions of monoamine oxidase (MAO) and catechol
lowed by more detailed investigations, including serum phe- O-methyltransferase (COMT). COMT transfers a methyl
group from SAM to catecholamines, initiating a degradation
nylalanine and tyrosine levels.
process. In the absence of sufficient SAM, COMT, or other
Tyrosinemia types I and II are rare but can be detected
degradation enzymes, accumulations in degradation
by molecular analysis. Type I, also known as hepatorenal products occur and are seen in several neurologic
tyrosinemia, is potentially fatal. Present in either acute or disorders.
chronic form, it is associated with liver failure or with liver
dysfunction and renal nephropathy. Type II, also known as
oculocutaneous tyrosinemia, usually presents during the first An intermediate in the methionine-to-cysteine pathway is
year as eye irritations in the form of photophobia, conjunc- homocysteine, a thiol compound required to regenerate
tivitis, or pain resulting from tyrosine crystals within the methionine. Classic homocystinuria is a recessive metabolic
cornea. Skin lesions begin as blisters on fingertips and toes; disorder due to deficiency of cystathionine β-synthase (CBS)
later keratotic plaques appear on palms and soles. Both types that produces increased urinary homocystine and methionine.
result from the accumulation of tyrosine. Mental retardation About 50% of homocysteine is recycled to methionine.
may occur in these individuals, but it is unclear whether it As shown in Figure 4-5, two other enzymes may result
results from elevated tyrosine or is secondary to the enzyme in hyperhomocystinuria: methionine synthase and 5,10-
deficiency. methylenetetrahydrofolate reductase.
 Aminoacidopathies 59

Methionine THF
Serine

S-adenosylmethionine
Methionine Glycine
synthase Methyl-B12
S-adenosylhomocysteine
N5, N10-Methylene-THF

Homocysteine
N5-methyl-THF 5,10-Methylenetetrahydrofolate
Pyridoxine reductase (MTHFR)
Classic Cystathionine-
homocystinuria b-synthase
Cystathionine

Cysteine

Figure 4-5. Classic homocystinuria results from a deficiency of cystathionine β-synthase. Other mutations can affect the
pathway but have less damaging effects.

There are two forms of homocystinuria: vitamin B6 respon-

BIOCHEMISTRY sive and vitamin B6 nonresponsive. Pyridoxine (B6) respon-
siveness is the ability to enhance transsulfuration of
Homocysteine, Homocystine, and
homocysteine upon pyridoxine administration. Vitamin B6–
Homocysteine-Cysteine
responsive homocystinuria is typically milder than the non-
Homocysteine, homocystine, and homocysteine-cysteine are responsive form. Pyridoxine responsiveness determined by a
sulfur-containing thiols. Homocysteine can form a disulfide challenge test is defined as a decrease of total homocysteine
with another homocysteine or cysteine to form homocystine
below 50 μmol/L, whereas nonresponsiveness is no change in
or a mixed disulfide, respectively. The disulfides are the
plasma total homocysteine after a dose of up to 10 mg/kg of
component that is primarily measured in standard amino
acid analysis, since this form accounts for about 98% of
pyridoxine per day administered for at least 2 weeks. About
homocysteine. Of this about 75% is bound to protein, such 40% of homocystinuria patients respond to B6 supplementa-
as albumin, and the remainder occurs in a non-protein- tion. Those who fail to respond require a diet restricted in
bound form as homocystine, homocysteine-cysteine methionine.
disulfide, and other minor forms. Normally, only about 1% to
2% occurs as homocysteine and homocystine; however, in
homocystinuria, these may represent 10% to 25% of the BIOCHEMISTRY & PATHOLOGY
total concentration, and referred to as homocyst(e)ine.
Pyridoxine
Pyridoxine, or vitamin B6, is an essential cofactor for many
transamination reactions, including gluconeogenesis. It is
also involved in the synthesis of niacin (vitamin B3) from
tryptophan, the synthesis of several neurotransmitters, and
Clinical manifestations of homocystinuria include lens dis-
the synthesis of δ-aminolevulinic acid, a precursor of heme.
location (ectopia lentis), excessive height and length of limbs, Deficiencies in this vitamin are usually seen along with
and vascular abnormalities that may lead to myocardial deficiencies of other water-soluble vitamins. Characteristics
infarction, stroke, or pulmonary embolism. Developmental of a deficiency include stomatitis, angular cheilosis, glossitis,
and mental delay may be present, but a third of individuals irritability, and depression; in moderate to severe
with homocystinuria may have normal intelligence. In many deficiencies, confusion may occur.
ways, homocystinuria resembles Marfan syndrome (see Deficiencies are often associated with hypochromic,
Chapter 7), including the phenotypes mentioned above plus microcytic anemia because sideroblastic anemias are much
arachnodactyly, pectus excavatum, and scoliosis. Phenotypic more common than inherited forms. Sideroblastic anemias
differences between the two are normal intelligence in Marfan have diverse etiologies but have the common association of
nonheme iron “encrustations” of the mitochondria of
syndrome and upward lens dislocation in Marfan syndrome
erythroid precursors. Mitochondria are the site where heme
versus downward in homocystinuria. Lens dislocation is the
biosynthesis begins. Some drugs, toxins, or nutritional
primary symptom leading to diagnosis in 80% of patients deficiencies may antagonize B6 metabolism, thereby
with undiagnosed homocystinuria; it is usually diagnosed producing a reversible sideroblastic anemia.
between ages 4 and 6.
 60 Genetics of Metabolic Disorders

homocystinuria if vitamin B12 deficiency is sufficient to also

PATHOLOGY affect homocysteine-to-methionine recycling. Metabolic disor-
ders of B12 synthesis result in the same biochemical presenta-
Pyridoxine Challenge Test tion but with different symptoms. Similarly, a deficiency of
B6 responsiveness is determined by a pyridoxine challenge. methionine synthase will mimic a deficiency of folic acid as a
Baseline amino acids are determined during a normal diet. result of the inability to convert homocysteine to methionine.
Pyridoxine is then given orally (100 mg), and amino acid A deficiency of 5,10-methylenetetrahydrofolate reductase
concentrations are determined 24 hours later. A 30% (MTHFR) is the most common inborn error of folate metabo-
reduction in homocysteine or methionine or both suggests
lism. It results in homocystinuria and hypomethioninemia.
B6 responsiveness. If no change is observed in amino acids,
These disorders present in infancy or adolescence with devel-
pyridoxine is increased to 200 mg and then 500 mg. No
significant decrease following these challenges indicates B6
opmental delay, motor dysfunction, seizures, psychiatric dis-
nonresponsiveness. turbances, and other neurologic abnormalities. Patients also
have an increased risk for coronary artery disease.

Most of the 132 mutations found in the CBS gene are mis-
sense mutations, with the majority being unique mutations. PATHOLOGY & PHYSIOLOGY
However, among these the two most frequent mutations are Homocysteine (Hcy)
I278T and G307S, both of which are found in exon 8 of the
Elevated Hcy causes oxidative stress and injury to vascular
gene. I278T, in which isoleucine is replaced by threonine at
cells through the auto-oxidation of Hcy, formation of Hcy
codon 278, accounts for about 25% of all homocystinuric
mixed disulfides, interaction of Hcy thiolactones, and protein
alleles. G307S, in which glycine replaces serine, is the leading homocysteinylation. Oxidation of Hcy produces reactive
cause of homocystinuria in Ireland, where it accounts for oxygen species (ROS), primarily superoxide, hydroxyl
more than 70% of the mutations, suggesting that at least in radicals, and peroxynitrites. These lead to the formation of
this population a founder effect for this allele may exist. The peroxide and hypochlorous acid. All are toxic to tissues and
I278T mutation is almost always B6 responsive, whereas the cause damage to lipids, nucleic acids, and proteins.
G307S mutation is almost always B6 nonresponsive. This type The formation of mixed disulfides leads to the formation of
of information is necessary for knowing which tests to perform additional ROS. In addition, Hcy may undergo
and similarly which therapies might be effective. rearrangements to form Hcy thiolactones that will acylate
As noted, there are several causes of hyperhomocystinuria lysines in proteins. These homocysteinylate proteins initiate
further ROS damage and loss of biological activity. In
in addition to the classic form (see Fig. 4-5). These may be
particular, low-density lipoproteins may be affected, leading
benign or associated with mental retardation. Liver disease
to inflammation and the formation of foamy cells associated
due to hepatitis or tyrosinemia type I will cause elevated with atherosclerosis. The ability to inactivate these ROS is
serum methionine. A deficiency of folic acid or vitamin B12 the function of antioxidants. However, even with
resulting in methylmalonic acidemia will also cause up-regulation of glutathionine peroxidase and superoxide
dismutase, there is evidence that they are depleted in
chronic situations (see Fig. 6-6 and Biochemistry Box,
p. 114).
Vascular cells do not express cystathionine β-synthase
(CBS), so they are more vulnerable to elevated Hcy. These
cells are therefore more dependent on the methionine
synthase and methyl-B12 remethylation pathway.

●●● A CARBOHYDRATE
METABOLISM DISORDER
Galactosemia
Galactosemia is an extraordinary metabolic disorder in which
Figure 4-6. Galactosemia cataract. The accumulation of affected infants are unable to utilize galactose found in milk.
galactose in the lens leads to the production of galactitol. Galactose is important beyond its ability to provide glucose.
This sugar alcohol exerts increased osmotic pressure within It is also a component of cerebrosides, gangliosides, and
the lens because it diffuses very slowly. The induced swelling glycoproteins. These are constituents of cell membranes, and
is not solely responsible for subsequent cataract formation; cerebrosides and gangliosides are a significant portion of
however, evidence supports its role in cataract formation brain lipid. Galactose ordinarily is converted to glucose and,
rather than galactose-1-phosphate because a galactokinase
deficiency in which galactose-1-phosphate is absent will still
eventually, oxidized to provide energy. In affected infants,
yield cataracts. (From Kanski J. Clinical Ophthalmology: A galactose is not transformed and accumulates in various
Systemic Approach, 4th ed. London, Butterworth Heinemann, tissues. The infant suffers from malnutrition, becomes
1999, p 177.) severely mentally retarded, and develops cataracts (Fig. 4-6).
 Organic Acidemias 61

Characteristically the liver becomes grossly enlarged. returns to normal size, vomiting ceases, and nutrition and
Untreated, the infant usually dies. growth improve markedly. Unfortunately, unless a galactose-
Classic galactosemia, an autosomal recessive disorder, free diet is instituted promptly at birth, there is usually no
results from a block in the conversion of galactose-1- recovery from the mentally retarded state. Damage to the
phosphate to glucose 1-phosphate by galactose-1-phos- liver, brain, and eyes occurs in the very first days of life.
phate uridyltransferase (Fig. 4-7). As a result of the defect, Accordingly, if a newborn infant is a first-degree relative of
galactose-1-phosphate and galactose metabolites accumu- an individual with galactosemia, pre- or perinatal testing
late in tissues and are elevated in the blood and urine. can identify the condition. Estimates for the prevalence of
Galactose-1-phosphate competes with the UTP-dependent galactosemia vary widely, from 1 in 18,000 to 1 in 70,000
glucose-1-phosphate to reduce UDP glucose production. infants.
Two mutations account for 62% of the mutations identi-
fied: Q188R (54%) and S135L (8.4%). The remaining
mutations are either private (unique to a specific family)
or unknown.
●●● ORGANIC ACIDEMIAS
In a nonclassic form of galactosemia, galactokinase is defi- During normal degradation of many amino acids, intermedi-
cient. This rare form demonstrates elevated levels of galactose ate metabolites known as organic acids are formed. Enzyme
but does not have deposition of galactose-1-phosphate in deficiencies causing the accumulation of organic acids can
tissues. Excess galactose is metabolized by alternative path- lead to severe metabolic acidosis. Failure to recognize the
ways to metabolites that are normally present in only trace underlying cause of metabolic acidosis can have serious con-
amounts. Both forms of galactosemia result in cataract forma- sequences to the individual. Typically, infants appear normal
tion from galactitol, the reduced sugar alcohol of galactose, during the first few days prior to the onset of symptoms.
by aldose reductase. Galactitol accumulates in the lens, Clinical symptoms of organic acid disorders may include
causing a change in osmotic pressure as water is absorbed and vomiting, metabolic acidosis, ketosis, dehydration or coma,
swelling occurs. hyperammonemia, lactic acidosis, hypoglycemia, failure to
If the diagnosis of galactosemia is made before the disease thrive, hypotonia, global developmental delay, sepsis, and
is too far advanced, nearly all the symptoms will disappear hematologic disorders.
if galactose is restricted from the infant’s diet. The liver

Maple Syrup Urine Disease

Maple syrup urine disease (MSUD) was one of the earliest
and best known of the inborn errors of amino acid metabo-
lism primarily because it was associated with an odor in
Galactitol Galactose Nonclassical the urine strongly reminiscent of maple syrup. It represents
Aldose galactosemia
reductase a disturbance of metabolism of the branched-chain amino
acids (BCAAs) leucine, isoleucine, and valine (Fig. 4-8).
Galactokinase The normal degradation of these three essential amino acids
is initiated in muscle and yields NADH and FADH2 that
can be utilized for ATP generation. Branched-chain keto
Galactose-1-phosphate
acid decarboxylase is involved in each of these reactions.
UDP-glucose Galactose-1- This enzyme is a complex of four enzymes, implying that
phosphate uridyl mutations in different genes can affect the overall activity
UDP-galactose transferase
of the complex—an example of locus heterogeneity. Enzyme
Galactosemia
deficiencies result in the accumulation of high concentra-
tions of both BCAAs and α-keto acids in the blood and
Glucose-1-phosphate urine.
Infants with MSUD appear normal at birth but quickly
Phosphoglucomutase demonstrate failure to thrive. Because essential amino acids
are involved, the goal of dietary management is normal-
Glucose-6-phosphate
izing concentrations rather than restricting concentrations.
Glucose-6-phosphatase Infants who survive usually demonstrate neurologic damage
due to cerebral edema and degeneration. Symptoms begin
Glucose
3 to 4 days after birth in infants with the classic form
of this disease. If it is not detected, severe acidosis and
Figure 4-7. Pathway of glucose formation from galactose. A
hypoglycemia occur; clinical progression is rapid, and death
defect in galactokinase or galactose-1-phosphate can occur. Neurologic deterioration is apparent with flac-
uridyltransferase results in the formation of galactitol from cidity, hypertonicity, a high-pitched voice, and opisthotonic
aldose reductase. posturing.
 62 Genetics of Metabolic Disorders

L-Valine L-Isoleucine L-Leucine

Transamination in the
cytoplasm
α-Ketoisovalerate α-Keto-β-methylvalerate α-Ketoisocaproate
α-Ketoacid
decarboxylase
complex
Isobutyryl-CoA 2-Methylbutyryl-CoA Isovaleryl-CoA

Propionyl-CoA Acetyl-CoA HMG-CoA

Glyconeogenic Ketogenic Acetoacetate

Figure 4-8. Branched-chain amino acid metabolism. A mutation in the α-keto acid decarboxylase enzyme complex results in
the accumulation of valine, isoleucine, leucine, and the α-keto analogs of these branched-chain amino acids in the urine,
resulting in maple syrup urine disease.

several branched-chain amino acids, the first step of gluco-

PHYSIOLOGY neogenesis, and fatty acid synthesis. Biotinidase is responsible
for cleaving biocytin in food to form biotin and lysine. It is
Metabolic Acidosis also necessary for the recycling of biotin from the enzymes.
Metabolic acidosis, one of four major acid-base disorders, is The carboxylases dependent on biotin are pyruvate carbox-
a sign of an underlying problem and occurs when total body ylase, propionyl-CoA carboxylase, 3-methylcrotonyl-CoA
acid increases because of an imbalance between acids and carboxylase, and acetyl-CoA carboxylase. Without biotin, the
alkali. Clinically, this can occur with a decrease in urinary functions of these enzymes are dramatically reduced because
secretion of H+ from renal failure, ketoacidosis from
biotin normally forms a transient covalent bond to the COO−
diabetes, lactic acidosis seen in shock, or HCO3− loss
group to be transferred and thus facilitates the carboxylase
occurring with severe diarrhea. These lead to decreases in
pH and HCO3−. Metabolic response to acidosis occurs by activity.
extracellular buffering, especially by hemoglobin, intracellular Individuals with profound biotinidase deficiency have less
and bone buffering, respiratory compensation, and renal than 10% enzyme activity. Those with a partial deficiency
excretion of H+. have enzymatic activity of 10% to 30%. Most affected indi-
viduals have profound biotinidase deficiency and become
biotin deficient during infancy or early childhood. Biotinidase
deficiency can occur from two different gene mutations.
Milder forms of MSUD may occur with a later onset after Mutations in the biotinidase gene are usually referred to as
the neonatal period. These individuals are usually only mildly the late-onset form while the early-onset form is attributed
retarded and have elevated plasma levels of BCAAs. Periodic to a mutation in the holocarboxylase synthetase gene. Both
ketoacidosis may occur with mild MSUD after infections or are similar in presentation, although the early-onset form
with high-protein diets. As might be expected, many diagno- generally appears before 3 months of age and the late-onset
ses of mild MSUD are made during an illness. As with most form after 3 months of age. Symptoms may occur indepen-
diseases, the nature of the mutation will probably be dently or in concert, including neurologic and cutaneous
displayed in variable degrees of severity depending on the symptoms. Among the neurologic symptoms, seizures and
percentage of activity in the enzyme complex. hypotonia are the most common, although mental retardation
As with all the organic acidemias listed in Table 4-5, MSUD and coma may also occur. Some cases of sudden infant death
is inherited in an autosomal recessive manner. In the general have been attributed to biotinidase deficiency. Cutaneous
population, MSUD is not the most common organic aca- symptoms are striking and include alopecia and an eczema-
demia; however, among the Mennonites it is quite common, tous, scaly, perioral or facial rash. As a metabolic disorder, the
with a general incidence of 1 in 1000. Some isolated popula- symptoms do not respond to dermatologic treatments. Other
tions of Mennonites with a high incidence of consanguinity symptoms may include breathing difficulties, ataxia, hearing
have an incidence as high as 1 in 176. loss, and developmental delay. As an autosomal recessive
disorder, biotinidase deficiency can be diagnosed prenatally
or with newborn screening.
Biotinidase Deficiency Propionyl-CoA carboxylase and 3-methylcrotonyl-CoA
Biotin is a water-soluble vitamin that acts as a prosthetic carboxylase deficiencies result in organic acidemias (see Table
group for four carboxylases required for the degradation of 4-5). These conditions, however, are caused by mutations in
 Treatment of Genetic Disease 63

TABLE 4-5. Features of Organic Acidemias

CLINICAL FEATURES
AMINO ACID
PATHWAY
DISORDER INCIDENCE AFFECTED DEFECTIVE ENZYME KETOSIS ACIDOSIS OTHER

Maple syrup urine 1 in 150,000 Leucine, Branched-chain keto Yes Maple syrup
disease (MSUD) (general isoleucine, acid dehydrogenase odor to urine
population) valine
1 in 1000
(Mennonites)
Propionic acidemia 1 in 20,000 Isoleucine, Propionyl-CoA Yes Yes Neutropenia
methionine, carboxylase
threonine
Methylmalonic 1 in 20,000 Isoleucine, Methylmalonyl-CoA Yes Yes Neutropenia
acidemia (MMA) valine, mutase
methionine,
threonine
Isovaleric acidemia 1 in 150,000 Leucine Isovaleryl-CoA Yes Sweaty feet
dehydrogenase odor
Biotin-unresponsive 1 in 15,500 Leucine 3-Methylcrotonyl-CoA Yes Hypoglycemia
3-methylcrotonyl- carboxylase
CoA carboxylase
deficiency
3-Hydroxy-3- Unknown Leucine Hydroxymethylglutaryl- No Reye syndrome,
methylglutaryl- CoA lyase hypoglycemia
CoA (HMG-CoA)
lyase deficiency
Ketothiolase Unknown Isoleucine Mitochondrial Yes Yes Hypoglycemia
deficiency acetoacetyl-CoA
thiolase
Glutaric acidemia 1 in 150,000 Lysine, Glutaryl-CoA No Basal ganglia
type I (GA I) hydroxylysine, dehydrogenase injury with
tryptophan movement
disorder

enzymes within a pathway and not by availability of biotin. defective enzyme. However, it is important to remember that
In fact, treatment with biotin has little effect in these disor- products downstream of the defective enzyme may be critical
ders but is very effective in biotinidase deficiency. Biotinidase to health. Early recognition is important to minimize effects
deficiency results in the deficiencies of multiple carboxylases of the disease.
rather than one. Ketoacidosis, organic aciduria, and mild Treatments for inborn errors of metabolism are generally
hyperammonemia may occur with biotinidase deficiency, but found in one or more of several categories: dietary restriction
in partial deficiency these symptoms may occur only during of substrate, replacement of end product, depletion of storage
metabolic stress. Partial biotinidase deficiency, in particular, substrate, amplification of mutant protein, replacement of
is associated with metabolic stresses such as illness, fever, and mutant protein, organ or bone marrow transplantation, and
fasting. Urinary organic acid analysis will distinguish between surgical removal (Table 4-6). As discussed in this chapter,
isolated and multiple carboxylase deficiencies. many of the disorders identified at birth can be treated by
dietary restriction and supplementation. A classic example of
dietary restriction is PKU. Phenylalanine should be restricted
soon after birth to achieve normal growth and cognitive
●●● TREATMENT OF
development. Special formulas are used and adjusted as the
GENETIC DISEASE infant grows. Early in the history of PKU treatment, many
The essential principles underlying treatment of many inher- patients were taken off the diet in childhood or as teenagers.
ited metabolic disorders are avoidance of the harmful envi- Likewise, other individuals lacked continual compliance with
ronmental factors that exacerbate the condition and the diet as they got older. Adherence to a low-phenylalanine
restoration of a normal internal metabolism by modifications diet should be lifelong and particularly so for women of
of the diet. In the examples described, the environmental childbearing age. A range of plasma phenylalanine levels from
factor is often a component of the diet. Removing this com- 2 to 6 mg/dL is recommended until age 12 years. Afterward,
ponent removes the substrate that accumulates because of the the suggested range is from 2 to 15 mg/dL.
 64 Genetics of Metabolic Disorders

TABLE 4-6. Strategies of Treatment for Inborn Errors of Metabolism

STRATEGY EXAMPLES TREATMENT

Dietary restriction of substrate Restrict:

Galactosemia Galactose
Fructosemia Fructose
Maple syrup urine disease Isoleucine, leucine, valine
Phenylketonuria Phenylalanine
Tyrosinemia Tyrosine
End-product replacement Replace:
Menkes syndrome Copper
Cystic fibrosis Pancreatic enzymes
Familial goiters Thyroxine
Orotic aciduria Uridine
Depletion of storage substance Deplete:
Wilson disease Copper
Hypercholesterolemia Cholesterol
Hemochromatosis Iron
Replacement of mutant protein Replace:
Hemophilia Factor VIII
Gaucher disease, type 1 Glucocerebrosidase
Fabry disease Galactosidase
Adenosine deaminase deficiency Adenosine deaminase
Transplantation Transplant:
Fabry disease Kidney
Cystinosis Kidney
Alport syndrome Kidney
Mucopolysaccharidoses Bone marrow
Tyrosinemia Liver
Surgical removal Remove:
Hereditary spherocytosis Spleen
Multiple polyposis of the colon Colon
Medullary thyroid carcinoma syndrome Thyroid

Some inborn errors of metabolism, as well as other types ●●● QUESTIONS

of genetic disorders, may not present symptoms until months
to years after birth even though the genetic defect is present 1. At a 6-month well-baby check, the mother mentions
at birth. For example, infants normal at birth will begin to the infant’s diapers turn dark before washing. All
developmental milestones were normal and there
demonstrate the devastation of mucopolysaccharidoses (see
were no abnormalities on physical examination. Uri-
Chapter 8) around age 2. Hemochromatosis (see Chapter 10)
nalysis was negative for sugar, protein, blood, bile
generally is not diagnosed until the ages of 30 to 50 years in
salts, or pigments. Tests for homogentisic acid were
men and beyond age 50 in women.
positive. Which of the following is the best diagnosis
For many disorders, several treatment options may be avail- for this infant?
able, but risks vary. For example, in maple syrup urine disease,
liver transplantation has been successful in several patients. A. Alkaptonuria
However, the risk and potential complications of this therapy B. Neonatal tyrosinemia
are considerably higher than the low risk of dietary restric- C. Phenylketonuria
D. Tyrosinemia type I
tion, with which individuals have good outcomes.
E. Tyrosinemia type II
Answer. A
KEY CONCEPTS
■ Metabolic disorders are also referred to as inborn errors of Explanation: Historically, urine has been important in diag-
metabolism. noses of metabolic disorders. Alkaptonuria is a defect in
homogentisic acid oxidase, and urine gradually darkens
■ The basic biochemical premise in these disorders is that sub-
when exposed to air. Other metabolic disorders, such as
strate + enzyme yields product.
phenylketonuria and maple syrup urine disease, have
■ Mutations in enzymes caused increased substrate and insuffi- distinctive smells to urine.
cient product.
■ Identification early through newborn screening tests may reduce
or eliminate clinical expression of the disease. Additional Self-assessment Questions can be Accessed
at www.StudentConsult.com
Cancer Genetics 5
CONTENTS accelerating cell division, whereas tumor suppressor genes
allow uncontrolled growth that was normally suppressed.
When tumor suppressor genes suffer incapacitating muta-
GENETIC REGULATION OF THE CELL CYCLE
tions, the cell loses the capacity to guard against tumor
G1-to-S Cell Cycle Transition formation.
Genetic Paradigm for Cancer Oncogenes and tumor suppressor genes are activated or
inactivated by various mutational mechanisms, including
ONCOGENES
point mutation, deletion, and chromosomal rearrangement,
Oncogene Activation by Translocation and each has a prominent role in sporadic and familial
Gene Amplification cancers. Although small subsets of cancers are associated
TUMOR SUPPRESSOR GENES
with inherited genetic mutations passed from generation to
generation in a family, overall, cancer appears to result from
Cell Division–Controlling Genes an accumulation of such deleterious mutations in somatic
Neurofibromatosis Type 1 tissues. While some mutations are found across multiple
DNA REPAIR GENES cancer types, other mutations are generally specific to certain
cancer syndromes, demonstrating the importance of such
Colorectal Cancer and the Genetic Paradigm for Cancer genes to particular tissues. With the identification of an
Revisited increasing number of cancer genes, attention is focused on
Breast Cancer
molecular genetic screening for at-risk individuals. This
Ovarian Cancer
has the advantage of providing early and presymptomatic
APOPTOTIC GENES detection, thus allowing appropriate cancer surveillance and
treatment.
p53 Protein and Li-Fraumeni Syndrome
Prostate Cancer

TELOMERES AND TELOMERASE

●●● GENETIC REGULATION OF
CYTOGENETIC ALTERATIONS IN CANCER AND TUMOR THE CELL CYCLE
HETEROGENEITY
Many genes control the complex and critical balance between
DNA-BASED CANCER SCREENING cell growth and the factors preventing unchecked growth. In
general, these genes are involved in cell cycle regulation, cel-
lular adhesion, contact inhibition, DNA repair and mainte-
nance, and programmed cell death (known as apoptosis).
Mutations in such genes can thereby initiate or perpetuate
cancer. This is particularly true for those gene products
involved in cell cycle regulation.
The eukaryotic cell cycle has four phases (Fig. 5-1). Three
It became clear that cancer is a genetic disease several of these, G1, S, and G2, comprise interphase—the period
decades ago. Under normal conditions, cells balance cell between two successive mitoses. The cell spends the great
division and cell death, and these processes are accom- majority of time in interphase, typically 16 to 24 hours.
plished and preserved by several biochemical “checkpoints” The fourth phase, M (“mitosis”), is relatively fast, taking
during the cell cycle. When these checkpoints are impinged, 1 to 2 hours. During the G1 (“gap 1”) phase, the cell
uncontrolled cellular proliferation and cancer can result. undertakes normal metabolic activities and prepares for
Because gene products regulate the life-death balance, DNA synthesis. Cells are diploid, and each chromosome
mutations can destroy the checkpoints and promote cellular consists of a centromere and a single chromatid. This phase
transformation into a neoplasia. Two general classes of cancer generally shows the most variability in length, lasting only
genes have been identified: oncogenes and tumor suppres- a few hours or for years. DNA replication occurs in the S
sor genes. Oncogenes facilitate tumor formation by directly (“synthesis”) phase. At this point, the cell is diploid and
 66 Cancer Genetics

External signals (growth

factors, integrins)

MYC, RAS, and other genes

DNA damage,
Cyclin D cell stress
Feedback
regulation
+CDK4
p53
Cyclin D/CDK4 (active complex) MDM2
p16INK4 p21

RB phosphorylation in p14ARF
E2F/DP1/RB complex

Active E2F

Cyclin E transcription (also

cyclin A, DNA polymerases,
and many other genes)
Figure 5-1. Movement from one
phase of the cell cycle to another is
dependent upon the regulating factors.
Cyclin E
MYC and RAS genes, activated by
external factors, initiate the synthesis +CDK2
and stabilization of cyclin D. In the
nucleus, the active cyclin D–CDK4
complex phosphorylates RB in the Cyclin E/CDK2 (active complex)
E2F-DP1-RB complex, causing the
p27
activation of E2F. E2F is a transcription
factor for cyclin E and cyclin A. The cell
cycle can be blocked by p21 and p27 S
(CIP/KIP inhibitors) and by p16INK4A and
p14ARF (INK4A/ARF inhibitors). In the Cell
event of DNA damage and cell stress, cycle
p53 causes cell arrest. (Redrawn from G1
Kumar V, Abbas A, Fausto N. Robbins G2
& Cotran Pathologic Basis of Disease, M
7th ed. Philadelphia, WB Saunders,
2004, p 291.)

each chromosome is represented as a bivalent, with two

HISTOLOGY
sister chromatids sharing a centromere. Here, it is important
to note that the cell expends a considerable amount of Mitotic Spindle
resources to provide regulatory checkpoints at the G1-to-S
The mitotic spindle is a network of microtubules formed of α- and
transition. This is because the S phase represents a com- β-tubulin during prophase. Microtubular organizing centers,
mitment to DNA synthesis and the subsequent cell division. such as the mitotic spindle apparatus that includes basal bodies
During G2 (“gap 2”) phase, cells prepare for mitosis by and centrioles, direct the assembly and disassembly of
manufacturing tubulin, necessary for microtubule formation microtubules and organize them in such a fashion that they are
as a component of the mitotic spindle apparatus. Mitosis oriented perpendicular to the plane of division.
concludes the cycle, with some cells reentering the G1 phase Microtubules must attach to the kinetochore, a point within
and recapitulating the cycle. Certain cells such as neurons, the centromere of the chromatids of the metaphase
skeletal muscle, and erythrocytes, however, are postmitotic chromosome, for metaphase to proceed correctly. MAD
in that they do not divide again after they have differenti- proteins, which block the beginning of anaphase, remain
attached to the kinetochore until microtubules attach. The
ated. These cells are suspended in a perpetual G1 state,
kinesin spindle protein drives the formation of the spindle
sometimes termed G0. Occasionally, such cells can be
and enables chromosome segregation.
induced—for example, by growth factor or hormone—to The mitotic spindle is a target for antimitotic drugs, such
reenter the cycle. as paclitaxel (Taxol) and vinblastine.
 Genetic Regulation of the Cell Cycle 67

Cells are provoked into cell division by the initiating actions

G1-to-S Cell Cycle Transition
of growth factors and hormones that permit transitions
through the cell cycle. This process typically begins at the cell For the cell to survive, all criteria for survival must be met
membrane by receptor-ligand, or growth factor–receptor, before the cell enters S phase. Fundamentally, this means the
interaction and is controlled by polypeptides called cyclins genome must be prepared and ready to replicate. Otherwise,
and their associated cyclin-dependent kinases (CDKs). Differ- damaging mutations are likely and the cell will either die or
ent cyclins are present within the cell at different stages of progress toward a pathology such as cancer. Thus, several
the cell cycle (Fig. 5-2). For example, at the all-important checkpoints, in the form of regulatory proteins, are in place.
transition from G1 to S, which marks a commitment to DNA Principal among these are cyclin D, CDK4, and CDK6, the
synthesis and mitosis, cyclin D is expressed following growth retinoblastoma gene product (pRB), and the E2F transcription
factor stimulation. Inhibitors of CDKs (cyclin kinase inhibi- factor (Fig. 5-3). Prior to transition to S phase, the pRB
tors, CKIs) regulate cyclin-CDK complexes. Because they protein is “active” by virtue of its hypophosphorylated state
inhibit the action of cyclin-CDK complexes, CKIs also tend and is bound to the E2F factor, thereby inhibiting the expres-
to slow cell cycle progression. Since the G1-to-S transition sion of genes necessary for DNA replication and cell prolifera-
hallmarks a commitment to cell division, the molecular tion. Here, the pRB protein acts as the “master brake” of the
genetic events that regulate this transition will be examined cell cycle progression. As the cell signals the time for replica-
in greater detail. tion, growth factors stimulate the expression of cyclin D.
Cyclin D, in turn, is bound by CD4 and CD6, resulting in
an activated protein kinase complex. An important target of
this complex is the RB1 gene. When phosphorylated by the
BIOCHEMISTRY cyclin D–CDK complex, pRB is inactivated and unable to
bind E2F. This allows the released E2F transcription factor to
Cyclins and Cyclin-dependent Kinases translocate to the nucleus, where it promotes the expression
The cyclin superfamily consists of proteins that bind to and of genes important to DNA replication and cell growth.
activate cyclin-dependent kinases (CDKs). Activation is Other cell cycle “brakes” exist besides pRB. As mentioned
required for progression through the cell cycle. The major above, CKIs can regulate the cell cycle by inhibiting the activ-
cyclins controlling the cell cycle are: ity of the cyclin-CDK complex. CKIs are classified into two
■ Cyclin D—G1 phase
groups: the CIP/KIP group and the INK4 group. CIP/KIP
■ Cyclins A and E—S phase
CKIs include the p21, p27, and p57 gene products and inhibit
■ Cyclins A and B—M phase
all cyclin-CDK complexes. INK4 members include the p15,
Major CDKs are:
■ CDK4, CDK6—G1 phase
p16, p18, and p19 gene products, and these proteins inhibit
■ CDK2—S phase
only the cyclin D–CDK4/6 complexes. In general, the CKIs
■ CDK1—M phase act as cell cycle inhibitors by inactivating the cyclin-CDK
complexes, which in turn keep pRB unphosphorylated and

Cyclin D Constitutively
degradation produced CDK2

Cyclin B
synthesis

S
CDK2/cyclin B
Cyclin D (P)
G2 ;
Cell CDK25 Figure 5-2. The cell cycle requires
P G1 cycle specific cyclins and cyclin-dependent
kinases (CDKs) to progress through
+Inactive each stage. The transition from G1 to S
M
kinase Active phase is regulated by cyclin D (D1-3)
CDK2/cyclin B and cyclin E that are synthesized in G1
complex and active when phosphorylated.
Cyclins A and B1-2 control transition
Cyclin D
from G2 to M. Cyclin B binds to CDK2,
synthesis Cyclin B
and this complex is activated by
G0 degradation
phosphorylation by CDK25 and other
kinases.
 68 Cancer Genetics

complexed to the E2F transcription factor. Interestingly,

certain CKIs (p21, for example) can be expressed preferen-
tially during times of cellular stress, including DNA damage. p16INK4a Cyclin D1
Inhibits
This has the benefit of halting entrance into the S phase so CDK4
that the cell has time to repair any genome damage prior to or CDK6
DNA replication. Stated differently, if a cell is stressed, p21 P P P P
is expressed and cyclin-CDK complexes remain inactive and
Hyperphosphorylation pRB
thereby the phosphorylation of pRB is slowed. Since more
pRB remains unphosphorylated and complexed to E2F,
E2F is unavailable as a transcription factor, slowing or halting
the G1-to-S transition. pRB Dissociation

E2F
Inactive

E2F
Repression of Active
BIOCHEMISTRY transcription

Cyclin Kinase Inhibitors

Cyclin kinase inhibitors (CKIs) inhibit cyclin-CDK complexes. Activation of
E2F-dependent
There are two classes of CKI: the INK4 family (p15/p16/p19) DNA transcription
and the CIP/KIP family (p21, p27) G1 phase
The INK4 family is specific for CDK4–cyclin D and Resting
phase S phase
regulates cell cycle entry in response to growth factors. Cell proliferation
Cyclin D activates CDK4, the kinase that phosphorylates RB. phase
This phosphorylation inhibits the normal growth-suppressive
function of RB and allows cell proliferation. Cell cycle progression
The CIP/KIP family is induced by p53 and can regulate all
classes of CDK-cyclins by inhibiting all cell cycle regulatory
kinases (CDKs). Thus, this inhibition prevents progression
through the cell cycle. Therefore, the loss of p53 and CIP/
KIP CDKs allows uncontrolled cell proliferation. These Figure 5-3. Control of cellular proliferation by
proteins may also mediate normal control and cell cycle retinoblastoma. When the retinoblastoma protein (pRB) exists
response to damage. in the underphosphorylated state, it can block exit from G1
and progression of the cell cycle.

It can be appreciated now that tumors arise from alterations Intense molecular genetic and family studies over the last
in the cell cycle resulting from abnormal proteins that dra- two decades have revealed that the initial mutation leading
matically alter the balance between cell growth and cell to cancer either can occur spontaneously (de novo) in certain
death. The principal mechanisms for cell cycle perturbation sporadic patients or can be inherited, thus prescribing predis-
are mutations in those genes that regulate it. Predictably, the position to heritable cancer. In either case, a single mutational
majority of genes associated with cancer are important in event is rarely sufficient for neoplasia. Rather, a genetic para-
normal cell growth, proliferation, differentiation, death, and digm has emerged in which cancer results from a progressive
maintenance of the genome. or sequential accumulation of mutations (Fig. 5-4). Often
mutations occur in genes necessary for DNA maintenance
and repair. Such mutations may facilitate the process by
increasing the likelihood of mutation within the cell. Accord-
Genetic Paradigm for Cancer ingly, with each new mutational “step,” cellular regulation of
Most of the time, cancer originates following genetic changes growth versus stasis or death diminishes, ultimately resulting
in a single cell; hence, cancers are monoclonal and all tumor in a cell lineage that divides without constraint. At this point,
cells are derived from a single progenitor. This cell exhibits normal cell metabolism is profoundly altered, and cells may
altered physiologic properties that upset normal cellular regu- obtain the ability, through further genetic change, to infiltrate
lation of proliferation and death. Because daughter cells will other tissues and metastasize to other organs, thus complet-
carry the same mutation or mutations, the propensity for ing the transformation into a malignant neoplasm (see Fig.
unchecked cellular growth is perpetuated during subsequent 5-4). Below, the salient features of oncogenes and tumor
cell divisions. Thus, all cells in a tumor have a common suppressor genes are discussed along with examples of their
origin. role in cancer.
 Oncogenes 69

PATHOLOGY MICROBIOLOGY
Types of Cancers Retroviruses
Major types of cancer can be described as: Retroviruses give rise to transposable elements. There are
■ Carcinoma (85% of all cancers): derived from epithelial usually three genes flanked by direct repeats. The genes of
tissues; includes cancers of glands, breast, skin, and retroviruses include:
most internal organs ■ gag: codes for core and structural proteins
■ Sarcoma: cancer of connective tissue origin, such as ■ pol: codes for reverse transcriptase, protease, and

muscle, bone, adipose tissue, cartilage, blood vessels, or integrase

other connective or supportive tissue ■ env: codes for viral coat proteins
■ Leukemia: uncontrolled proliferation of leukocytes by Reverse transcriptase transcribes the viral RNA into DNA
either lymphoid organs or bone marrow in the cytosol. The DNA then enters the nucleus and
■ Lymphoma: malignant cancer of lymphoid organs, integrates into the genome, where it may cause insertional
particularly the spleen and lymph nodes mutation. It may also translocate to another region
(transposition) and carry additional genome sequences.

A large number of oncogenes have been identified (Table

PATHOLOGY
5-1). They are named by three-letter abbreviations that indi-
Metastasis cate their origin and the type of resultant tumor. For example,
Metastasis is the movement or spreading of cancer cells from
the RAS oncogene was initially identified through retroviral
one organ or tissue to another, usually through the studies of rat sarcomas. Normally, RAS functions as a G
bloodstream or lymph, and is affected by type, stage, and protein, a small protein that binds and hydrolyzes GTP. G
location. Metastasis is characteristic of all malignancies; proteins have a number of roles within the cell, including
benign tumors are not metastatic. Cancer at a secondary site regulating cellular proliferation, morphogenesis, cell motility,
usually has the same characteristics as the primary cancer. and cytokinesis. These proteins act as cell regulators or
switches via an intrinsic activation mechanism. They are
activated to regulate cellular processes when bound to GTP,
but these proteins slowly hydrolyze the GTP to GDP and
Pi using an intrinsic GTPase activity (Fig. 5-5). In the acti-
vated (GTP-bound) state, G proteins are able to bind target
proteins, but this capacity diminishes as GTP is slowly con-
verted to GDP. Overall, the activities of G proteins are
regulated by a number of G protein–related accessory pro-
teins that serve to exchange GDP for GTP (GEF, or guanine
●●● ONCOGENES
nucleotide exchange factor); stimulate the rate of GTP hydro-
Oncogenes (“cancer genes”) are typically mutated genes that lysis (GAP, or GTPase activating proteins), thus inactivating
produce an aberrant protein that stimulates cell division, pro- the protein; or inhibit the dissociation of GDP from the
liferation, or differentiation, even in the absence of proper protein (GDI, or GDP dissociation inhibitor), which is also
growth signals. The majority of oncogenes are derived from an inactivating mechanism.
proto-oncogenes, which are normal genes that participate in
growth regulation. Because they regulate cellular prolifera-
tion, such genes are often growth factors, growth factor recep-
tors, transcription factors, nuclear protein regulators, or cell
signaling molecules. Mutations within proto-oncogenes occur BIOCHEMISTRY
either by spontaneous mutation or via a mutagen that pro-
motes genetic change within the cell. Alternatively, a cell can G Proteins
undergo cancerous “transformation” from a normal to an G proteins are heterotrimers that bind GDP and GTP. These
abnormal state through the introduction of an oncogene from heterotrimers are found in all cells, and include three
a virus. Retroviruses, for example, are capable of transporting subunits: Gα, which has a binding site for nucleotide; Gβ;
oncogenic versions of genes into human host cells via integra- and Gγ. Heterotrimers associate with the inner cell
membrane surface as well as transmembrane receptors,
tion of their genome into the human chromosome. Even if a
such as receptors for polypeptide hormones.
transforming retrovirus carries a proto-oncogene, it is quite
GDP is bound to Gα in the inactive G protein state.
possible that, given the high rate of mutation observed in Allosteric change occurs in Gα, and GTP replaces GDP. The
retroviruses, a mutation will occur that alters the properties activated Gα then activates an effector molecule.
of the gene and turns it into an oncogene.
 70 Cancer Genetics

Acquired DNA Normal cell

mutations from
environmental agents: Successful
(Chemicals, irradiation, DNA repair
viruses)
DNA damage

Failure of DNA repair Inherited mutations in

genes affecting DNA
repair and genes
affecting cell growth
or apoptosis
Mutation in the genome
of somatic cells

Activation of Inactivation of Alterations in

growth-promoting tumor suppressor genes that regulate
oncogenes genes apoptosis

Unregulated cell Decreased

proliferation apoptosis

Clonal expansion

Angiogenesis
Additional mutations
Escape from immunity

Tumor progression
Figure 5-4. Simplified schematic for
the molecular basis of cancer.
(Redrawn from Kumar V, Abbas A, Invasion and
Fausto N. Robbins & Cotran Pathologic Malignant neoplasm metastasis
Basis of Disease, 8th ed. Philadelphia,
WB Saunders, 2010, p 280.)

Specifically, RAS is located in the plasma membrane and is mutations. As discussed later, tumor suppressor genes do not
involved in regulation of cellular proliferation through inter- share this genetic mechanism.
action with growth factors (see Fig. 5-5). Normally, when RAS
binds GTP, it undergoes a conformational change to its acti-
vated state and binds a protein kinase called RAF, a serine
Oncogene Activation by Translocation
protein kinase also called MAPKKK (for mitogen-activated Burkitt Lymphoma
protein kinase kinase kinase), initiating the MAP kinase– Burkitt lymphoma is a rare cancer of the jaw found in African
mediated protein phosphorylation cascade that ultimately children. Cytogenetic analysis confirms that the majority of
results in the activation of at least one nuclear transcription patients have a balanced translocation involving the MYC
factor that regulates the expression of genes important in cell proto-oncogene from chromosome 8q24 to chromosome
proliferation. A mutation altering a single base pair in RAS 14q32 [t(8;14)(q24;q32)] (Fig. 5-6). Occasionally, the MYC
is all that is necessary to produce an abnormal RAS that is gene is translocated to chromosome 2q11 or chromosome
constantly in the “active” conformation. In this case, the 22q11. In each position, the MYC gene is placed in a new
abnormal RAS protein promotes cellular growth and is con- genomic environment that features the genes for immuno-
strained by normal cellular checkpoints, thus promoting globulin (Ig) heavy chains or κ light and λ light chains, respec-
tumor formation. In fact, RAS mutations are found in many tively. It is clear that this new genetic address results in
tumor types, including colon cancer, as will be discussed later. uncontrolled MYC expression. Though MYC is constitutively
Functionally, oncogenes exact their transforming capacity expressed at low levels in cells, its translocation places it
through dominant transmission, since only a single allele under different promoter control—that of the immuno-
needs to be mutated in order to deregulate cell proliferation. globulin genes with higher expression levels—resulting in its
Hence, oncogenes typically act as dominant, gain-of-function overexpression. This is critical to cancer formation since the
 Oncogenes 71

TABLE 5-1. Examples of Oncogenes and Associated Cancers

ONCOGENE LOCATION FUNCTION ASSOCIATED CANCERS

Growth Factor Genes

HST 11q13 Fibroblast growth factor Stomach carcinoma

SIS 22q12 β Subunit of platelet-derived growth Glioma
factor

Growth Factor Receptor Genes

RET 10q Receptor tyrosine kinase Multiple endocrine neoplasia, thyroid carcinomas
ERB-B 7p12 Epidermal growth factor receptor Glioblastoma, breast cancer
ERB-A 17q11 Thyroid hormone receptor Acute promyelocytic leukemia
NEU (ERB-B2) 17q21 Receptor protein kinase Neuroblastoma, breast cancer
MET 7q31 Receptor tyrosine kinase Hereditary papillary renal carcinoma, hepatocellular
carcinoma
KIT 4q12 Receptor tyrosine kinase Gastrointestinal stromal tumor syndrome

Signal Transduction Genes

H-RAS 11p15 GTPase Carcinoma of colon, lung, pancreas

K-RAS 12p12 GTPase Melanoma, thyroid carcinoma, acute monocytic leukemia,
colon carcinoma
B-RAF 7q34 Serine/threonine kinase Malignant melanoma, colon cancer
ABL 9q34 Protein kinase Chronic myelogenous leukemia, acute lymphocytic
leukemia
CDK4 12q14 Cyclin-dependent kinase Malignant melanoma

Transcription Factor Genes

N-MYC 2p24 DNA-binding protein Neuroblastoma, lung carcinoma

MYB 6q22 DNA-binding protein Malignant melanoma, lymphoma, leukemia
FOS 14q24 Interacts with JUN oncogene to Osteosarcoma
regulate transcription

From Jorde LB, Carey CC, Bamshad MJ, White RL. Medical Genetics, 3rd ed. St. Louis, Mosby, 2003, p 236.

MYC protein is a transcription factor whose targets are a translocations seen in the endemic form of Burkitt lymphoma,
number of genes important in cell proliferation and cell cycle in the sporadic form c-MYC exons 2 and 3 are translocated
control. Hence, Burkitt lymphoma is an example of a chro- to the switch region of the Ig heavy chain locus; a cryptic
mosomal rearrangement that places a normal gene in a new promoter next to exon 2 is used to overexpress c-MYC.
genetic environment, altering its expression and thereby
promoting cellular transformation.
Burkitt lymphoma is endemic in Africa, and most people
associate it with an African B-cell neoplasm involving the PATHOLOGY
maxilla or mandible. However, there is also a sporadic form
Hodgkin versus Non-Hodgkin Lymphomas
that usually does not involve the maxilla or mandible. This
form, appearing in Caucasians, involves the abdominal organs Hodgkin lymphomas and non-Hodgkin lymphomas (NHLs)
most frequently and presents with tumors causing swelling differ in several respects. Hodgkin lymphomas arise in a
single or related nodes, spread continuously, and contain
and pain. The distal ileum, cecum, or mesentery are more
Reed-Sternberg cells.
often involved than other abdominal organs, pelvic organs,
Non-Hodgkin lymphomas may arise at extranodal sites
or facial bones. Epstein-Barr virus is strongly implicated in and spread in an unpredictable manner. There are about 20
the etiology of the African form, but this correlation is types of NHL, including Burkitt lymphoma.
less clearly understood in the sporadic form. Unlike the
 72 Cancer Genetics

Growth factor ligand

Receptor-ligand complex

RAS
RAS
GDP
P GTP
Growth factor P
receptor RAF (MKKK)

Other substrates: Bridging protein GAP

Cytoplasmic PI3 kinase complex
effects PLC-γ (GRB2, SOS) MAP
SRC kinase
MEK cascade
Cytoplasmic (MKK)
effects
ERK
(MK)
Transcription factors P
Cytoplasm

Nucleus Gene activation

Figure 5-5. Tyrosine receptor signaling. Growth factor ligand causes the dimerization and autophosphorylation of tyrosines.
Bridging proteins (BRB2/SOS) bring the receptor into the vicinity of RAS, where it catalyzes guanine exchange on RAS. The
activated RAS associates with RAF1, a serine/threonine protein kinase, and RAF1 then activates and phosphorylates MEK. The
MAP kinase cascade interacts with proteins that are translocated to the nucleus, where transcription factors are activated.
(Redrawn from Kumar V, Abbas A, Fausto N. Robbins & Cotran Pathologic Basis of Disease, 7th ed. Philadelphia, WB Saunders,
2004, p 99.)

Translocation

H chain enhancer
H chain genes C-MYC gene
C-MYC gene
Translocation
Chr 8 Chr 14

Figure 5-6. Burkitt lymphoma. The c-MYC gene is

translocated downstream of the immunoglobulin heavy chain
enhancer. Expression of c-MYC is up-regulated by its position
BCR gene Fused
near the enhancer. ABL gene ABL-BCR gene

Chronic Myeloid Leukemia

Chronic myeloid (myelogenous) leukemia (CML) is a form of Chr 9 Chr 22 Philadelphia
chromosome
leukemia hallmarked by a uniquely abnormal chromosome,
the Philadelphia (Ph) chromosome (Fig. 5-7). The Ph chromo- Figure 5-7. Chronic myeloid leukemia. The ABL gene on
some results from a translocation from the long arm of chro- chromosome 9 inserts into the BCR gene on chromosome 22
mosome 9 to the long arm of chromosome 22. This t(9;22) and creates an altered gene and protein.
 Oncogenes 73

(q34;q11) rearrangement is a balanced, reciprocal transloca-

tion that is found in 90% of patients with CML. The result of PHARMACOLOGY
the translocation is a slightly longer chromosome 9 and a
smaller chromosome 22. The translocation relocates the ABL Imatinib Mesylate (Gleevec)
proto-oncogene from 9q to 22q, where it is positioned within Imatinib mesylate (Gleevec) is a specific inhibitor of tyrosine
the “breakpoint cluster region 9,” or BCR, consisting of genes kinases, including BCR-ABL. It blocks the binding site for
of unknown function. Here, unlike Burkitt lymphoma, the ATP on the kinase and prevents phosphorylation of tyrosines
genetic juxtaposition results in a novel fusion protein com- in proteins, which prevents signal transduction pathways
leading to transformation in CML. It is metabolized by
posed of BCR-encoded amino acids at the N-terminus and
CYP3A4 of the P-450 enzyme system, and it is most effective
ABL-encoded amino acids at the C-terminus. This constitu-
in the chronic phase of CML.
tively expressed BCR-ABL protein chimera is a 210-kDa
protein with an enhanced tyrosine kinase activity compared
with the normal ABL gene product, a change in activity that
promotes neoplasia. While the 210-kDa chimera is the
Gene Amplification
primary protein expressed, other forms may be expressed
also depending on the exact breakpoint in BCR (Fig. 5-8). The phenomenon of gene duplication is another mechanism
Product p230 is associated with chronic neutrophilic leuke- of altering the expression patterns of otherwise normal genes
mia and thrombocytosis. such that they become oncogenes. In gene amplification,
There are three phases of CML, which are progressive in certain regions of DNA are duplicated and amplified, result-
symptomology as well as in increasing resistance to therapies. ing in as many as several hundred additional copies of genes
Understanding the tyrosine kinase activity of the chimeric contained within the amplified DNA. These changes are often
protein led to the development of the first in a new class of visible at the microscopic level or are readily detected by
pharmacologic agents specifically targeted to a cancer. Ima- modern cytogenetic techniques such as fluorescent in situ
tinib mesylate (Gleevec) is a specific and potent inhibitor of hybridization (FISH) or comparative genome hybridization
the BCR-ABL tyrosine kinase. Its efficacy has been demon- (CGH) (Fig. 5-9).
strated in different phases of CML disease (Box 5-1). It is also Two general patterns of chromosomal alterations are
effective against other tyrosine kinases, such as ABL, platelet- observed with gene amplifications: multiple small
derived growth factor receptor, and the stem cell factor chromosome-like structures called double minutes (dmin) and
receptor known as c-KIT. homogeneously staining regions (HSRs). Together, dmin and

BCR
b1 b2 b3 b4 b5
e1 2 3 4–8 9–11 12–16 17 19 24

m-BCR (~55 kb) M-BCR (5.8 kb) m-BCR

ABL
a1 a2 a3 a4 a5 a6 a11 a12

Breakpoint region (~200 kb)

BCR ABL
e1 b2 a2
b2a2-p210
e1 b2 b3 a2
b3a2-p210
e1 a2
e1a2-p190
e1 e19 a2
e19a2-p230

Figure 5-8. The major forms of the BCR-ABL fusion gene. Translocations involving the m-BCR region of the BCR gene lead to
joining of the second or third exons of this region (b2, b3) with the second exon of the ABL gene (a2) to form the b2a2 or b3a2
transcripts and the p210 product. Breakpoints in the m-BCR region of the BCR gene result in fusion of the first exon of this
gene (e1) with the second exon of the ABL gene (a2) to form the e1a2 BCR-ABL transcript that encodes the p190 hybrid
protein. Breakpoints in the m-BCR region of the BCR gene juxtapose the exon 19 (e19) of this gene to the second exon of the
ABL gene (a2) to produce the e19a2 transcript that encodes the p230 protein. (Redrawn courtesy of Rajyalakshmi Luthra, PhD,
University of Texas MD Anderson Cancer Center, Houston, Texas.)
 74 Cancer Genetics

Box 5-1. FEATURES OF CHRONIC MYELOID ●●● TUMOR SUPPRESSOR GENES

LEUKEMIA Whereas oncogenes are typically dominant, gain-of-function
mutations, tumor suppressor genes facilitate neoplastic trans-
Chronic Phase formation via a cellular recessive model where there is a loss
Less than 10% to 15% blast cells of function in both alleles. In the normal state, these genes
WBC count ≥20 × 103/μL are active in “putting the brakes on” cellular proliferation. In
Symptoms mild this way, they function oppositely to growth-promoting
25% to 40% progress directly to blast crisis oncogenes.
Average duration: 5 to 6 years Tumor suppressor genes (Table 5-2) can be subclassified
Accelerated Phase into three general groups: genes that control cell division,
genes that repair DNA, and genes that destroy cells. The first
10% to 15% blast cells (<30%)
Symptoms may include fever, bone pain, splenomegaly, group is also referred to as “gatekeeper” genes that are
hepatomegaly actively and directly involved in cell cycle, cell growth, or
Cytogenetic abnormalities increase—duplication of Ph contact inhibition regulation. The second group is also referred
chromosome, isochromosomes, trisomies to as “caretaker” genes that are responsible for DNA repair
Average duration: 6 to 9 months and genomic maintenance; thus, when inactivated by muta-
tion, these genes indirectly facilitate cancerous cellular change
Blast Crisis
by enabling genetic change. If too much DNA damage accu-
More than 30% blast cells mulates within a cell, the cell undergoes apoptosis controlled
50%—Myeloid blast crisis by the third group of genes.
25%—Lymphoid blast crisis
25%—Mixed
Symptoms may include anemia and infection, CNS disease, Cell Division–Controlling Genes
lymphadenopathy, bleeding
Poor prognosis Retinoblastoma
Average duration: 3 to 6 months Retinoblastoma (Rb) is a model for tumor suppressor genes
in both inherited and sporadic cancer and is the most
common intraocular tumor in early childhood, with an
incidence of 1 in 20,000 children. Unless treated, it is fatal.
HSRs are seen in roughly 10% of tumors and are most fre- Retinoblastoma serves as the paradigm for tumor suppressor
quently detected in the latter stages of cancer. The mechanism gene–related malignancies. Rb is caused by the genetic
for dmin formation is poorly understood. HSRs represent inactivation of the RB1 gene on chromosome 13q14.3. RB1
regions of amplification that do not label normally with chro- mutations inactivate the RB1 gene product, a 110-kDa
mosomal identification techniques; hence, they lack a normal phosphorylated protein that regulates the cell cycle at the
banding pattern in karyotypic analysis. These amplified point of the G1-to-S transition (see Fig. 5-3). Absence of
regions often contain proto-oncogenes such as RAS and pRB deconstrains this mitotic checkpoint, thus allowing cell
members of the MYC family. For example, the n-MYC gene proliferation. Because of its role in cell cycle control, the
is amplified over 100-fold in 25% to 40% of late-stage neu- pRB protein provides an excellent example of a gatekeeper
roblastoma, a malignancy of neuroblasts found in infants or tumor suppressor gene.
children that has widespread metastatic potential (Fig. 5-10). The most common clinical presentation of retinoblastoma
However, n-MYC amplification is rare in early-stage neuro- in children is leukocoria, a white pupillary reflex; likewise,
blastoma. Hence, the appearance of amplified n-MYC appears over 50% of leukocoria is associated with retinoblastoma
to signify or promote an aggressive tumor and therefore (Fig. 5-11). Retinoblastoma originates from primitive retinal
predicts a poor prognosis. cells and may grow either over the retina and into the vitre-
ous humor or under the retina, causing detachment. If unde-
tected or untreated, the tumor may extend into the sclera,
into the orbital lymphatics, or through the optic nerve (Fig.
BIOCHEMISTRY 5-12). It is the replacement of the vitreous humor with tumor
that causes leukocoria. Another common presentation in
MYC Protein Family
children is strabismus.
The MYC family of oncogenes functions in a sequence- The existence of inherited cancer syndromes such as Rb
specific manner to regulate transcription and includes heralded the discovery of tumor suppressor genes. The rela-
c-MYC, n-MYC, l-MYC, s-MYC, and b-MYC. The MYC tionship between the two was first elucidated in 1971 by
proteins function in cell proliferation, differentiation,
A. G. Knudson, who observed the rare Rb tumor in patients
apoptosis, and transformation as components of the cell
with and without a family history of retinal tumors. Knudson
cycle. However, if regulation of these genes is altered, they
can lose the status of proto-oncogenes and become proposed that two separate mutations were necessary for
oncogenes associated with cancer. tumor development. One mutation might present in the
germline cells and therefore was termed a “constitutional”
 Tumor Suppressor Genes 75

1. Labeling of genomic
tumor DNA and normal
genomic control DNA
by nick translocation
Biotin-labeled tumor DNA Digoxigenin-labeled
control DNA

2. Simultaneous hybridization of
differentially labeled tumor and
control DNAs to normal human
metaphase spreads

3. Fluorescence detection of
the hybridized DNAs

Figure 5-9. Comparative genomic

hybridization (CGH). This cytogenetic
technique demonstrates a change in
4. Result DNA content of tumor cells. CGH can
detect unbalanced chromosome
changes only such as seen with double
minutes (dmin) and homogeneously
staining regions (HSRs) by comparing
the ratio of the intensities of the two
Balanced Overrepresentation Underrepresentation High-level fluorochromes along target
DNA content of the whole of the long arm amplification chromosomes to indicate regions of
chromosome within within the DNA gain or loss. It will not detect
the tumor DNA tumor DNA
balanced changes such as reciprocal
translocations.

mutation. This mutation would be present in every somatic ANATOMY

cell in the body but would also be passed on to subsequent
generations. The second mutational event would be a new The Eye
mutation in the other, nonmutated RB1 allele. To effect Rb, The eye is formed as an outgrowth of the optic vesicle and
this second mutation would occur within a retinal cell—a consists of several layers:
■ External fibrous region: sclera, cornea
somatic cell. The combination of the first—inherited—“hit”
■ Middle vascular region: choroid, ciliary body, iris
and the second—somatic—“hit” effectively eliminates the
■ Internal region: retina
protein of both alleles of the RB1 gene in an individual and
The internal region includes both the neural retina (optic
thus permits tumor formation. This paradigm of tumor sup-
disk, macula, fovea centralis, rods, and cones) and the
pressor gene action is known as Knudson’s “two-hit” hypoth- pigmented retina. Retinal detachment occurs when the
esis and serves as a model for cancers resulting from tumor neural retina becomes separated from the pigmented retina.
suppressor genes.
 76 Cancer Genetics

A B
Figure 5-10. Fluorescence in situ
hybridization with the probe MYCN.
A, Interphase nucleus with dmin and
one HSR from patient 1. B, Interphase
nucleus with dmin and five HSRs from
the same patient. C, Metaphase cell
with dmin and one HSR from patient 2.
D, Metaphase cell from the same case
with dmin and two HSRs. (From
Yoshimoto M, de Toledo SRC, Caranet
EMM, et al: MYCN gene amplification:
identification of cell populations
containing double minutes and
homogeneously staining regions in
neuroblastoma tumors. Am J Pathol
1999;155:1439–1443. With permission
from the American Society for C D
Investigative Pathology.)

TABLE 5-2. Examples of Tumor Suppressor Genes and Associated Cancers

TYPE OF TUMOR
SUPPRESSOR GENE GENE INHERITED CANCER NONINHERITED CANCERS

Cell division–controlling genes RB1 Retinoblastoma Many cancers

VHL Von Hippel–Lindau kidney Kidney cancers, hemangioblastomas of
cancer CNS, colon cancers
NF1 Nerve tumors, including brain Colon cancers, melanomas, neuroblastoma
NF2
APC Familial adenomatous polyposis Colorectal cancers
DNA repair genes MLH1 Colorectal, gastric, endometrial cancers
MSH2 Colorectal cancer (without
MSH6 polyposis)
BRCA1 Breast and/or ovarian cancer Rare ovarian cancers
BRCA2
Apoptosis genes TP53 Li-Fraumeni syndrome Many cancers
INK4a (p16) Melanoma Many cancers
HPC1 Prostate cancer Unknown
MSR
 Tumor Suppressor Genes 77

Figure 5-11. Leukocoria in a child with retinoblastoma. The

normal red reflex to light, which may be red, orange, or
yellow, is replaced by a white reflex in 60% of patients with
retinoblastoma. The normal red reflex should be symmetric
between both eyes. Leukocoria may be unilateral or bilateral
and may occur with conditions other than retinoblastoma,
including congenital cataracts, Coats disease, persistent
hyperplastic primary vitreous (PHPV), and toxocariasis. (From
Augsburger JJ, Bornfeld N, Giblin ME. Retinoblastoma. In
Yanoff M, Duker JS, eds. Ophthalmology, 2nd ed. St. Louis,
Mosby, 2004.)

A
With Rb as a model, it can now be seen how tumor sup-
pressor genes are key etiologic factors in familial cancers. Rb
can present as sporadic or familial cases (Fig. 5-13). In familial
(or heritable) cases, a mutation of one RB1 allele is passed
through generations in an autosomal dominant fashion. As
noted above, Rb expression requires a second hit in the other,
normal allele. This would seemingly be an extremely rare
event, but the 1 million or so retinoblasts are mitotically
active, giving ample opportunity for genetic error. Hence, Rb
clinical expression is quite common in families with a germ-
line RB1 mutation, although penetrance is not complete
owing to the random nature of the second, somatic mutation
(see Fig. 5-13). It is proper, then, to suggest that the predis-
position for Rb is high in families carrying the mutation,
although some individuals heterozygous for an RB1 mutation
may not develop retinal tumors.
An apparent paradox regarding tumor suppressor genes is
voiced by the question: How can cancer due to tumor sup-
pressor genes be autosomal dominant when both copies of
the gene must be inactivated to permit tumor formation? In
B
fact, the inherited or constitutional deleterious allele is trans-
mitted in an autosomal dominant manner and most hetero- Figure 5-12. CT scan (A) and MRI (B) of retinoblastoma in
zygotes do manifest the disease. So, while the predisposition a child with leukocoria showing a calcified mass in the globe
for cancer is inherited in a dominant fashion, pathology at of the eye. (Courtesy of Simin Dadparvar, MD.)
the cellular level requires the loss of both alleles—a decidedly
recessive mechanism. This explains why penetrance is incom-
plete in Rb, since only about 90% of heterozygotes ever including the RB1 locus. In familial Rb, the remaining RB1
undergo the second, somatic hit to their normal RB1 allele. allele was the inherited and deleterious gene, and the loss of
the nonmutated allele equaled a second hit and precipitated
Loss of Heterozygosity tumor formation. Several mechanisms may account for LOH,
Intensive molecular genetic analysis of the Rb locus revealed including interstitial deletion in chromosome 13q14, mitotic
that, in a significant proportion of cases, individuals who were nondisjunction resulting in the loss of one chromosome 13,
otherwise heterozygous in normal tissues contained tumors or mitotic recombination. In any case, LOH represents the
that harbored only a single RB1 allele. In other words, most common genetic mechanism for loss of the normal RB1
one allele seemingly vanished. The tumor cells underwent a allele in patients. If LOH is not found, the most likely expla-
loss of heterozygosity (LOH) for part of chromosome 13q, nation is a point mutation inactivation of the second RB1
 78 Cancer Genetics

Sporadic and Familial (Mendelian) Forms of Cancer

Knudson’s Two-Hit Hypothesis

Sporadic* Normal tumor

suppressor gene

Somatic
mutation
in one allele

Somatic
mutation
in other allele

Single tumors, unilateral,

later-onset

Familial † Tumor suppressor gene

containing a germline
mutation in one allele —
heterozygous for the mutation

Somatic
mutation
in other allele

Multiple tumors, bilateral,

Figure 5-13. Comparison between early-onset
sporadic and inherited forms of
*Two somatic mutations (two hits) are required for loss of tumor suppressor function.
cancers, as seen with retinoblastoma †The first “hit” is inherited and the second “hit” is somatic.
and other cancers.

TABLE 5-3. Examples of LOH and Associated Tumor Suppressor Gene Cancers

CHROMOSOME TUMOR SUPPRESSOR GENE GENE-ASSOCIATED CANCER

Unknown Breast carcinoma

1q HPC1 Prostate cancer
3p Unknown Small cell lung carcinoma, renal cell carcinoma
VHL CNS hemangioblastoma
5q APC Familial polyposis coli, colorectal carcinoma
8p MSR1 Prostate carcinoma, breast carcinoma
9p INK4A Non–small cell lung carcinoma, melanoma
13q RB1 Retinoblastoma, osteosarcoma
17p TP53 Colorectal carcinoma, breast carcinoma
18q DCC Colorectal carcinoma

allele. LOH is considered an important genetic phenomenon TABLE 5-4. Comparison Between Heritable and
in cancer and has been observed in a number of cancers that Nonheritable Retinoblastoma
are perpetuated by the loss of tumor suppressor genes (Table
5-3). HERITABLE NONHERITABLE
FEATURE RB RB
Spontaneous Retinoblastoma
Spontaneous retinoblastoma accounts for roughly 60% of Tumor Usually Unilateral
bilateral
cases and results from the concerted action of two indepen-
Family history 20% of cases None
dent somatic RB1 mutations in a single retinal cell (Table 5-4). Average age <1 year About 2 years or
Hence, these tumors are unifocal and unilateral, since they at diagnosis later
are truly monoclonal in origin. This can be contrasted to Increased risk Osteosarcoma, None
familial Rb, in which tumors can be multifocal and are often of second other
primary sarcomas,
bilateral—reflecting the need for only a single additional
tumors melanoma,
somatic mutational event within the normal RB1 allele in a others
retinal cell.
 Tumor Suppressor Genes 79

Neurofibromatosis Type 1
PATHOLOGY
Neurofibromatosis type 1 (NF1) is an autosomal dominant
Retinoblastoma disease of the nervous system that features the cardinal signs
Tumors may contain both differentiated and undifferentiated of multiple benign neurofibroma tumors in the skin (Fig.
cells. Differentiated cells may contain Flexner-Wintersteiner 5-14), irregularly pigmented patches of the skin (café-au-lait
rosettes and fleurettes, whereas undifferentiated cells have spots), and small benign tumors on the iris known as Lisch
hyperchromatic nuclei and appear as collections of small, nodules (Box 5-2). Occasionally, NF patients will develop
round cells.
cancer related to the nervous system, including neurofibro-
Calcification is characteristic of retinoblastoma and
sarcoma, cancer of Schwann cells, or astrocytoma. The NF1
differentiates retinoblastoma from other tumors or lesions that
mimic retinoblastoma in children under age 3 years. In older
gene is located at chromosome 17q11.2, and its nucleotide
children, calcification may occur in other lesions, such as and protein sequence analyses showed it has extensive simi-
retinopathy of prematurity and Toxocara infections from pets. larity with a GTPase-activating protein (GAP). This, in turn,
is significant to carcinogenesis because one of the GAP func-
tions is to accelerate the hydrolysis of GTP bound to the RAS
protein. Thus, the NF1 protein, neurofibromin, can be envi-
Using Rb as a model, the key differences between the sioned as a GAP specific to neural-related cells that regulates
heritable cancer predisposing syndromes and sporadic cancer RAS activity in neuronal tissues. Normally, neurofibromin
can be examined. Typically, the inherited cancer predisposi-
tion is passed through a family by autosomal dominant trans-
mission. Because offspring of a carrier have a 50% chance Box 5-2. DIAGNOSTIC FEATURES OF
to inherit the predisposing allele, the risk is high within NEUROFIBROMATOSIS TYPE 1
families. There is a much lower risk for sporadic cases of a
certain cancer in the general population. Individuals harbor- Café-au-lait spots
ing a cancer predisposition allele tend to have an earlier Prepubertal—6 or more >5 mm
onset, develop second tumors that tend to be bilateral as Postpubertal—diameter >15 mm
opposed to unilateral, and/or exhibit cancers in other organ Neurofibroma
systems more frequently than do sporadic cases. Although Two or more of any type of neurofibroma or
the sporadic form is much more common than the familial One plexiform neurofibroma
form for any particular cancer, there is still great value in Freckling in the axillary or inguinal regions
forging a deep understanding of the molecular pathophysi- Optic glioma
Two or more Lisch nodules (iris hamartomas)
ologic mechanisms of heritable cancers. Advances in diagno-
A distinctive osseous lesion such as sphenoid dysplasia or
sis, prognosis, presymptomatic detection and surveillance,
thinning of the long bone cortex with or without
and treatment will not only help those patients with the pseudarthrosis
heritable form but also may lead to mechanistic insights into A first-degree relative with NF1
the sporadic forms.

Figure 5-14. Neurofibroma removed

from a child. Most neurofibromas
present as active stage 2 tumors (G0,
T0, M0) in the skin or associated with
nerves. Occasionally they can be seen
as stage 3 (M0, T1–2, M2) aggressive
infiltrative forms. (Courtesy of Dr. Rahul
A B Nath, Texas Nerve and Paralysis
Institute, Houston, Texas.)
 80 Cancer Genetics

interacts with RAS in the signal transduction pathway by carcinomas. Molecular genetic dissection of colorectal cancer
down-regulating the activity of RAS. As the neurofibromato- has demonstrated that the inactivation of the APC (“adeno-
sis gene product is diminished or lost by mutation, RAS matous polyposis coli”) gene on chromosome 5q and the
activity may increase in such a way to promote cell growth DCC (“deleted in colon cancer”) gene on chromosome 18q
and inhibit cellular differentiation. In this case, the loss of is common in colorectal cancer. This, in turn, explains the
NF1 serves as the “trigger” for increasing the activity of the increased frequency of LOH on chromosomes 5q and 18q in
RAS oncogene. this form of malignancy—the APC and/or the DCC genes
When a deleterious NF1 mutation occurs, the heterozygote have been lost via LOH. The general genetic model for cancer
exhibits typical neurofibromatosis. If the second NF1 allele evolution to colorectal cancer can now be applied (Fig. 5-16).
is inactivated, however, RAS activity may increase further It is important to remember that it is the accumulation of
and the chance for malignancy increases. This scenario sug- mutations that is key in this model—not necessarily the order.
gests that the NF1 gene is a tumor suppressor gene. Indeed, Empirically, for example, it has been reported that greater
LOH for the normal NF1 allele has been observed in a than 90% of carcinomas contain at least two of these genetic
number of NF1-related malignancies. changes and roughly 40% show at least three accumulated
mutations.
This genetic paradigm can be applied to the two forms of
●●● DNA REPAIR GENES familial colorectal cancer syndromes, familial adenomatous
polyposis and hereditary nonpolyposis of the colon cancer.
Colorectal Cancer and the Genetic
Like many of the autosomal dominant cancer syndromes,
Paradigm for Cancer Revisited the familial form of colorectal cancer is recognized by an
Nearly 2% of individuals in Europe and North America increased incidence of cancer in people with a family history
develop colorectal cancers, making it one of the most common of colorectal cancer and families exhibiting a number of
cancers in the Western world. Most of these will be sporadic
cases, but up to 30% will be familial cases (Fig. 5-15). Two
subclasses of familial colorectal cancers have been identified
Familial adenomatous polyposis
that are associated with the loss of tumor suppressor genes: (FAP)1%–5%
familial adenomatous polyposis and hereditary nonpolyposis Hereditary nonpolyposis
Rare CRC syndromes
colon cancer. Although the primary genes associated with <0.1% colorectal cancer
5% (HNPCC)
these colorectal cancers have radically differing functions, the
overall mechanism for carcinogenesis is quite similar.

Applying the Genetic Paradigm for Familial

10%–30%
Cancer Evolution
Most colorectal cancers proceed from benign adenomatous Sporadic
polyps to malignant tumors. This transition is associated with 65%–85%
increasing polyp size and an accumulation of genetic altera-
tions. For example, polyps less than 1 cm in diameter rarely
appear cancerous histologically and harbor RAS mutations in
only a few percent of examined cases. As polyps increase in
size to 2 cm, however, RAS mutations appear in 40% of
cases. Likewise, p53 mutations and LOH for regions of Figure 5-15. Causes of hereditary susceptibility to colorectal
chromosomes 5q and 18q seem to accumulate or increase cancer. Single-gene mutations have been identified for
in frequency as benign adenomas progress to malignant HNPCC and FAP.

Chromosome Chromosome
18 17

APC gene loss RAS gene DCC gene loss TP53 gene loss
mutation

Increased cell Early-stage Intermediate- Late-stage Metastasis

Normal cell Carcinoma
growth adenoma stage adenoma adenoma occurs

DNA loses PRL3

methyl groups overexpression

Figure 5-16. Multiple-step progression of colon cancer. Different genes are mutated at different stages of tumorigenesis.
 DNA Repair Genes 81

affected individuals consistent with autosomal dominant and LOH for the APC region is found in 70% of adenomatous
transmission. polyps in non-FAP patients. Most APC mutations are non-
sense and frameshift mutations, and molecular genetic diag-
Familial Adenomatous Polyposis (FAP) nostics can identify carriers at an early age, permitting
Approximately 1% to 5% of colorectal cancers are caused by appropriate monitoring and treatment.
FAP, an autosomal dominant disorder. FAP is a serious dis-
order in which penetrance is greater than 90% by age 35, Hereditary Nonpolyposis Colon Cancer
reflecting the near-100% risk in untreated individuals. There (HNPCC)
is also an increased risk for extracolonic tumors in FAP, A small percentage of colorectal cancer is described by the
including upper gastrointestinal (GI) tumors, osteomas, autosomal dominant HNPCC, which is characterized by the
thyroid tumors, and brain tumors. The gene responsible for presence of only one or a few adenomas in the proximal
FAP is the APC gene. The APC gene product is a cytoplasmic colon. Cancer is usually diagnosed by age 45, and the lifetime
protein that regulates the activity of β-catenin, one of three penetrance risk for cancer is 80%. Extracolonic tumors are
ubiquitously expressed cytoplasmic catenins. β-Catenin has not rare and include cancers of the endometrium, biliary and
two functions within the cell. First, it permits cell-cell interac- urinary tracts, ovary, and stomach.
tions by anchoring cadherins, which are extracellular glyco- HNPCC is due to mutations in one of the four genes
proteins that form intercellular complexes necessary for cell (MLH1, MSH2, PMS2, and MSH6) involved in DNA mis-
adhesion. Second, β-catenin is also a transcription factor that match repair (MMR)—a class of genes that is highly evolu-
promotes the expression of MYC and cyclin D1 proteins. tionarily conserved, supporting their importance in
Under normal conditions, APC regulates β-catenin levels by maintaining genome viability (Fig. 5-17). MLH1 and MSH2
binding to it and marking it for proteolytic degradation, thus mutations are most common and found in 60% to 80% of
preventing excessive levels of β-catenin. The loss of APC individuals developing HNPCC. Typical of the tumor sup-
permits accumulation of β-catenin in the nucleus, where pressor gene model of carcinogenesis, in individuals hetero-
it facilitates cellular proliferation by activating cyclin D1 zygous for mutations in these genes, only a second somatic
and MYC. As noted previously, cyclin D proteins are impor- mutation in the other allele via an LOH mechanism or point
tant in the transition from G1 to S and MYC proteins are mutation is needed in order to promote cancer. Specifically,
transcription factors for cell proliferation and cell cycle the loss of MLH1 or MSH2 proteins renders DNA mismatch
control genes. repair inefficient and thus promotes genetic instability and
an increased mutation rate. This instability is referred to as
microsatellite instability. In regions of di- or mononucleotide
repeats, mismatching during DNA replication can occur, pro-
BIOCHEMISTRY ducing deletions or additions of the repeats. Mutations in
the repair enzymes decrease effective repair, leading to
β-Catenin
disease. Two examples seen in patients with HNPCC are in
Most β-catenins are bound to cadherins, a class of calcium- the BAX gene, which is involved in apoptosis, and the
dependent proteins important in cell adhesion, at the cell TGFBR2 (transforming growth factor-β receptor 2) gene
membrane. The remaining β-catenins either occur in a free
involved in signaling. Indeed, much somatic variation in DNA
form and enter the nucleus or are part of a complex
microsatellite regions is observed in these patients owing to
associated with APC (adenomatous polyposis coli) and GSK
(glycogen synthase kinase) proteins. Nuclear β-catenins
errors in DNA replication accompanied by decreased repair.
recruit transcription factors to regulate gene expression. In Obviously an increased mutation rate could facilitate the
resting cells, however, cytoplasmic β-catenins turn over second genetic hit in other tumor suppressor genes, or the
rapidly and few enter the nucleus. The APC-GSK complex initial activating mutation in an oncogene, again promoting
controls degradation. GSK phosphorylates β-catenins, malignancy.
leading to ubiquination and degradation by proteosomes. The Amsterdam criteria were established in 1991 to facili-
WNT proteins control β-catenin expression. WNTs bind to tate identification of HNPCC families. More recently, these
a seven-helix receptor at the cell membrane, leading to the have been modified to assist in diagnosing the disease.
inactivation of GSK phosphorylation activity. In the absence HNPCC should be considered in families with: (1) two cases
of GSK and β-catenin phosphorylation, β-catenin is not
of colorectal cancer where families are small (one age under
degraded. The increased concentration of free β-catenin can
55) or (2) two cases of colorectal cancer and one case of
then enter the cell and affect transcription.
endometrial cancer or other early-onset cancer.

Breast Cancer
Individuals inheriting a deleterious APC allele develop
numerous (hundreds or thousands of) adenomatous polyps at Breast cancer is a malignancy that originates in the glandular
an early age. In some polyps, a second, somatic genetic hit tissue of the breast, primarily the milk-producing lobules and
occurs, thus effectively knocking out the APC gene product. the milk ducts. It is, like colorectal cancer, one of the most
This is possibly an early event in the pathway to malignancy; common malignancies in humans. Roughly 10% of North
the association between colorectal cancer and FAP is strong, American women will develop cancer in their lifetime. This
 82 Cancer Genetics

Hereditary nonpolyposis
colorectal cancer (HNPCC)

PMS2,
MSH6 (rare)

MSH2 ∼30%
5%

Unknown MLH1
Figure 5-17. Five mismatch repair
∼30% ∼30%
genes have been identified in HNPCC.
Two of these are responsible for a
majority of cases, and genetics
laboratories that offer HNPCC testing
examine only these two genes: MLH1
and MSH2.

translates to greater than 220,000 newly diagnosed cases per

year—and greater than 40,000 deaths per year—in women ANATOMY
living in North America. Overall, this form of cancer is the
Breast Cancer
most commonly diagnosed malignancy in this group of
women, and it is second only to lung cancer for cancer-related Sixty percent of breast cancer cases occur in the upper
mortality. lateral quadrant. The cancer attaches to suspensory
Although most breast cancer is sporadic, the disease is ligaments as progression occurs, shortening them and
causing dimpling. The cancer may also attach to lactiferous
noted to cluster within families. In addition, a woman’s risk
ducts, causing inverted nipples, or invade the deep fascia of
for developing this cancer is 3-fold higher if a first-degree
the pectoralis major muscle. Mastectomy may be performed
relative has had breast cancer; the risk is 10-fold higher if to varying degrees:
more than one first-degree relative has developed breast ■ Radical: removal of breast and related structures—
cancer. This suggests a strong heritable genetic component pectoralis major and minor muscles, axillary lymph nodes/
in a subset of families. In fact, 15% to 20% of cases have fascia, part of thoracic wall
been labeled as “familial clustering” and another 5% to ■ Modified radical: removal of breast and axillary lymph nodes

10% have been classified as heritable. Although the familial ■ Lumpectomy: removal of palpable mass

clustering cohort likely has a genetic etiologic component,

there is no clear evidence of mendelian or nonmendelian
transmission within such families and no specific mutations
have been associated with this group. Thus, any genetic com-
ponent must be considered a multifactorial mechanism in
which the gene or genes involved impart a predisposition to
develop breast cancer, while other factors such as environ- PATHOLOGY
ment, socioeconomic status, diet, and more, also influence
disease expression. Breast Cancer Classification
In the heritable form, the propensity for developing breast Breast cancer is classified by histologic presentation.
cancer appears to be transmitted among families in an auto- Noninvasive (in situ) cancer growth occurs within the
somal dominant fashion. Besides multiple affected individuals ducts and without penetration of the basement membrane.
in the pedigree, such families share certain clinical features, This includes ductal carcinoma in situ (DCIS) and lobular
including early onset (5–15 years before the average age of carcinoma in situ (LCIS).
sporadic case onset), bilateral involvement, and the occur- Invasive carcinoma penetrates the basement membrane
rence of other cancers, primarily ovarian. The availability of of a duct containing ductal carcinoma in situ and extends
such families greatly facilitated genetic linkage analysis that into the stroma. Ductal carcinoma accounts for 75% of all
invasive breast cancer. Other examples include invasive
led to the identification of several susceptibility genes, includ-
lobular carcinoma, mucinous carcinoma, medullary
ing BRCA1, BRCA2, TP53, and PTEN/MMAC1. Because
carcinoma, papillary carcinoma, tubular carcinoma,
BRCA1 and BRCA2 mutations are much more commonly apocrine carcinoma, squamous cell carcinoma, and spindle
associated with breast cancer, these genes are featured in the cell–type carcinoma.
subsequent discussion.
 DNA Repair Genes 83

BRCA1 and BRCA2 Box 5-3. RISK FACTORS FOR BREAST CANCER
The BRCA1 gene maps to chromosome 17q21 and encodes
a 1863-amino-acid polypeptide. Mutations in this gene are Nationality
Most common in white women
responsible for 40% to 50% of the autosomal dominant
Least common in Hispanic, Asian, and black women
breast cancer families. For patients harboring a mutation, the
Age
risk for developing a breast cancer is 80% by age 70. Hence, Uncommon before menopause
penetrance in these families is not 100%. A second primary Positive family history
breast tumor forms in roughly 50% of the cases. As for other Early menarche—before age 12 years
tumor suppressor genes, one deleterious BRCA1 allele is Late menopause—after age 55 years
inherited and the loss of both alleles is required for neoplasia. Atypical epithelial hyperplasia of breast on biopsy
Most identified mutations are nonsense or frameshift muta- Dense breast parenchyma on mammography
tions, predicting a truncated or nonfunctional gene product. Hormone replacement therapy
Again in keeping with the tumor suppressor gene motif in Radiation exposure to breasts—before age 30 years
cancer, the normal BRCA1 allele is frequently absent in Lifestyle
Physical activity and weight management reduce risk
tumor tissue but is present in adjacent normal breast tissue,
Alcohol use
signaling LOH (Fig. 5-18).
BRCA2 localizes to chromosome 13q12 and produces a
large protein of 3418 amino acids. Greater than 100 BRCA2 of mutations, consistent with the genetic model of cancer
variants have been characterized, and these account for progression.
roughly 30% of families with breast cancer. The risk for Worldwide, approximately 1 in 800 persons carries a del-
breast cancer associated with this gene is 50% to 85% in eterious BRCA1 allele (Box 5-3). This carrier frequency for
females and 6% for the rare male breast cancer. This gene is breast cancer predisposition varies from population to popu-
also associated with an increased risk for prostate, pancreatic, lation. Affected families also rarely share the same pathogenic
and laryngeal cancer. Just like BRCA1, this gene is a tumor mutation; BRCA1 mutations are therefore “private” to a
suppressor gene and shares a similar profile of mutations family. In certain cases, however, seemingly unrelated breast
(protein termination mutations and LOH resulting in no cancer families have been found to have a deleterious allele
protein production) in tumor tissues. Hence, they share in common.This phenomenon has been observed, for example,
pathogenic characteristics with the RB1 and APC tumor sup- in the Ashkenazi Jewish population, where two BRCA1
pressor genes already discussed. However, unlike these genes, alleles, 187delAG and 5385insC, and the 6174delT BRCA2
BRCA1 and BRCA2 mutations are not generally found in allele are common among families (Fig. 5-19). These three
sporadic breast cancer cases. variants are found in 1%, 0.13%, and 1.5% of Ashkenazi
Recent data suggest that BRCA1 and BRCA2 may be Jews, respectively. Taken together, these three mutations
involved in more sporadic cancers than previously thought. result in an overall carrier frequency of 1 in 40 and account
Many of these are not due to mutations in the coding for 25% of early-onset breast cancer and 90% of familial
region of BRCA1, but rather are promoter mutations that breast and ovarian cancer in this population. The unexpected
increase promoter methylation and lead to transcriptional high frequency of certain mutations within a subpopulation
silencing of the gene. As expected, the prognosis for disease is consistent with a genetic founder effect whereby a modern
with these mutations is poor. The mean survival of women population has derived recently from a small group of found-
with ovarian cancer with BRCA1 promoter hypermethyl- ers. For BRCA1 and BRCA2, additional founder mutations
ation was 36.1 months compared with 63.3 months for have been characterized in northern European and Icelandic
women with ovarian cancer but with a normal BRCA1 populations. The presence of certain disease-causing alleles in
gene. particular population subgroups can streamline DNA-based
The BRCA1 and BRCA2 proteins do not share significant testing for the BRCA1 and BRCA2 mutations.
DNA or amino acid homology, but they do participate in the
same molecular complex. BRCA1 is a phosphorylated nuclear
protein that has two functional motifs: a BRCT domain at the
Ovarian Cancer
C-terminus and a ring-finger domain at the N-terminus. It is Ovarian cancer is the fifth leading cause of death from cancer
expressed in a variety of tissues, particularly in G1 through S among women and is the deadliest of all gynecologic cancers,
phase of the cell cycle. BRCA1 and also BRCA2 apparently with a 5-year survival rate of less than 50%. Like breast
interact with the RAD51 protein, a gene product involved in cancer and most other cancers, the majority of ovarian
the repair of double-stranded breaks in the DNA. BRCA1 cancers appear sporadic, and only 5% to 10% can be called
may also activate the p21 CDK inhibitor, which, as discussed hereditary on the basis of multiple affected family members
for p53 later in this chapter, is involved in suppressing growth and an age of onset 10 to 15 years before sporadic cases (Fig.
at the G1-to-S cell cycle transition, presumably to repair DNA 5-20). BRCA1 (70%) and BRCA2 (20%) mutations account
damage. BRCA1 and BRCA2 are therefore caretaker tumor for 90% of inherited ovarian cancer. In these families, both
suppressor genes, since the homozygous loss of these alleles breast and ovarian cancer segregate among affected individu-
predicts genome instability that promotes the accumulation als. Mutations in BRCA1 are associated with an earlier age
 84 Cancer Genetics

A B

C D

Figure 5-18. Invasive and noninvasive ductal carcinomas. A, Carcinoma has poorly defined edges that have begun to extend
into surrounding tissue. Fibrous, or scar-like, tissue may form. Depending on the location, symptoms may include dimpling,
retraction of the nipple, and nipple discharge. B, Histology of sample from patient in part A shows a random configuration of
cells extending through the periductal connective tissue. C, Noninvasive ductal carcinoma (also known as interductal carcinoma
or ductal carcinoma in situ) contains breast duct cells that have malignant characteristics but have not invaded surrounding
tissue. D, Histology of sample from patient in part C shows proliferating malignant ductal cells limited to existing ductal units
without invasion through the basement membrane. (Courtesy of Dr. Juan Lee, Mercer University School of Medicine, Georgia;
and Dr. Emil Goergi, Dodge County Hospital, Georgia.)
 Apoptotic Genes 85

BRCA1
185delAG 5382insC Figure 5-19. One in 40 Ashkenazi
prevalence ~1% 1 in 100 women prevalence ~0.13%
Jews carries a BRCA1 or BRCA2
mutation, demonstrating a founder
effect. Since the prevalence of these
specific mutations is high, population
Founder mutations traced to a BRCA2
screening can be done in this
common ancestor who lived more 6174delT
than 600 years ago prevalence ~1.5%
population with a three-mutation test.
Frequencies are for carriers of this allele
in the population.

Hereditary
5%–10%

BRCA2 HNPCC ~2%

~20%
Other single
genes ~8%
Sporadic

BRCA1 ~70% Figure 5-20. Causes of ovarian

cancer. Most genetic causes of ovarian
cancer are associated with BRCA1
mutations. Unlike breast cancer, familial
clustering does not appear to occur in
ovarian cancer families.

of onset than is seen with BRCA2 mutations: 54.6 years ●●● APOPTOTIC GENES
versus 64 years, respectively. A lifetime risk of developing
p53 Protein and Li-Fraumeni Syndrome
ovarian cancer by age 75 years for heterozygous carriers of
either mutated gene is 63%, even though women with BRCA1 The TP53 gene, producing the p53 protein involved in the
mutations will develop ovarian cancer earlier. cell cycle, holds a special place in cancer genetics; somatic
Ovarian cancer is also not uncommon in the previously mutations in this gene are found in roughly half of all human
discussed HNPCC due to mutations in the MLH1 and MSH2 cancers, easily making it the most frequently altered gene in
tumor suppressor genes involved in DNA mismatch repair. cancer. Over 50% of colorectal, lung, and bladder tumors
Up to 8% of familial ovarian cancer families do not have have p53 mutations, as do roughly 40% of breast cancers.
BRCA1, BRCA2, or HNPCC-related genes. These cases may The extremely high frequency of p53 variants in various
be caused by an unidentified site-specific major allele. tumor types obviously suggests an important role in cell
growth and differentiation.
The TP53 gene maps to chromosome 17p and encodes a
transcription factor that is expressed when the cell is stressed
or damaged by such factors as ionizing radiation, environ-
ANATOMY mental mutagens that damage the DNA, or ultraviolet light.
Because p53 levels rise when DNA is damaged, this protein
Ovaries
is often called the “guardian of the genome.” One of the
The ovaries lie on the posterior side of the broad ligament targets for p53 is the promoter for the CDKN1A gene encod-
projecting into the ovarian fossae. They are supported by: ing p21 (Fig. 5-21). p21 is a cyclin-dependent kinase inhibitor
■ Mesovarium: attaches ovary to the broad ligament
(CDI) in the CIP/KIP family of CDIs. It inhibits the activity
■ Suspensory (infundibulopelvic) ligament of the ovary:
of the cyclin/CDK complex, thereby disallowing inactivation
attaches ovary to the pelvic brim
■ Ovarian ligament: remnant of the gubernaculum lying
of the pRB protein by phosphorylation. pRB therefore remains
within the mesovarium that attaches the ovary to the active in its hypophosphorylated state, resulting in halting the
lateral surface of the uterus beneath the uterine tube cell cycle prior to the S phase, as discussed previously. This,
in turn, gives the cell time to repair any DNA damage prior
 86 Cancer Genetics

to DNA replication. p53 also increases the expression of affected children, while breast cancer is most common in
GADD45 (for growth arrest and DNA damage 45), a DNA adult females.
repair enzyme. If the genome is successfully repaired and The TP53 gene product is a tumor suppressor. Following
ready for subsequent replication, p53 down-regulates itself. the model previously discussed for retinoblastoma, the mech-
In this way, p53 acts to preserve the integrity of the genome anism of cancer in Li-Fraumeni syndrome is clear. Family
by inhibiting tumor formation. However, if the cell is unable members transmit the constitutional TP53 mutation in an
to respond appropriately to excessive damage, p53 triggers autosomal dominant fashion. Those who inherit the deleteri-
apoptosis by inducing the proapoptotic BAX and IGFBP3 ous allele need only one additional mutational hit in the other
genes. So, in a sense, if p53 cannot safeguard the cell by TP53 allele. This second hit may occur in any somatic cell,
permitting DNA repair, it provokes death rather than allow thus accounting for the wide variety of tumor types found in
continued genetic damage that would promote cancer. Li-Fraumeni syndrome.

Prostate Cancer
BIOCHEMISTRY & PATHOLOGY Prostate cancer is the most common malignancy in men and
the second leading cause of death due to cancer in men.
Apoptosis The lifetime risk is 10% to 14%, although the diagnosis,
Apoptosis is a cascade of events leading to the activation of surveillance, and prognostication efficiency of prostate cancer
caspases and ending with cell death. There are three have been greatly facilitated by laboratory testing for the
phases to apoptosis: initiation, intrinsic, and extrinsic. In the prostate-specific antigen (PSA) (Box 5-4).
initiation phase, the caspases become active.
In the intrinsic pathway, BCL2 and BCLX antiapoptotic
proteins are located on mitochondrial membranes and
ANATOMY
recognize internal damage to the cell such as that caused
by reactive oxygen species. The balance of BCL2 and BCLX Prostate
with the proapoptotic proteins BAK, BAX, and BIM changes,
and the increase in the proapoptotic proteins causes an The prostate is located between the base of the urinary
increase in mitochondria permeability. Cytochrome c then bladder and the sphincter urethrae muscle, and it consists
leaves the mitochondria and forms a complex with apoptotic of five lobes:
■ Anterior
protease-activating factor 1 (APAF1). These cytochrome c
■ Median—prone to benign hypertrophy causing obstruction
molecules and APAF1 molecules form apoptosomes that
activate caspase-9. This leads to activation of caspases 3, 6, of urethral orifice
■ Posterior—prone to cancer transformation
and 7 and to cell death through cleavage of cell substrates.
■ Right lateral
The extrinsic pathway involves signals from receptors that
■ Left lateral
are members of the tumor necrosis factor (TNF) family,
containing a “death domain.” When bound to ligands, It secretes PSA, prostaglandins, citric acid, acid
caspases are activated. For example, the type 1 TNF phosphatase, and proteolytic enzymes.
receptor and FAS located on the cell surface can bind TNF
and FAS ligands and activate caspase-8. The proteolytic
cascade is thus activated, as is caspase-9, leading to cell
death followed by phagocytosis of debris. PATHOLOGY
Prostate-specific Antigen
Prostate-specific antigen (PSA) is an enzyme produced by
the prostate and normally secreted in semen from prostatic
Because somatic TP53 mutations are clearly a key etiologic epithelium. It functions as a protease to liquify seminal fluid.
event in many cancers, the presence of germline TP53 muta- PSA is not specific to cancer; it is found in an increased
tions predicts dire consequences. In fact, this condition is seen amount in the blood during
in Li-Fraumeni syndrome, a familial cancer disorder in which ■ Prostate cancer

a deleterious TP53 allele is transmitted in an autosomal dom- ■ Benign prostatic hyperplasia

■ Infections or inflammations of the prostate
inant manner (Fig. 5-22). Li-Fraumeni families have a higher
risk for early-onset cancer, predicting multiple affected family Serum levels below normal (4.0 ng/mL) may occur in 20% to
40% of patients with cancer.
members commonly found in pedigrees. By age 30, roughly
50% of individuals harboring such a mutation will develop
one of a variety of malignancies, including soft-tissue sarco-
mas, osteosarcomas, brain tumors, breast or colon carcino- Typical of cancers, most cases of prostate cancer are spo-
mas, adrenal carcinomas, and leukemia (see Fig. 5-22). radic. However, up to 20% of cases may be considered
Development of multiple primary tumors, however, is not “familial” on the basis of at least one of the following criteria:
uncommon, and 15% of individuals develop a second cancer. (1) family history is positive for three or more first-degree
Bone and soft-tissue sarcomas are more commonly found in relatives with the disease, (2) three or more generations
 Apoptotic Genes 87

Ionizing radiation
Carcinogens
Mutagens Cell with p53
mutations or
Normal cell loss of
(p53 normal) normal p53

Figure 5-21. The role of p53 is pivotal

to the integrity of DNA in each cell. In
normal cells, p53 is present in low
DNA damage DNA damage
levels; however, DNA damage increases
TP53 gene transcription, leading to
Hypoxia p53 Activated and p53-Dependent genes increased transcription of genes for p21
binds to DNA not activated and GADD45, a CDK inhibitor and a
DNA repair gene, respectively. The BAX
No cell No DNA gene, which produces a proapoptotic
Transcriptional up-regulation cycle repair protein, is up-regulated by p53 binding
of target genes arrest to its promoter and is subsequently
expressed on mitochondrial surfaces.
Mutant cells BAX interacts with other proteins to
form pores that release cytochrome c
p21 GADD45 from the mitochondria into the cytosol,
(CDK inhibitor) (DNA repair) Expansion and leading to activation of caspases and
additional
BAX mutations cell death. (Not shown: BCL2 inhibits
G1 arrest p53-mediated cell death, but increased
(Apoptosis gene)
pathway expression of TP53 represses BCL2
expression.) Shown at right are the
Successful repair Repair fails consequences of inactivation of p53
through mutation or deletion. DNA
repair does not occur in the presence
of damage, and abnormal cells
continue to expand unchecked.
(Redrawn from Kumar V, Abbas A,
Fausto N. Robbins & Cotran Pathologic
Normal cells Apoptosis Malignant tumor
Basis of Disease, 8th ed. Philadelphia,
WB Saunders, 2010, p 291.)

exhibit the disease on the same side of the family, or (3) two Box 5-4. RISK FACTORS FOR PROSTATE CANCER
or more close relatives have early-onset prostate cancer.
Among the familial forms, 5% to 9% may be hereditary, and Age
these seem to follow an autosomal dominant mode of trans- Risk increases over age 55
mission within families (Fig. 5-23). Examination of tumors 75% of cases are diagnosed after age 65
from such families reveals LOH at several loci, consistent with Positive family history
the tumor suppressor model of cancer. Diet—risk increases with high saturated fat intake
Molecular genetic investigation of hereditary prostate Nationality/race
cancer indicates two loci linked with the disease. The two Most common in North America and northwestern Europe
hereditary prostate cancer genes, HPC1 and HPC2, are linked Highest risk for blacks
to chromosomes 1q24 and 17p11, respectively. Originally, it 70% more common in blacks than in whites
Lowest risk for Asians
was believed that these two loci represented major suscepti-
Exposure to heavy metals
bility genes for this form of cancer. Subsequent detailed
Lifestyle
analysis of heritable prostate cancer, however, revealed that Physical activity and weight management reduce risk
only HPC1 represented a strong association with prostate Smoking
cancer. Another gene, MSR1, also demonstrates a strong
 88 Cancer Genetics

to incomplete double-stranded DNA synthesis during DNA

Bilateral breast replication. Because DNA replication in mammals is bidirec-
I
d 40 tional and DNA polymerase can process only in a 5′-to-3′
direction, the lagging strand of DNA must be replicated in a
discontinuous fashion. This means that, for DNA polymerase
II to initiate lagging strand discontinuous replication, an RNA
Age 50 Breast dx 41 Leukemia primer must be utilized well 5′ of the replication fork. Repli-
osteosarcoma d 35
Dx 43 cating this strand at the terminus of the chromosome, however,
is not possible using this mechanism because there is no DNA
III
template for an RNA primer to pair with. Therefore, the
lagging strands at the end of chromosomes cannot be repli-
Breast Brain tumor
Dx 35 Dx 32
cated in the normal fashion, and this has been termed the
“end replication problem.” The telomeric ends of chromo-
somes would get progressively shorter at each DNA replica-
IV
tion, ultimately damaging coding sequences on chromosomal
Soft tissue Leukemia tips. Telomerase evolved as a solution to this problem. It uses
sarcoma Dx 5
Dx 7 its internal RNA moiety that is complementary to the single-
stranded telomeric overhang as a template to synthesize
Figure 5-22. Pedigree of a Li-Fraumeni family telomeric DNA on the ends of the lagging strand (Fig. 5-24).
demonstrating multiple types of cancers within a family. As This mechanism corrects for the continued erosion of
shown here, male cancer may have a later onset (II-2) and chromosomal ends that occur in its absence.
multiple primary tumors can be seen in one person (II-3).
Telomerase is composed of two major components: the
d, died; Dx, diagnosis.
catalytic subunit (hTERT) and the template RNA (hTER). The
hTERT component is a reverse transcriptase that is involved
in synthesizing DNA from the RNA template. Other proteins
association with prostate cancer risk. The HPC1 protein par- and kinases are additional components of the holoenzyme.
ticipates in apoptosis, and mutations in the gene result in Activation of the holoenzyme requires transcriptional and
apoptotic inhibition. MSR is a macrophage scavenger receptor posttranscriptional levels of the hTERT. A number of tran-
localized to chromosome 8p22 and within an area frequently scriptional factors, including c-MYC, BCL2, and RAS, and
recognized for LOH, chromosome 8p21-25. The association acetylating and methylating changes in the chromatin struc-
of prostate cancer with LOH is greater for chromosome 8 ture also play a role in the control of hTERT. To illustrate
than for any other chromosome. their role, the overexpression of c-MYC is correlated with
increased hTERT expression in several cancers, including pros-
tate, neuroblastoma, and cervical. In studies using cells with
●●● TELOMERES AND TELOMERASE down-regulated c-MYC expression, cell proliferation decreased;
Telomerase is a ribonucleoprotein enzyme that acts as a cel- subsequently increasing hTERT resulted in increased prolifera-
lular reverse transcriptase to maintain the length and integ- tion through restored telomerase activity. A few approaches
rity of chromosomal ends, or telomeres. In humans, telomeres to directly target either telomerase and telomeres or the
are represented by hexameric DNA repeats of TTAGGG telomerase-associated regulatory mechanisms are shown in
that are present in thousands of copies at the ends of chro- Table 5-5. It is worth noting that an increase in hTERT activity
mosomes. Telomeres, in partnership with telomerase, serve to is regulated by androgens and so removing androgens is
protect the ends of chromosomes from degradation due reflected in decreased expression of hTERT.

II

Dx 75 Dx 65

III
Dx 67 65 69 Dx 67 Dx 65 Dx 60 Dx 64 Dx 62 Dx 60

Figure 5-23. Family with prostate cancer linked to HNPC1 mutations. Hereditary prostate cancer accounts for about 10% of all
prostate cancer. About 34% of cases are linked to mutations in this gene. The ages of two unaffected brothers (III-2 and III-3)
are shown. Dx, diagnosis.
 Telomeres and Telomerase 89

3'
TTGGGGTTGGGGTTGGGGTTG Parental strand
AACCCC Incomplete, newly synthesized
5' lagging strand
Telomerase
binds

3'
TTGGGGTTGGGGTTGGGGTTG
AACCCC ACCCCAAC Telomerase
5' 5' synthesis
3'
Telomerase
Telomerase with bound RNA template
extends 3' end
(RNA-templated
DNA synthesis)

3'
TTGGGGTTGGGGTTGGGGTTGGGGTTGGGGTTG
AACCCC ACCCCAAC
5' 3' 5'

Completion of
lagging strand by
DNA polymerase
Figure 5-24. Telomere replication and
(DNA-templated
DNA synthesis)
the role of telomerase. Telomerase
binds to the 3′ end of a parental
3' strand—the terminal sequences of the
TTGGGGTTGGGGTTGGGGTTGGGGTTGGGGTTG chromosome—and provides a template
AACCCC CCCCAACCCCAACCCC (ACCCCAAC) for synthesis in the 5′ →
5' 3′ direction. The newly extended
DNA polymerase
sequence serves as the template for
synthesis on the opposite strand.

In normal cells, telomerase activity decreases with age and telomerase is active in tumor cells and may facilitate prolifera-
the number of cell divisions, yielding progressively shorter tion provides a diagnostic substrate as well as a potential new
telomeres over time. Normal cells also have a limited poten- target for anticancer drugs (Fig. 5-25).
tial for cell division, and it is believed that the shortening of
the telomeres is associated with the limited proliferative
capacity of the typical somatic cell. Indeed, after hundreds of PHARMACOLOGY
cell divisions, certain telomeres may be dangerously short
Antitelomerase Therapy
and terminal genes are threatened. Such damage may signal
p53 and pRB expression to halt the cell cycle and place the Telomeric DNA and the core telomerase components, hTR
cell into a G0 state. At this point, the cell is said to be in and hTERT, are good targets for antitelomerase
pharmacologic strategies.
“senescence.”
■ Approaches to targeting hTR
In many cancer cells, telomerase activity is reactivated, ■ Antisense 2′-5′ oligoadenylate inhibits telomerase and
allowing tumor cells to proliferate indefinitely. Experimen- increases apoptosis
tally, two lines of data underscore the potential importance ■ N3′-P5′ thiophosphoramidate oligonucleotides
of telomerase to cancer. First, tumor cells show no telomeric (GRN163) increase apoptosis and senescence
shortening over repeated cell divisions, strongly suggesting ■ Ribozymes
that this enzyme is required for indefinite proliferation. ■ Peptide nucleic acids to stop growth and initiate

Second, 90% of tumors show telomerase activity but adja- apoptosis

cent normal tissues do not. Hence, in sharp contrast with ■ Approaches to targeting hTERT
■ Ribozyme inhibits telomerase
normal cells, a reactivation of telomerase activity is a common
■ Peptide nucleic acids
finding in highly proliferative and immortal tumor cells.
■ Small molecules such as BIBR1532 induce senescence-
Telomerase expression, and thereby availability, may be
like growth arrest
enhanced in tumor cells due to mutations that enhance activ- ■ Approach to targeting the telomeric G-quadruplex
ity or by an oncogenic transcription factor. Telomerase ■ Acridine compounds bind the G-quadruplex and induce
activity has been correlated with aggressiveness in several growth arrest and increase sensitivity to paclitaxel
cancers, including prostate. In any case, the observation that
 90 Cancer Genetics

TABLE 5-5. Examples of Several Therapeutic Approaches Targeting Components of the Telomerase Core

TARGET APPROACH MOLECULAR EFFECT BIOLOGICAL EFFECT APPLICATION

hTER 2′-5′ Oligoadenylate Inhibit telomerase Apoptosis; increased sensitivity to Prostate, ovarian,
antisense cisplatin bladder, cervical
cancers
N3′-P5′ Apoptosis, senescence Prostate cancer
thiophosphoramidate
oligonucleotides
hTERT Ribozyme Inhibition of proliferation; sensitivity to Breast and
topoisomerase inhibitors ovarian cancer
Peptide nucleic acids Decreased survival Prostate cancer

Data from Birrocio A, Leonetti C. Telomerase as a new target for the treatment of hormone-refractory prostate cancer. Endocr Relat Cancer 2004;11:407–421.

HDAC
inhibitors
Demethylating
agents hTER
CH3
hTERT Gene
HDAC Transcription
MYC
SP1
c-MYC
antisense
CAAUCCCAAUC
AAAAAA mRNA
hTERT antisense
or ribozyme Translation
hTR antisense
or ribozyme
HAP90
Small hTERT
molecules p23

Assembly and
dimerization

DN-hTERT
hTERT HAP90 hTERT HAP90
P
Telomeres hTERT p23 P p23
hTERT
5' TTAGGGTTAGGGTTAGGG 3'
3' CAAUCCCAAUG
CAAUCCCAAUC
Kinase 3' 3' 5'
inhibitors 5'
Posttranslation
modification G-quadruplex
PKC ligand
AkT P hTERT HAP90
P
hTERT p23 5' TTAGGGTTAGGG
Telomeres
5' 3' AATCCCAAUT
TTAGGGTTAGGGTTAGGG 3'
3' 5' CAAUCCCAAUC
3'
5'

Figure 5-25. Some of the most promising approaches that directly target either telomerase and telomeres or the telomerase-
associated regulatory mechanisms are reported in the boxes at the targeting sites. HDAC, histone deacetylase; DN-hTERT,
dominant-negative hTERT; PKC, protein kinase C. (Reproduced by permission from Biroccio A, Leonetti C. Telomerase as a new
target for treatment of hormone-refractory prostate cancer. Endocr Relat Cancer 2004;11:407–421 [Fig. 1]. © Society for
Endocrinology, 2004.)
 Cytogenetic Alterations in Cancer and Tumor Heterogeneity 91

●●● CYTOGENETIC ALTERATIONS types are generated. Because a hallmark of tumor growth
IN CANCER AND TUMOR is genetic instability, each sublineage differs in the accumula-
tion of genetic alterations ranging from an accumulation of
HETEROGENEITY point mutations to karyotypic differences. In turn, these
Cytogenetic changes such as translocations, deletions, inver- genetic changes provoke variation in invasiveness, growth
sions, and aneuploidy are common in tumor cells and decid- rate, hormonal responsiveness, and metastatic abilities. From
edly uncommon in normal cells. Certain of these play a a genome point of view, the most frequent chromosomal
strong causal role in certain cancers such as Burkitt lym- alteration in solid tumors, such as colon cancers, is a change
phoma, chronic myeloid leukemia, and neuroblastoma—all of in chromosome number. Colon cancers are frequently hyper-
which result from earlier discussed chromosomal abnormali- diploid (i.e., possessing chromosomes in excess of the diploid
ties that transform proto-oncogenes into oncogenes. Some number). However, not all chromosomes are fully intact,
chromosomal numerical defects such as aneuploidy appear and it can be speculated that tumors exhibiting LOH not
later in the more malignant stages of cancer. Such changes only suffer from the functional loss of suppressor genes but
suggest either that the loss of genetic control of chromosomal also have lost growth-promoting genes. The unintended dele-
stability and number is key to late-stage cancer or that chro- tion of growth-promoting genes might restrict the cell’s
mosomal instability is secondary to the cellular deregulation growth potential. The cancer cell would thus be expected
found late in tumor progression. to obtain a selective advantage from an endomitosis, or
Tumors are clonal and derived from a single progenitor chromosomal replication within a cell nucleus that does not
cell. However, as the tumor grows, the constituent cells divide, leading to hyperdiploidy; this mechanism will restore
become extremely heterogeneous and sublineages of variant copies of the growth-promoting genes necessary to maintain

Types of Breast Carcinomas

(Each column represents a single carcinoma)

Basal-like HER2- ER-positive

postive
Lobular Ductal
HFN3
AR
GATA-3
LIV-1
ER
Ker 19 Cluster of ER genes
TFF3 and luminal keratins
Cyclin D1
Ker 8
(Each row is a separate gene)

Ker 18
E-cadherin
mRNA Transcripts

PPARBP
GRB-7 17q11-21 amplicon
HER2 (including HER2)
MLN64
Ker 17
Ker 5
P-cadherin
CDK 2
P53
Basal keratins
Ker 16 and cluster of
Cyclin A2 proliferation genes
Cyclin E1
Myc
HDGF
AC133
Cyclin B1
PCNA

Figure 5-26. Selected data from mRNA expression profiling of 26 breast carcinomas. Each vertical column represents one
carcinoma, and each horizontal row represents the data for a gene identified at the left. Red circle, increase in expression;
green circle, decrease in expression; black circle, no difference in expression between normal and carcinoma cells. (Redrawn
from Kumar V, Abbas A, Faust N. Robbins & Cotran Pathologic Basis of Disease, 7th ed. Philadelphia, WB Saunders, 2004,
p 1137.)
 92 Cancer Genetics

tumor progression. This extreme chromosomal heterogeneity In certain cases, such as FAP (colectomy), familial ovarian
may or may not facilitate certain features, such as metastasis, cancer (oophorectomy), and familial breast cancer (bilateral
of a tumor, but at the very least, it demonstrates the dys- mastectomy), prophylactic surgery is considered a viable
functional biology of the cancer cell while serving as a option to eliminate the genetically identified and verified
cancer biomarker. malignancy risk.
A new technique called microarray analysis, which com-
pares patterns of gene expression in normal and abnormal
●●● DNA-BASED CANCER
tissues, is demonstrating great promise for diagnostic use
SCREENING (see Chapter 13). This gene expression profiling has proved
As the number of cancer-related genes increases, attention is most useful in cancer diagnosis and surveillance (Fig. 5-26).
turning to the role of DNA testing for early detection and Here, complementary DNA (cDNA) probes are used that
prevention of cancer. This method is primarily applicable to are derived from tumor and adjacent normal tissues. Gene
familial cancers with a known mutational association, since chip expression studies permit the classification of new sub-
sporadic cancer implies unknown molecular etiology. Until types of breast cancers and produce a rapid diagnostic tool
recently, cancer surveillance in families predisposed to the for the distinction between acute lymphoblastic leukemia
disease relied on phenotypic or symptomatic monitoring with and acute myeloid leukemia. Prior to such tests, it was
or without indirect diagnostic tests. As familial cancer genes often difficult and time consuming to classify tumor subtypes.
and mutations are identified, however, cancer screening is Overall, gene expression analysis can be applied to neoplasia
performed noninvasively and well before the onset of early classification, diagnosis of malignant versus benign tumors,
symptoms. The identification of a cancer-predisposing muta- prognosis (including metastatic potential), and the develop-
tion therefore permits an aggressive surveillance or treatment ment of a cell-based surveillance platform to monitor the
plan for at-risk individuals as well as reproductive planning. response to therapy.

KEY CONCEPTS ■ Tumor suppressor genes can be grouped as cell division–

■ Cancer develops through a progression of cellular events that controlling genes, DNA repair genes, and apoptotic genes.
escape detection and correction or cell elimination. ■ Tumor suppressor genes are the model for the two-hit
■ The cell cycle is finely coordinated actions of cyclins, cyclin- hypothesis.
dependent kinases, transcription factors, enzymes, and other ■ Tumor suppressor gene mutations can demonstrate loss of
factors with checkpoints leading to apoptosis to protect the
heterozygosity because of a “second hit.”
individual from abnormal cell proliferation.
■ Telomeres protect ends of chromosomes from degradation.
■ Genes leading to cancer are oncogenes and tumor suppressor
Normally, telomerase activity decreases with age. In cancers,
genes. The normal function of these genes does not cause
telomerase is reactivated, allowing continued proliferation.
progression to cancer.
■ Rapid proliferation of cancer cells results in heterogeneous
■ Mutations in oncogenes are usually gain-of-function changes.
chromosome complements, with many different presentations
■ Mutations in tumor suppressor genes are generally loss-of- in a single tumor even though it had a clonal origin.
function changes. ■ mRNA expression profiling demonstrates differences in normal
and abnormal cells of the same or different types.

●●● QUESTIONS in the p53 protein, which is activated by DNA damage or

hypoxia to up-regulate expression of proteins participating
1. An 11-year-old male is diagnosed with leukemia 5 in apoptosis. If p53 is mutated, this up-regulation cannot
years after having an osteosarcoma removed from occur effectively, and the cell continues to divide, leading to
his femur and undergoing chemotherapy. His mother expansion and possibly to malignancy. APC gene expres-
died from breast cancer and five other family sion is involved in familial adenomatous polyposis. BRCA1
members in his mother’s family had different expression is associated with breast and ovarian cancers.
cancers. Which of the following genes is the most The RB1 protein is also involved in the cell cycle. It binds to
likely candidate for the cancer in this family? the E2F transcription factor to inhibit transcription, and
A. APC
therefore a mutation will decrease the inhibition. These
B. BRCA1
cancers generally present as retinoblastomas but can also
C. DCC
occur in other cells that receive either two spontaneous
D. RB1
mutations or a single spontaneous mutation along with one
E. TP53
inherited mutation (two-hit hypothesis). DCC is the “deleted
in colon cancer” gene and is one of the genes mutated
Answer. E in the progression model of cells going from normal to
Explanation: This patient has Li-Fraumeni syndrome, sug- abnormal to malignant.
gested by multiple types of cancers in the same family. The Additional Self-assessment Questions can be Accessed
patient has had two different cancers. The mutation occurs at www.StudentConsult.com
Hematologic Genetics 6
and Disorders
●●● BLOOD GROUP ANTIGENS
CONTENTS
In the early 1900s, the Austrian pathologist Karl Landsteiner
discovered that, when red blood cells of one person were
BLOOD GROUP ANTIGENS
mixed with the blood serum of another person, the cells
ABO Blood Groups frequently agglutinated. Agglutination reactions did not
Rh Blood Group Antigens occur in every combination of serum and cells, leading to a
Molecular Genetics of ABO Blood Groups hypothesis that the serum of certain persons reacts to the cells
Inheritance of others as if the cells were a foreign substance, or antigen.
Importance of Blood Group Antigens Red blood cell (RBC) antigens are inherited carbohydrate or
RED BLOOD CELL MEMBRANE protein structures located on the cell membrane. The serum
contains antibodies that bind specifically to the antigen, much
HEMOLYTIC ANEMIAS in the same manner that antibodies attack and destroy infec-
Red Blood Cell Membrane Defects tious microorganisms and foreign grafts. RBCs have many cell
Erythrocyte Metabolic Defects surface structures that are recognized as antigens by the
immune system.
ERYTHROCYTE HEMOGLOBIN DEFECTS
Two distinct types of antigens, A and B, are located on the
Hemoglobinopathies surface of human blood cells. These antigens are controlled
Sickle Cell Anemia by different alleles and can therefore be expressed indepen-
Thalassemias dently and observed together on the surface of some cells.
Blood groups not only illustrate clearly and simply the basic
BLEEDING DISORDERS
mechanism of the inheritance of multiple alleles, but also
Hemophilia A permit an appreciation of a uniquely human disorder known
Hemophilia B as hemolytic disease of the newborn (HDN).
von Willebrand Disease Most people are familiar with the ABO blood groups, but
THROMBOPHILIA there are other ways to identify blood cells by the antigens
present on the cell surface. There are approximately 29 major
Activated Protein C Resistance and Factor V Leiden blood group systems; each system is defined as one or more
antigens expressed by a single locus or more than one antigen
expressed by two closely linked, homologous loci. There is no
organization to the names of these systems. They have been
The heritable blood disorders are a heterogeneous group of named predominantly for the family in which the antibody
diseases that historically account for significant morbidity was found or given an alphabetical designation (Table 6-1).
and mortality around the world. The advent of transfusion
techniques and appropriate surgical and pharmacologic
approaches and the emergence of DNA-based carrier and
ABO Blood Groups
prenatal testing have rendered most of these disorders man- A person may have one of two antigens (A or B) on blood
ageable through early detection and diagnosis. In this chapter, cells, or both or neither may be present (Table 6-2). The pres-
blood group antigens, blood types, and inheritance are dis- ence of the A antigen reflects the blood group A; the presence
cussed. This lays the foundation for understanding a number of the B antigen reflects the blood group B; the presence of
of the more common inherited hematologic disorders that both A and B antigens reflects blood group AB; and the
are genetically well characterized and therefore serve as absence of both A and B antigens is designated as blood group
paradigms for the genetic basis of blood disease that are O. As indicated above, there are two corresponding antibodies
then discussed. Disorders of white blood cells are more in the serum of the blood, designated anti-A and anti-B. When
appropriately addressed in other chapters. serum containing anti-A is mixed with blood cells expressing
 94 Hematologic Genetics and Disorders

TABLE 6-1. Examples of Major Blood Group Systems Expressed on Erythrocyte Membrane Surfaces*

COMMON NAME ABBREVIATION NUMBER OF ANTIGENS EPITOPE OR CARRIER, AND NOTES

ABO ABO 4 Carbohydrate (N-acetylgalactosamine, galactose); A, B,

and H antigens mainly elicit IgM antibody reactions
Rhesus Rh 47 Protein; C, c, D, E, e antigens (there is no “d” antigen;
a lowercase “d” indicates the absence of D)
MNS MNS 37 GPA/GPB (glycophorins A and B)
P P1 1 Glycolipid
Kell KEL 21 Glycoprotein; K1 can cause hemolytic disease of the
newborn (anti-Kell)
Hh H 1 Carbohydrate (fucose)
Lutheran LU 18 Protein (member of immunoglobulin superfamily)

*The most readily recognized are the ABO and Rh systems.

TABLE 6-2. Comparison of ABO Genotypes and Phenotypes

BLOOD TYPES THAT BLOOD TYPES THAT

ANTIGENS ANTIBODIES PRESENT CAN BE TOLERATED CAN ACCEPT BLOOD
PRESENT ON RBC ABO PHENOTYPE IN BLOOD IN TRANSFUSION* FOR TRANSFUSION*

A Type A Anti-B A&O A & AB

A Type A Anti-B A&O A & AB
B Type B Anti-A B&O B & AB
B Type B Anti-A B&O B & AB
A&B Type AB Neither anti-A nor anti-B A, B, AB & O AB only
Neither A nor B Type O Anti-A & anti-B O only A, B, AB & O

*Individuals with type AB are referred to as “universal recipients”; individuals with type O are the acknowledged “universal donors.”

the A antigen, cells clump together in large granular masses. encodes the D antigen (RHD) and the other encodes proteins
On the other hand, if anti-A is mixed with B blood cells, or carrying C or c and E or e antigens (RHCE). RHD and RHCE
anti-B with A blood cells, there is no reaction; the cells remain are 96% homologous, representing the consequence of gene
suspended without clumping. This simple principle is the basis duplication. Interestingly, these two genes are oriented in
for blood group tests. opposite directions with the 3′ ends facing each other (Fig.
There are three alleles responsible for four main ABO 6-2). Individuals who are Rh-positive have both RHD and
blood group system phenotypes. The phenotypes produced RHCE, whereas Rh-negative erythrocytes have only RHCE
are A, B, AB, and O. The carbohydrates defining A and B on their surfaces. Eight common antigen combinations are
antigens are added to the H antigen, fucose (Fig. 6-1). shown in Box 6-1. The lack of D is shown as d, though there
Together there are more than 90 alleles of the three major is no d antigen or anti-d.
gene blood types. The origin of these alleles is the gene
producing a glycosyltransferase enzyme that attaches a sugar
to a precursor.
Molecular Genetics of ABO
Blood Groups
Rh Blood Group Antigens The locus specifying different blood group types is called
The second most commonly known antigens are the Rh anti- the ABO locus. This locus encodes glycosyltransferases that
gens, named for the rhesus monkey in which they were dis- synthesize A and B antigens expressed on the erythrocyte
covered. These are transmembrane proteins with loops cell surface. For these antigens to be expressed, a precursor
exposed on cell surfaces. They appear to be used for the molecule called the H antigen, which is synthesized by
transport of carbon dioxide and/or ammonia across the cell fucosyltransferase (expressed by the FUT1 gene), must be
membrane. Among the ABO blood group antigens, there are present. Another gene, FUT2, synthesizes H antigens found
four phenotypes (A, B, AB, and O); the Rh blood group in saliva and other fluids.
system has 47 antigens that are determined by two closely The ABO locus is located at chromosome 9q34.1. It con-
linked genes located at chromosome 1p34-1p36. One gene tains 7 exons that span over 18 kb. Most of the coding
 Blood Group Antigens 95

N-acetylgalactosamine O
added to precursor O O
O O
O
O This is the A antigen;
A allele O
it reacts with
transferase anti-A antibody

O O
OH O O
O OH
O allele O
O transferase
O O
O
This is the H antigen;
it reacts with neither
Precursor carbohydrate anti-A nor anti-B antibody
B allele O
transferase
O O
O O
O
O
Galactose added O This is the B antigen;
to precursor it reacts with
anti-B antibody

Key: CH2OH CH2OH CH2OH H

H O H HO O H HO O H H O OH
H H H CH3
OH H OH H OH H H HO
HO OH H OH H OH HO H
H NHCOCH3 H OH H NHCOCH3 OH H
N-acetylglucosamine Galactose N-acetylgalactosamine Fucose

Figure 6-1. The ABO antigens on the surface of erythrocytes are carbohydrates. Note that the O-encoded transferase is
inactive due to a frameshift mutation. Therefore, the precursor, H antigen, remains unchanged. Failure to express the H antigen
results in the Bombay phenotype.

Box 6-1. EIGHT COMMON COMBINATIONS OF

30-kb RH ANTIGENS*

RHD SMP1 RHCE DCE

Dce
DcE
DCe
Figure 6-2. Schematic of the RH gene locus. The two RH dce
genes have opposite orientations and are separated by the dCe
SMP1 gene. The RHD gene is flanked by two highly dcE
homology Rhesus boxes (gray) that are thought to participate dCE
in nonhomologous recombination and the deletion of RHD
from one chromosome and its duplication on the *D/d antigens are expressed from the RHD gene and the CE/ce antigens are
homologous chromosome. This deletion explains the “d” expressed from the RHCE gene.
phenotype.

sequence of this gene is found in exon 7. A single, critical antigens, individuals with O blood type lack this ability. A
base deletion mutation in exon 6 results in a loss of glycos- and B alleles differ in a few single-base substitutions changing
yltransferases enzymatic activity that is found in most O four amino acid residues (R176G, G235S, L266M, G268A),
alleles. This frameshift deletion, of guanine at position 261 and these changes are reflected in the differences in A and B
(A261G), results in an inactive enzyme. Therefore, unlike transferase specificity.
individuals with A, B, or AB blood types that express glyco- The H locus encodes fucosyltransferase and is required for
syltransferases that convert the H antigen into A or B synthesis of the H antigen. The H antigen consists of a chain
 96 Hematologic Genetics and Disorders

of β-D-galactose, β-D-N-acetylglucosamine, and α-L-fucose. A, B, and AB (Table 6-5). Type O children do not arise from
Either HH or Hh genotypes occur from in the production of parents with type AB blood, and AB offspring occur twice as
H antigen. If a person has no H locus, or the expression of often as either the A or B child. The 1 4 A : 1 2 AB : 1 4 B pheno-
functional fucosyltransferase is so low that the phenotypic typic ratio is clearly the result expected from the mating of
effect is that no fucosyltransferase is present, the alleles are two heterozygous parents. Predicting the phenotypic ratio is
referred to as “null” alleles. In the situation where an indi- possible for any mating in which the blood type of the parents
vidual has no H locus on either chromosome 19, the lack of is known. Likewise, knowing the blood type of a child and
H antigen (hh) precludes the production of A and B antigens one parent provides information about what the other par-
and the serum contains anti-A, anti-B, and anti-H. These cells ent’s blood type might be. The blood types of several children
demonstrate a rare phenotype of H-deficient erythrocytes may yield even more definitive information about an unknown
called the “Bombay phenotype” (Oh). These individuals differ parental blood type. More recently, however, blood typing is
from those with O blood type who have H antigen present used less for parental identity than it once was, but it is still
on cells but who lack A and B antigens because of the lack very important for transfusions and organ transplantation.
of glycosyltransferases. Transfusions of individuals with the
Bombay phenotype should therefore be from someone with
the Bombay phenotype.
Importance of Blood Group Antigens
Antigens expressed from the ABO locus are universally rec-
ognized as “erythrocyte” antigens; however, they are found
Inheritance on most epithelial and endothelial cell surfaces, including
As discussed above, the ABO blood group antigens are other blood cells such as leukocytes and platelets. A soluble
expressed from one locus, which has three alleles yielding six form of the antigens is also present in all body fluids with the
possible genotypes and four possible phenotypes (Tables 6-3 exception of cerebrospinal fluid. The function of these anti-
and 6-4). gens is unknown, though their importance for transfusion
The presence of three alleles does not alter the rules of compatibility is critical. It has also been noted that some
inheritance presented in Chapter 3; an individual normally diseases are linked to specific ABO phenotypes. For example,
possesses only two of the three alleles. When both parents there is a correlation among individuals with type A blood
are AB, only three blood types can appear in the offspring: and gastric cancer, and individuals with type O blood have a
higher incidence of gastric and duodenal ulcers. Of interest
for this particular section is that individuals with type O
blood have about 25% less factor VIII and von Willebrand
factor than other individuals. Similarly, individuals who have
TABLE 6-3. All Possible Genotypes for other blood types (A, B, and AB) have an increased risk of
Offspring of Parents with
arterial and venous disease.
Each Allelic Contribution
An ABO incompatibility may occur in a fetus expressing an
A or B antigen inherited from the father that is not present
ALLELES INHERITED
in the mother. The mother, for example, may be type O and
FROM MOTHER
the baby may be type A. As shown in Table 6-2, the mother
ALLELES INHERITED has anti-A and anti-B antibodies that may enter the fetal
FROM FATHER A B O
circulation through the placenta and destroy fetal RBCs car-
rying the A antigen. This may result in hemolytic disease of
A AA AB AO
the newborn (HDN), in which the principal manifestation is
B AB BB BO
O AO BO OO jaundice. Unlike Rh incompatibility, which is discussed below,
ABO incompatibility may occur during a first pregnancy
because anti-A and anti-B antibodies are found early in life

TABLE 6-4. All Possible Phenotypes for

Offspring of Parents with TABLE 6-5. Possible Blood Types in Offspring
Each Allelic Contribution of Parents Who Each Have Blood
Type AB
ALLELES INHERITED
FROM MOTHER ALLELES FROM
MOTHER
ALLELES INHERITED
FROM FATHER A B O
ALLELES FROM FATHER A B
A Type A Type AB Type A
B Type AB Type B Type B A AA AB
O Type A Type B Type O B AB BB
 Red Blood Cell Membrane 97

from exposure to A- and B-like antigens found in many foods The C, c, E, and e antigens are less immunogenic and only
and bacteria. Hemolytic disease due to anti-A (or anti-B) is important after antibodies have been detected or when more
much milder than that caused by anti-Rh, and rarely requires precise haplotypes are required. The additional antigens,
an exchange transfusion of blood (<1 : 1000). those not shown in Box 6-1, represent different Rh protein
For most clinical purposes, testing for the presence of RhD epitopes that are very rare.
(Rh positive or Rh+) or its absence (Rh negative or Rh−) is
sufficient for compatibility testing. Approximately 85% of the
Caucasian population is Rh-positive. Individuals who are
●●● RED BLOOD CELL MEMBRANE
Rh-negative produce anti-D when given Rh-positive blood. If The red blood cell (RBC) membrane consists of a lipid bilayer
anti-D antibody is formed, hemolysis occurs in an adult. (primarily phospholipids and unesterified cholesterol), inte-
However, if this occurs in during pregnancy, where the mother gral membrane proteins, and structural proteins that form the
becomes sensitized to D antigens in fetal blood, hemolysis membrane infrastructure. Membrane proteins are often glyco-
(HDN) can occur that is much more serious than ABO incom- proteins that confer either functional or antigenic properties,
patibility (Fig. 6-3). Also known as erythroblastosis fetalis, illustrated by anion exchange band 3 protein and glycopho-
harm to a fetus can be prevented by administering anti-Rh rins, respectively. The structural proteins define an inner mem-
factor, known as Rh immune globulin, to the Rh-negative brane coating lattice that forms the typical biconcave shape
mother before exposure to Rh-positive blood and production and promotes the deformability of normal RBCs. Such pro-
of antibodies. The Rh immune globulin is administered when- teins include spectrin, ankyrin, actin, and protein 4.1. Spectrin
ever there is a risk of maternal and fetal blood mixing, such is composed of two chains, α and β, that form heterodimers by
as following childbirth, an abortion, a miscarriage, or prenatal wrapping around each other. These heterodimers associate in
testing. Once sensitized, the woman will always produce anti- a head-to-tail manner, resulting in heterotetramers. Spectrin
bodies to Rh-positive cells. heterotetramers interact with two other structural proteins,
actin and protein 4.1, at the spectrin “tail” end. At the “head”
end, the spectrin heterotetramer is attached to ankyrin, and
this complex is stabilized by protein 4.2. This proteinaceous
Rh-negative woman before pregnancy
“membrane skeleton” is connected to the lipid bilayer by an
association between ankyrin and band 3 proteins. Mutations
in these structural proteins affect the ability of RBCs to appro-
Pregnacy with Rh-positive fetus priately change shapes when circulating through narrow and
tortuous vessels. Decreasing the flexibility of RBCs can lead
to an increased opportunity for abnormal pathology.
Blood mixes at placental separation

BIOCHEMISTRY
Mother sensitized to Rh-positive blood
Membranes
Lipids that consist predominantly of aliphatic or aromatic
Mother develops anti-Rh antibodies
hydrocarbons form the framework of cell membranes. The
major lipid components are phosphoglycerides (sometimes
just referred to as phospholipids), sphingolipids, and sterols.
Second pregnacy with Rh-positive fetus Cholesterol is the major sterol. There are hundreds of minor
lipids, and differences in specific composition cause different
cell membrane fluidity; in some situations, differences are
Mother’s anti-Rh antibodies enter fetal circulation reflected by disease.
Lipids in membranes usually contain C16 saturated fatty
acids and longer fatty acids with the presence of double
Anti-Rh antibody to fetal Rh-positive RBCs bonds. The presence of double bonds is critical, since C18 fatty
acids are solid at physiologic temperatures without double
bonds. Double bonds create specific conformational changes
Hemolysis of fetal RBCs that affect membrane fluidity. Fluidity is particularly influenced
by the amount of cholesterol in membranes because the rigid
steroid ring binds and immobilizes other fatty acids. Thus,
Figure 6-3. Erythroblastosis fetalis. Hemolytic disease modest changes in cholesterol concentrations have great
caused by the mixing of fetal blood containing Rh-positive impact on membranes. Spur cell anemia illustrates the impact
antigens with maternal blood that is Rh-negative. The first of an increased ratio of cholesterol to phospholipids. This
pregnancy usually is not affected since blood from the fetus condition is usually seen in alcoholic cirrhosis, in which
is not mixed with that of the mother, which initiates an cholesterol content increases from 25% to 65%. The increased
immune response, until birth. Subsequent pregnancies, cholesterol leads to a progressive increase in RBC
however, are at risk for HDN unless the mother is given Rh deformation and ultimately hemolysis and anemia.
immune globulin to suppress antibody formation.
 98 Hematologic Genetics and Disorders

●●● HEMOLYTIC ANEMIAS HS HE HS

Anemia can result from many causes, some congenital and
some acquired. Among the most serious are the hemolytic
anemias resulting from increased RBC destruction. In general, Band 3 tetramer
hemolytic anemias share two characteristics: the life span of GPA
the red cell is reduced and iron is retained after hemolysis. GPC
Congenital hemolytic anemias in this chapter are classified
into three general etiologic categories: erythrocyte membrane 4.1 HE
defects, metabolic defects, and hemoglobin defects. 4.2 p55 4.9
Tropomodulin 4.1
Ankyrin Actin

PATHOLOGY HS, HE HPP HS β-Spectrin HS, HE

Hemolytic Disorders α-Spectrin

Hemolytic disorders are characterized by premature Figure 6-4. The red blood cell membrane has several
destruction of red cells. Hemolysis can occur either proteins that affect the structure of the cell. Mutations in
intravascularly or extravascularly. During intravascular proteins leading to hereditary spherocytosis (HS), hereditary
hemolysis, hemoglobin is released and bound to elliptocytosis (HE), and hereditary pyropoikilocytosis (HPP)
haptoglobin. The hemoglobin-haptoglobin complex is are shown.
removed by hepatic RES cells; thus, the haptoglobin levels
generally fall, and the resulting excess hemoglobin is
converted to ferritin and hemosiderin. With rapid
intravascular hemolysis, hemoglobinuria can be seen. screening tests; however, once a mutation is identified within
Most hemolytic disorders demonstrate extracellular
a family, other members may be screened. HS is most com-
hemolysis. Red cells are sequestered in the spleen and/or
monly an autosomal dominant disease with variable expres-
liver and phagocytized, with the hemoglobin escaping into
plasma and thus causing the haptoglobin to decrease. In sivity (Table 6-6). α-Spectrin, however, is a notable exception.
this case, hemosiderinuria and hemoglobinuria generally do Roughly four times more α-spectrin than β-spectrin is
not occur. expressed within an RBC. This seeming excess of α-spectrin
is reflected in heterozygotes for α-spectrin mutations. In these
individuals, enough α-spectrin protein is usually produced
without serious consequence. However, homozygous or com-
pound heterozygous α-spectrin mutations can precipitate a
Red Blood Cell Membrane Defects
severe HS presentation.
Hereditary Spherocytosis and HE is similar to HS in that both may have mutations in
Hereditary Elliptocytosis α-spectrin and β-spectrin genes. These disorders differ in the
Hereditary spherocytosis (HS) and hereditary elliptocytosis effect the mutations have on the membrane and its resulting
(HE) are the most common hemolytic anemias caused by shape. Typically in HE, spectrin heterodimers cannot self-
RBC membrane defects. HS is the most common hereditary associate into heterotetramers. This is consistent with the fact
hemolytic anemia found in individuals of Northern European that most HE mutations are found in the protein domains that
descent and results from structural defects linking the under- directly participate in the self-association process. HE is also
lying membrane skeleton with the lipid bilayer. Mutations typically an autosomal dominant disease (Table 6-7).
causing HS are in genes encoding ankyrin, spectrin, band 3 Patients with the most common form of HE are usually
protein, and protein 4.2 (Fig. 6-4). Such defects cause a loss asymptomatic. Only 5% to 20% develop hemolysis with
of membrane and an associated decreased surface area– anemia, splenomegaly, scleral icterus, pallor, gallstones, or
to-volume ratio. These changes account for the spheroidal occasional leg ulcers. In many cases, splenectomy eliminates
shape of the HS erythrocytes and the decreased ability of symptoms by decreasing the number of ruptured RBCs. This
RBCs to change shape as they circulate through small vessels defect is common in individuals of African and Mediterra-
and capillaries. The deformed erythrocytes, called sphero- nean descent, and many of these individuals harbor similar
cytes, become trapped in the splenic microcirculation and genetic backgrounds, or haplotypes. This indicates a genetic
undergo hemolysis (Fig. 6-5). founder effect and persistence within the population. Such
The mutational spectrum in ankyrin, α-spectrin, β-spectrin, persistence can be explained by selection, since elliptocytosis
band 3 protein, and protein 4.2 includes missense, nonsense, appears to confer some resistance to malaria. This contrasts
frameshift, splicing, and promoter variants. The majority of with the situation for HS, which is much more common in
patients have unique (or “private”) mutations. This indicates European-derived individuals than African-derived individu-
an absence of a mutational “hot spot.” Unique mutations als and does not exhibit a founder effect or allele persistence
are more difficult to identify and are not well suited for due to selection.
 Hemolytic Anemias 99

A B

Figure 6-5. A, Hereditary spherocytosis. Spherocytes lack central pallor, stain more darkly, and are smaller in diameter than
nonspherocytic RBCs. B, Hereditary elliptocytosis. Many cells are elliptical rather than oval. The marker ellipsoidal cell has
an axial ratio of >2 : 1. Cell fragments and microelliptocytes are present. Note that they maintain an area of central pallor.
C, Hereditary pyropoikilocytosis. Almost all cells are misshapen. Fragmented spheroidal cells and elliptical forms predominate.
(Courtesy of Dr. Anna Walker, Mercer University School of Medicine, Macon, Georgia.)

TABLE 6-6. Mutation Frequency in Hereditary

Hereditary Pyropoikilocytosis
Spherocytosis (HS) Hereditary pyropoikilocytosis (HPP) has been called a
subtype of homozygous elliptocytosis as well as an “aggra-
FREQUENCY
vated” form of elliptocytosis. Both HPP and HE result from
GENE IN HS mutations in α-spectrin, but the clinical presentations differ
PRODUCT TRANSMISSION PATIENTS (%) in severity. Hereditary elliptocytosis has a wide spectrum of
clinical manifestations ranging from no symptoms to severe.
Ankyrin AD, some AR 40–50 Pyropoikilocytosis is a severe form of hemolytic anemia with
Band 3 AD 20–30 thermal instability of red cells. The consequences of this
Spectrin α-Spectrin, AR 10 severe anemia are evident in children who exhibit growth
β-Spectrin, AD 10 retardation, frontal bossing, and gallbladder disease.
Protein 4.2 AR Rare
HPP is rare; however, it is worth considering along with
AD, autosomal dominant; AR, autosomal recessive. HE because it illustrates that some mutations affect the rate
 100 Hematologic Genetics and Disorders

TABLE 6-7. Red Blood Cell Membrane Defects Leading to Hemolysis

FEATURE ELLIPTOCYTOSIS SPHEROCYTOSIS PYROPOIKILOCYTOSIS

Inheritance Dominant Dominant Recessive

Incidence 1 in 2000–4000 1 in 5000 Rare
Ethnicity In all racial and ethnic groups; Common in people of Northern Predominantly in blacks
more common in blacks European descent, but found
in all people
Mutation Spectrin, glycophorin C, protein Spectrin, ankyrin, band 3, Compound heterozygous
4.1 protein 4.2 α-spectrin functional mutation,
reduced synthesis mutation
Clinical presentation Most asymptomatic or with Asymptomatic (80%) to Splenomegaly, intermittent
minimal (15%) compensated moderate anemia (20%); jaundice, aplastic crises
anemia jaundice, pallor, splenomegaly
Laboratory findings Elliptocytosis, few or no Reticulocytosis, spherocytosis, Severe hemolysis;
poikilocytes, no anemia, little elevated MCHC, increased microspherocytes, poikilocytes,
or no hemolysis, reticulocytes osmotic fragility, normal reticulocytosis, decreased MCV,
1–3%, normal osmotic fragility Coombs test increased osmotic fragility,
decreased red cell heat stability

MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume.

of expression rather than the structure or function of the (NADPH), a cofactor in glutathione metabolism in human
protein produced. The differences between HPP and HE in RBCs. The HMP shunt is tightly coupled to glutathione
disease severity are explained by allele specificity resulting metabolism, which serves to protect RBCs from oxidant
from mutations in the same gene. Specific mutations in injury. Accordingly, a marked deficiency of G6PD leaves the
α-spectrin associated with HPP are called “low-expression” red cells vulnerable to oxidant damage.
alleles. There are at least four of these alleles that produce
fewer α-spectrin chains. When any of these alleles combines
with other α-spectrin alleles that are more commonly associ-
ated with HE, fewer α-spectrin chains are produced from one BIOCHEMISTRY
allele and defective α-spectrin chains produced from the
other allele fail to form spectrin tetramers. Hexose Monophosphate Shunt
The hexose monophosphate shunt (HMP) is also called the
pentose phosphate pathway. It occurs in the cytoplasm and
Erythrocyte Metabolic Defects is a major source of NADPH and 5-carbon sugars. The HMP
Glucose-6-Phosphate consists of two irreversible oxidative reactions and a series
of reversible sugar-phosphate conversions. No ATP is
Dehydrogenase Deficiency
consumed or produced directly. Carbon 1 is released from
Glucose-6-phosphate dehydrogenase (G6PD) deficiency
glucose-6-phosphate (G6P) as CO2, and 2 NADPH are
reigns as the most prevalent enzyme disorder in the world. It produced for each G6P entering the pathway. The HMP also
occurs in an estimated 400 million people in the world popu- produces ribose-phosphate for nucleotide synthesis.
lation. As an X-linked disorder, it affects mostly males. The
highest frequencies occur in Mediterranean countries, Africa,
and China. The worldwide distribution of G6PD deficiency
parallels that of malaria, which suggests a genetic state of
balanced polymorphism associated with resistance to falci- HISTOLOGY
parum malaria. It has been observed that female heterozy-
gotes for G6PD deficiency, who have both normal and Heinz Bodies
G6PD-deficient RBCs, have lower parasite counts in G6PD- Heinz bodies are intracellular inclusions composed of
deficient red cells and are relatively resistant to malaria. This denatured hemoglobin. Found at the cell membranes of
selective advantage has been observed for other diseases such erythrocytes, they are seen in thalassemias, enzymopathies,
as sickle cell disease and β-thalassemia. hemoglobinopathies, and after splenectomy.
G6PD activity is essential to normal functioning of the
hexose monophosphate (HMP) shunt. This pathway generates
reduced nicotinamide adenine dinucleotide phosphate
 Hemolytic Anemias 101

MICROBIOLOGY Glucose-6-phosphate
dehydrogenase
Sickle Cell Trait and Malaria Resistance (G6PD)
Glucose
6-Phosphogluconate
Erythrocytes in a person with sickle cell trait (heterozygote) 6-phosphate
confer protection from infection with falciparum malaria.
RBCs develop “knobs” on the cell membrane surfaces that
cause the cells to stick to the endothelium of small vessels.
Other
This sticking occurs because of low O2 concentration, NADP; NADPH
reactions
presumably caused by the parasite.
The parasite requires a high K+ environment, and when
the RBC membrane is damaged, potassium is lost from the
RBC as well as the parasite. Infected RBCs are more acidic
and hypoxic. These conditions increase sickling, leading to GSSG 2 GSH
the sequestration of infected cells (but not uninfected cells) Glutathione
reductase
and elimination of the sickled cells by phagocytes.
Neonates have an increased resistance to malaria because Ribose
Hb F is very stable and resistant to malaria hemoglobinases.
Hb F cells are infected preferentially to Hb A cells.

BIOCHEMISTRY Glutathione
peroxidase
G6PD and Oxidative Stress
H2O H2O2
The metabolism in erythrocytes is almost entirely anaerobic,
and the major source of energy is derived from the glucose Antioxidant
that is metabolized by the glycolytic pathway and the activity
pentose phosphate pathway. These cells are very sensitive
to oxidative stress. Figure 6-6. Role of G6PD in oxidative stress. G6PD is
Glucose-6-phosphate dehydrogenase is the only RBC required to generate NADPH and break down H2O2. GSH,
enzyme that produces NADPH through glutathione reduced glutathione; GSSG, oxidized glutathione; NADP+,
reductase, and therefore any variation in the function of nicotinamide adenine dinucleotide phosphate; NADPH,
G6PD can decrease the amount of NADPH available to the reduced nicotinamide adenine dinucleotide phosphate.
cell. A deficiency in G6PD diminishes the amount of NADPH
available for glutathione reductase. This is not an issue in
other cells, because they have several enzymes. A reduction
in glutathione allows reactive oxygen products to damage enzyme. The G6PDA− enzyme is more rapidly degraded than
cell proteins, lipids, and DNA. the normal enzyme, but young G6PD A− cells are capable of
Individuals deficient in G6PD should not be given withstanding oxidant stresses. In stark contrast, both the cata-
oxidative drugs such as antimalarial drugs (primaquine), lytic ability and stability of the variant G6PDB− enzyme are
certain analgesics/antipyretics, cardiovascular drugs reduced so drastically that virtually the entire G6PDB− RBC
(procainamide, quinidine), sulfonamides, and cytotoxics/ population, young and old cells, is susceptible to oxidant-
antimicrobials. These oxidant drugs can induce immediate
induced hemolysis. The A form of G6PD deficiency is
acute hemolytic episodes characterized by progressive
contrasted with the B form in Table 6-8.
anemia, hemoglobinuria, and reticulocytosis caused by
hemolysis of cells with low G6PD activity. G6PD allelic changes result in varied forms of enzyme
deficiency: decreased enzyme synthesis, the presence of an
enzyme with abnormal kinetics, or the presence of an unsta-
ble enzyme whose catalytic activities become diminished as
There are two normal alleles of G6PD—the B allele cells age. Two prominent hemolytic conditions, primaquine
(G6PDB), which is widespread in the Mediterranean, Middle sensitivity and favism, illustrate effects of the mutated alleles.
East, and Orient, and the A allele (G6PD A), which is largely
confined to sub-Saharan Africans and their descendants. The Primaquine Sensitivity
normal enzyme products of these alleles are designated In the mid-1900s, a strain of vivax malaria with a long latent
G6PDB+ and G6PDA+, respectively, where the “+” denotes period was common in Korea. During the Korean War
normal enzyme activity. Both enzymes slowly degrade nor- (1950–1953), American soldiers were prophylactically admin-
mally over the life span of a normal red cell, but G6PD activ- istered an antimalarial drug, primaquine (a 6-methoxy-8-
ity is still sufficient in the oldest normal RBCs to withstand aminoquinoline). It was observed that about 10% of black
oxidant stresses (Fig. 6-6). The mutations causing G6PD A− and soldiers experienced an intravascular hemolytic reaction fol-
GGPDB− differentially affect the rate of degradation of the lowing administration of primaquine. Intravascular hemolytic
 102 Hematologic Genetics and Disorders

TABLE 6-8. Comparison between G6PD A− and G6PDB− NEUROSCIENCE

FEATURE G6PDA− G6PDB− Kernicterus

Kernicterus results from bilirubin deposition in the basal
Frequency Common in Common in ganglia and causes diffuse neuronal damage. Elevated
African Mediterranean bilirubin moves out of blood and into brain tissue, causing
populations populations lethargy, hypotonia, and poor sucking reflex in the first few
Enzymatic activity days of life, followed by marked hypertonia—especially of
G6PD A+ 100% — extensor muscles. Children are hypotonic for years before
G6PDB+ — 100% hypertonicity returns and have marked developmental and
G6PD A− 10–20% — motor delays in the form of choreoathetoid cerebral palsy.
G6PDB− — 0–5% Mental retardation may be present. Other sequelae include
Degree of acute Moderate Severe extrapyramidal disturbances, auditory abnormalities, gaze
hemolysis palsies, and dental dysplasias.
Abnormal G6PD Old RBCs All RBCs
activity
Hemolysis with
Fava beans Unusual Common Severe hemolysis following exposure to fava beans occurs
Primaquine Common Rare in G6PD-deficient whites and Asians; it is rarely seen in black
Other drugs Moderately Very common Africans. Acute infections, however, do trigger hemolytic
common
Infection Common Common
episodes in black Africans. The physiologic properties of
G6PDCanton, the variant common in Asians, are very similar to
Need for transfusion Rare Sometimes
those of G6PDMediterranean.
The experience of the black soldiers during the Korean War
and of favism among Mediterranean and Asian individuals
reactions had long been known to occur in some individuals clearly established differences in the expression of G6PD
from Mediterranean areas after eating broad beans (Vicia deficiency between Mediterranean and black males. In the
fava); however, soldiers of Mediterranean ancestry in Korea RBCs of blacks with primaquine-induced G6PD deficiency, a
were largely spared the hemolytic reaction to primaquine that residual enzyme activity of 10% to 20% is regularly found,
befell certain black soldiers. An important clinical observa- whereas affected Mediterranean males show only minimal,
tion was that this primaquine-induced hemolytic anemia was often barely detectable, activity below 5%. The young red
self-limited and not life threatening. It was discovered that cells of primaquine-induced G6PD deficiency have a suffi-
the differential sensitivity to primaquine was a function of cient level of catalytic activity to provide protection against
RBC age. Young RBCs were resistant to the hemolytic effect oxidative damage and hemolysis. In fava-induced hemolysis
of primaquine while older RBCs were sensitive. Hence, in an in Mediterranean males, virtually all RBCs are susceptible to
affected individual, once older cells are destroyed, hemolysis destruction, and the acute hemolytic episodes are thus life
stops despite continuation of drug treatment. threatening.
Primaquine sensitivity among sub-Saharan Africans and
their descendants is associated with a mutation of the normal Sex-linked Inheritance
G6PDA allele. The frequency of the normal G6PDA allele is The G6PD gene is located on the X chromosome. As a result,
about 10% to 15% in American black males and over 20% males are typically more severely affected than females, who
among males in many parts of Africa. have two X chromosomes and demonstrate lyonization. A
G6PD-deficient cell in a female is as vulnerable to hemolysis
Favism as an enzyme-deficient cell in a male. However, the presenta-
Fava beans are a staple of the diet in many Mediterranean tion of G6PD deficiency in female heterozygotes may be mild,
countries. A severe hemolytic anemia is associated with the moderate, or even severe, depending on the proportion of
ingestion of fava beans and can even be induced by inhalation RBCs in which the abnormal G6PD enzyme is expressed. A
of fava bean pollen. The culpable mutant allele is G6PDB− female may even have two different G6PD alleles and, accord-
(sometimes written G6PDMediterranean). This allele is responsible ingly, produce two different biochemical types of enzyme.
for severe hemolytic episodes when G6PD-deficient individu-
als acquire infections such as pneumonia, salmonellosis, and
hepatitis. During acute infections, phagocytic activity of mac- ●●● ERYTHROCYTE HEMOGLOBIN
rophages liberates oxidants that RBCs cannot adequately DEFECTS
degrade because of inadequate G6PD, and hemolysis occurs.
A dire consequence of G6PD deficiency in Mediterranean and
Hemoglobinopathies
Asian neonates is hyperbilirubinemia. Severe hyperbilirubine- The primary function of an RBC is its role in delivering
mia can result in kernicterus and severe neurologic sequelae. O2 to cells and tissues. As seen with HS, HE, and HPP, com-
The affected neonates manifest jaundice at 1 to 4 days of age. promising the integrity of the cell membrane can lead to
 Erythrocyte Hemoglobin Defects 103

hemolysis and thus affect oxygen delivery. The role of hemo-

globin molecules is equally or more critical. Globin is the Hemoglobin
protein that surrounds a heme molecule that mediates oxygen Different hemoglobins exist at various phases of human
binding. Several types of hemoglobins are produced, begin- development (Fig. 6-7). Two hemoglobins, Gower 1 and
ning in the fetus, before the “adult” form appears. Two dis- Gower 2, are found in embryos of up to 8 weeks of gestation.
tinct globin chains combine with each heme. The fetus and Hemoglobin Portland, which was first characterized in an
embryo have hemoglobins that differ from the mature “adult” infant with a chromosome abnormality, is a third normal
form. Adult hemoglobin, Hb A, is composed of α and β embryonic hemoglobin. The predominant hemoglobin from
chains. Generally speaking, mutations causing a structural the eighth week to term is fetal hemoglobin, Hb F, and is
change in the β-globin gene result in sickle cell anemia, composed of α2γ2. There are two distinct γ chains, one with
whereas mutations causing an absence or reduced amount glycine and the other with alanine at position 136. These two
of hemoglobin, from either the α- or the β-globin allele, γ chains are expressed at distinct loci. The rate of production
result in thalassemias. of β chains increases coincidentally with a decline in γ-chain
synthesis. In adult life, Hb A (α2β2) makes up about 97% of
Nomenclature the total hemoglobin, the remaining 2% to 3% being repre-
It is important to clarify certain terminologies that can be sented by Hb A2 and a small amount of Hb F.
confusing. Greek letters have been used historically by bio- Genetic studies established that the α-globin gene and the
chemists to designate protein chains with complex molecules. β-globin gene reside on different chromosomes. Indeed, a
Geneticists have used Greek letters to name genes within a cluster of α-like genes evolved from a single ancestral α gene
family that produce related proteins. For example, “α” and by a series of duplications on one chromosome, and a com-
“β” have been used to designate two different spectrins in parable family of β genes emerged on another chromosome.
RBC membranes. The gene symbols for spectrins are in the As shown in Figure 6-7, the linked group of α-like genes on
SPT gene family. Globin genes are another family of closely human chromosome 16 contains an active embryonic ζ (zeta)
related genes using Greek symbols to indicate loci. Several gene and two active α genes. The β-like cluster on human
letters, some less commonly used than others, designate the chromosome 11 comprises five active genes: one ε (epsilon),
gene, but these designations are not the gene name or symbol. two γ (gamma), one δ (delta), and one β.
The α-globin locus and protein may be shown as α or α-globin, The β and δ genes are nearly identical in composition,
but the genes are designated as HBA1 and HBA2. which reflects their recent duplication during evolution.
It is also important to recognize that types of hemoglobin Indeed, the δ gene arose only 40 million years ago and is
have a single letter designation or a combination of two found only in higher primates. The divergence of the δ gene
letters. This is demonstrated by normal adult hemoglobin, occurred prior to the separation of the phylogenetic lineage
which is Hb A, or sickle cell hemoglobin, which is Hb S. All leading to the Old World monkey assemblage and the line
hemoglobins consist of four polypeptide chains—two of one represented by the great apes and humans.
type and two of another type. For Hb A, these chains are two Both families of hemoglobin genes contain loci called
α chains and two β chains. For Hb S, mutations have occurred pseudogenes, depicted as psi (ψ), which do not encode func-
and the hemoglobin is composed of two normal α chains and tional polypeptides. Although each pseudogene shares many
two mutated β chains. These are discussed in greater detail base sequences with its corresponding normal gene, the
below. presence of frameshift mutations has shifted the triplet of

Various Globin Genes and Products

z a2 a1 q
a-Like genes
Chr 16

Gower I Gower II Portland F A2 A

e Gg Ag d b
b-Like genes
Chr 11
Embryonic Fetal Adult
Produced Yolk sac Fetal liver
Figure 6-7. Genetic control of various
globin genes and products in the =Y pseudogene
embryo, fetus, and adult.
 104 Hematologic Genetics and Disorders

bases so that polypeptide synthesis is prematurely halted.

Sickle Cell Anemia
Thus, pseudogenes are products of gene duplication that
have accumulated debilitating base changes during sequence Sickle cell anemia afflicts 1 in every 500 black children born
divergence. in the United States. This is an inherited disorder in which the
RBCs, normally discoidal, are contorted into rigid crescents
(“sickled”) (Fig. 6-8). Sickle cell disease is characterized by
PATHOLOGY chronic hemolytic anemia, recurrent vaso-occlusive painful
Hematologic Indices “crises” of variable duration and severity, and infarctions of
tissues and organs. Pain is the most frequent cause of recurrent
Red blood cell indices are part of the complete blood count
morbidity. Life expectancy has increased in recent years owing
(CBC):
■ Mean corpuscular volume (MCV): average RBC size
to pharmacologic advances; however, the mean survival age
■ Mean corpuscular hemoglobin (MCH): amount of remains at less than 50 years (Table 6-9). A disproportionate
hemoglobin per RBC number of deaths occur in infancy or early childhood, usually
■ Mean corpuscular hemoglobin concentration (MCHC): resulting from overwhelming bacterial infections, a sudden
amount of hemoglobin relative to the size of the cell severe splenic crisis, or acute cerebrovascular occlusion.

A B

Figure 6-8. A, Sickle cell disease. Crescent- and cigar-shaped cells are present along with target cells and teardrop cells.
B, Hemoglobin sickle cell disease. The striking sickle forms of sickle cell are not evident, but densely staining, elongated
RBCs with rather blunt ends are present. (It has been said these are trying to “sickle like S, and crystallize like C.”) Cells appear
dense on the smear, with many target cells. C, Hemoglobin C disease. Target cells are present as are some spherocytic cells.
Hemoglobin crystals can be seen within intact cell membranes. These are pathognomonic. (Courtesy of Dr. Anna Walker,
Mercer University School of Medicine, Macon, Georgia.)
 Erythrocyte Hemoglobin Defects 105

MICROBIOLOGY TABLE 6-9. Mean Survival Age of Individuals with

Sickle Cell Disease
Infection in Sickle Cell Disease
Splenic dysfunction can occur in sickle cell disease by age GENOTYPE MEAN SURVIVAL
3 months. These infants have a high risk for septicemia and
meningitis, pneumococci, and infections by other Male Hb SS 42 years
encapsulated bacteria. The most common cause of death in Female Hb SS 48 years
children with sickle cell disease is Streptococcus Male Hb SC 60 years
pneumoniae sepsis. There is also an increased risk of Female Hb SC 68 years
osteomyelitis caused by Staphylococcus aureus, Salmonella
Data from Office of Genomics and Disease Prevention, Centers for Disease
species, and others. Control and Prevention (CDC), May 5, 2005.

Normal
hemoglobin A
PHARMACOLOGY
Penicillin Prophylaxis in Infants with G A A
Sickle Cell Anemia DNA
Prophylactic penicillin therapy prevents 80% of life- C T T
threatening Streptococcus pneumoniae sepsis. Infants
should receive 125 mg of penicillin V PO4 prophylaxis orally
G A A mRNA
twice a day. Children ages 3 to 5 years should receive
250 mg of penicillin V PO4 prophylaxis orally twice a day.
Erythromycin prophylaxis is an alternative for individuals Glutamic acid
allergic to penicillin. Folic acid supplementation may also be
considered.

Hemoglobin S Hemoglobin C

G T A A A A

PHYSIOLOGY DNA
C A T T T T
Hemoglobin and O2 Binding
Hemoglobin is a tetramer with each monomer composed
of a heme and a globin. Heme is a general term for a G U A mRNA A A A
metal ion chelated to a porphyrin ring. Central to the
pyrrole rings of porphyrin is Fe++. Oxygen binds to
Valine Lysine
hemoglobin only when iron is in the ferrous state (Fe++),
and therefore hemoglobin in the Fe+++ state
Figure 6-9. Hemoglobin A mutations. Two different
(methemoglobin) does not bind oxygen. However, O2
missense mutations at the same codon result in two different
interaction can bind reversibly to Fe++ because of the
proteins. The mutations are allelic.
interaction of heme with specific amino acids in
hemoglobin. The interaction of histidine with Fe++ stabilizes
the Fe-O2 complex. When O2 binds to Fe++, the shape of
the hemoglobin molecule is changed to a planar
Genetic Aspects of Sickle Cell Anemia
conformation. This change in the shape of hemoglobin Sickle cell anemia results from the single substitution of
corresponds to a change from the tense (T) form to the valine for glutamic acid at amino acid 6 in the 146-amino-acid
relaxed (R) form. Oxygen binding is sterically inhibited in chain of the β-hemoglobin chain. This abnormal hemoglobin
the T form. In the R form, the affinity for O2 is is known as hemoglobin S (Hb S), and its cause is an altera-
approximately 150-fold greater than in the T form. The tion of a single base of the triplet of DNA that specifies an
binding of the first O2 is energy dependent, but affinity for amino acid, as depicted in Figure 6-9. Another abnormal
binding increases after the first O2 is bound. These affinity hemoglobin molecule, hemoglobin C, results from a mutation
changes account for the S shape of the initial slope of the at the same DNA triplet; however, whereas the glutamine
oxyhemoglobin dissociation curve. The capacity to bind O2 residue is replaced by valine in hemoglobin S, it is replaced
is dependent on the availability of Fe+++. Pao2 determines
by lysine via a different missense mutation in hemoglobin C.
the binding of hemoglobin with O2, or the hemoglobin
Hemoglobin S and hemoglobin C are allelic; two indepen-
saturation level, and this oxyhemoglobin saturation level
can be influenced by alterations in Paco2, pH, and dent mutations occurred in the same sequence of bases in the
temperature. DNA that make up alleles of a single gene (Table 6-10). This
demonstrates that there may be more than one mutation site
 106 Hematologic Genetics and Disorders

of age. After 6 months of age, the sickling of RBCs is a con-

TABLE 6-10. Mutations in β-Globin Gene stant finding.
Producing Sickle Cell Anemia, In individuals with the sickle cell trait, the proportion of
Hemoglobin C Disease, and Hb SC
Disease Hb S is between 35% and 45%. Of course, two types of
hemoglobin are expected to be synthesized in equal amounts.
However, it appears that Hb S is synthesized at a lower rate
HEMOGLOBIN GENOTYPE CLINICAL STATUS
than Hb A. It has been suggested that this difference is the
result of a specific delay in translation of mRNA on the
Hb A αα/αα Normal
polyribosome.
β/β
Hb AS αα/αα Sickle cell trait The substitution of valine for glutamic acid causes the
β/βS abnormal hemoglobin S molecules to aggregate, or polymer-
Hb CC αα/αα Hemoglobin C ize, into strands that are laid down to form cable-like fibers.
βC/βC disease As greater numbers of fibers accumulate, the large aggregates,
Hb SC αα/αα Hb SC disease
or polymers, of linearly arranged fibers attain sufficient length
βS/βC
Hb SS αα/αα Sickle cell disease and rigidity to distort the cell membrane into a crescent
βS/βS shape. Polymerization of Hb S occurs at low oxygen tensions.
In the fully oxygenated state, Hb S behaves like normal
Note: all α globin genes are normal.
hemoglobin (Hb A) and remains in solution. The sickling phe-
nomenon is reversible with reoxygenation of Hb S; the aggre-
gated molecule dissociates and the distended cell returns to
within a single allelic locus, which can lead to different altera- its normal shape. However, the continual dual process of
tions in the function of the gene. Hemoglobin C disease is polymerization and depolymerization ultimately takes its
usually a benign hemolytic anemia, whereas sickle cell anemia toll, and many RBCs become irreversibly sickled—even in the
can have severe consequences. Because Hb S and Hb C are fully oxygenated state.
allelic, an allele could be inherited from each parent, resulting The sickling of RBCs leads to the obstruction of microvas-
in Hb SC disease (see Fig. 6-9). The severity of this condition culature that can result in vaso-occlusion, blocking of blood
is between that of sickle cell disease and Hb C except that flow, and perhaps infections in postoccluded areas. In lungs,
visual damage due to retinal vascular lesions is worse. gas exchange becomes difficult and individuals may suffer
Sickle cell anemia is an autosomal recessive disease. Indi- breathing difficulties (dyspnea). Increased pressure from fluid
viduals with one normal and one defective allele are gener- seeping into the lung parenchyma from capillaries will also
ally healthy carriers. Such heterozygous individuals are said activate cough receptors. These physiologic alterations induce
to have a sickle cell trait. Two million African Americans (8% intensely acute problems, such as acute chest syndrome, and
to 9%) have sickle cell trait. Although heterozygous individu- may require intervention with oxygen either through inhala-
als are typically asymptomatic, even RBCs of heterozygotes tion or extracorporeal administration. Transfusions may also
can undergo sickling under certain circumstances, such as low be necessary. If microemboli are trapped in bone marrow,
O2 tension, and produce clinical manifestations. Since the infections may develop and fat emboli may be released into
detrimental allele can occasionally express itself in the het- the blood. These emboli may then also be trapped in the lung
erozygous state, the gene should be considered dominant. and exacerbate the crisis.
Thus, dominance and recessiveness are somewhat arbitrary
concepts that depend on the point of view. From a molecular
standpoint, the relation between the normal and the defective
allele in this instance may best be described as codominant,
since the heterozygote produces both normal and abnormal PATHOLOGY & PHYSIOLOGY
hemoglobin. As with many heterozygous conditions, both
normal and abnormal proteins are produced with possible Acute Chest Syndrome (ACS)
consequences due to reduced amounts of normal protein and ACS is a common complication of sickling disorders
increased amounts of abnormal protein. such as Hb SS, Hb SC, Hb S β+-thalassemia, and Hb S
β0-thalassemia. It is responsible for considerable morbidity
Hemoglobin and Pathophysiology of and mortality in these patients owing to the altered
Sickle Cell Anemia hemoglobin that causes erythrocytes to accumulate on
Sickle cell anemia does not manifest itself in the first few endothelial microvasculature surfaces. Because of these
months of neonatal life. There is a protective action of Hb F accumulations, ACS presents with a pulmonary infiltrate
to the very low levels of the disease-causing abnormal hemo- (infection or infarction) on radiography that may have been
induced by or associated with cough, fever, sputum,
globin (Hb S) during early life. The percentage of Hb F at birth
dyspnea, or hypoxia.
is high, often as high as 85%, but the quantity drops pre-
Major clinical problems are distinguishing between
cipitously as the synthesis of the adult form of hemoglobin infection and infarction and establishing the clinical
accelerates. In sickle-cell anemic infants, the proportion of significance of fat embolism.
abnormal hemoglobin rises to near-adult levels by 6 months
 Erythrocyte Hemoglobin Defects 107

A B C
Size Heterozygote Homozygous
marker Normal sickle trait affected

Figure 6-10. The region of the

β-globin gene corresponding to codon
6 was amplified by polymerase chain 500 bp
reaction (PCR). For sickle cell, the 250 bp
knowledge that the mutation changes a
restriction enzyme recognition site is
used to identify the mutation. As shown 100 bp 110 bp
here, a 110-base-pair fragment of the
normal β-globin gene was amplified in
56 bp
lane A; in lane B, there is a normal
fragment and one fragment that was 50 bp 54 bp
cleaved by the restriction enzyme MstII.
Amplified and digested products are
visualized after separation gel by
electrophoresis. This pattern (B)
represents the heterozygote Hb AS. If
both alleles are cleaved by MstII, sickle
cell disease occurs (Hb S), as shown in
lane C.

DNA Analysis of Sickle Cell Anemia

Thalassemias
Technology has made it feasible to examine DNA directly and
to identify sickle cell anemia with a high degree of precision Thalassemia is a potentially fatal blood disorder associated
during early development.A variety of polymerase chain reac- with a marked suppression or absence of hemoglobin produc-
tion (PCR)–based techniques is used, yielding 99% to 100% tion. This differs from sickle cell and hemoglobin C diseases,
detection of specific mutations in the β-hemoglobin gene. which result from a structural alteration of hemoglobin.
As noted previously, an A-to-T substitution in the sixth
triplet of the globin gene causes the substitution of valine for α-Thalassemia
glutamine. This results in the subsequent production of sickle The α-thalassemias are characterized by reduced synthesis of
cell hemoglobin. The sequence CCTGAGG in the region α chains. Anemia stems both from the lack of adequate hemo-
coding for amino acids 5 to 7 in the abnormal globin sequence globin and from the effects of excess unpaired non-α chains.
is recognized by the restriction enzyme MstII. A PCR test can Since different non-α chains are synthesized at different times
be designed to utilize this ability to cleave a normal DNA of development, different conditions prevail. In the newborn
fragment containing this site. Failure of the restriction enzyme with α-thalassemia, the excess unpaired γ chains form γ tet-
to cleave the DNA demonstrates an amplified fragment ramers called Hb Barts. In the adult, the excess β chains
containing the unaltered, CCTGTGG, site (Fig. 6-10). aggregate to form tetramers called Hb H (Fig. 6-11). Reflec-
tion on Figure 6-4 and the transition of Hb F to Hb A, along
with Figure 6-9, demonstrates how an affected individual may
BIOCHEMISTRY have both Hb Barts at birth and Hb H at a later time. This
can be confusing because both form aggregates because of
Techniques to Demonstrate Hemoglobin Variation inadequate α chains; however, in Hb Barts the aggregates are
Various techniques are used to differentiate between γ-chain tetramers and in Hb H the aggregates are β-chain
different hemoglobins. tetramers.
Electrophoresis is used for quick screening. Bands may The α-thalassemias most often result from deletions of one
overlap, and quantitation is inaccurate at low concentrations. or more α genes (Fig. 6-12). These deletions occur by unequal
It is being replaced by high-performance liquid crossing-over between homologous sequences in the α-goblin
chromatography (HPLC). gene cluster. When this occurs, one chromosome will have
Isoelectric focusing (IEF) has better resolution and only one α gene (α-) and the other chromosome will have
quantitation than does electrophoresis.
three (ααα). Since there are normally four α-globin genes, the
HPLC resolves protein bands that are not separated by
severity of α-thalassemia depends on the number of available
other tests. It can achieve accurate quantitation even at low
concentrations but does not enable the identification of Hb S and normally functioning α-globin genes. Each α gene nor-
β0-thalassemia, which requires hemoglobin electrophoresis. mally is responsible for 25% of the α chains, and each may be
deleted independently of the other α genes. If only one of
 108 Hematologic Genetics and Disorders

Figure 6-11. α-Thalassemia. RBCs are hypochromic and Figure 6-13. β-Thalassemia. Marked anisocytosis and
microcytic and vary considerably in shape. Target cells are poikilocytosis are present. Along with the bizarre forms are
present. (Courtesy of Dr. Anna Walker, Mercer University occasional teardrop cells and target cells. (Courtesy of Dr. Anna
School of Medicine, Macon, Georgia.) Walker, Mercer University School of Medicine, Macon, Georgia.)

witnessed more often in those of African origin. Although

Chr 16 both genetic patterns are identical clinically, the position of
the deleted genes is important in terms of the likelihood of
Chr 16
severe α-thalassemia in the offspring. Accordingly, progeny
Normal Silent carrier with hydrops fetalis, who have no α-globin chains, rarely
1 a-Thalassemia
occur in black African populations because each parent
contributes a chromosome containing one functional α gene.
Hemoglobin H disease, most commonly found in Asiatic
populations, is associated with the loss of three of the four α
genes. The outcome is a significant imbalance in globin synthe-
a-Thalassemia trait a-Thalassemia trait
2 a-Thalassemia 2 a-Thalassemia sis. Although β chains are produced in normal amounts, they
are actually present in relative excess owing to the marked
suppression of α-chain production. The excess β chains form
unstable β4 tetramers (Hb H). These tetramers form insoluble
inclusions in mature red cells (see Fig. 6-11). The spleen
Hemoglobin H disease Hydrops fetalis removes the older red cells with precipitates of Hb H.
3 a-Thalassemia 4 a-Thalassemia The most severe form of α-thalassemia, hydrops fetalis,
results from the deletion of all four α genes. In the fetus,
Figure 6-12. α-Thalassemia results from reduced synthesis excess γ chains form tetramers (Hb Barts) that have extremely
of α-globin chains. This occurs most often from deletion of high oxygen affinity but are unable to deliver oxygen to
one to all four genes found on chromosome 16. Open boxes
tissues. Severe tissue anoxia invariably leads to intrauterine
represent deleted genes.
fetal death. Presently there is no effective therapy for the
hydropic fetus. Exchange transfusions will fail because the
these genes is lost, there is no detectable clinical abnormality. fetus has no capacity for endogenous production of functional
Such a “silent” carrier of α-thalassemia is asymptomatic, with hemoglobin.
a hematologic profile that is within normal limits. The silent
carrier can transmit the deletion to offspring, who could β -Thalassemia
manifest a symptomatic form of α-thalassemia if the other β-Thalassemia is the second most common cause of hypo-
parent also transmits a chromosome with one or more chromic, microcytic anemia; iron deficiency anemia is the
α-deleted genes. most common. Homozygous β-thalassemia, in which the
The loss of two of the four genes is referred to as patient has inherited two defective β alleles, results in
α-thalassemia trait. The pair of deleted genes may be from impaired β-chain synthesis. The production of α chains con-
the same chromosome, or one α-globin gene may be deleted tinues at normal, or elevated, levels in β-thalassemia, but the
from each of the two chromosomes. The former situation is unmatched α chains accumulate and precipitate as inclusion
more common in Asian populations, whereas the latter is bodies in the RBC precursors (Fig. 6-13). Most damaged RBC
 Erythrocyte Hemoglobin Defects 109

precursors remain in the marrow; those that are released to β-Thalassemia is a heterogeneous disorder. Some patients
the circulation are disadvantaged and rapidly destroyed by with homozygous β-thalassemia are unable to synthesize
the spleen. The suppressed synthesis of β chains is compen- any β chains; this is known as β0-thalassemia. The produc-
sated by overproduction of fetal hemoglobin. The compensa- tion of some β chains is known as β+-thalassemia. In
tion, however, is incomplete, since the blood cells are still either event, the lack of or marked reduction in β-chain
deficient in hemoglobin. synthesis is accompanied by the unimpaired synthesis of
To reiterate the role of Hb F in the etiology of the disease, α chains. The clinical severity of β-thalassemia reflects the
as Hb F (α2γ2) γ-chain production switches off postpartum, extreme insolubility of α chains, which are present in
the deficit of β chains in β-thalassemia becomes a significant relative excess because of the deficiency of β-chain syn-
problem. Anemia is compounded by the death of RBC precur- thesis. Therefore, the fewer functional β chains present,
sors, which leads to compensatory erythropoietin-induced the more insoluble α-chain aggregates occur and the more
marrow hypertrophy. This, in turn, leads to a hypermetabolic severe the disease.
state, skeletal changes, and increased intestinal absorption of The loss of β-chain gene function results from a variety of
iron and iron overload. Iron overload is compounded by different structural mutations within or surrounding the β
administration of transfusions to treat the anemia. gene. The level of β-chain synthesis is determined by the
specific manner in which gene expression is altered. Unlike
α-thalassemia, in which α-globin genes are deleted, the
PATHOLOGY
β-globin gene is present in most cases, and defects in gene
Erythropoietin-induced Marrow Hypertrophy expression have been identified that alter gene transcription,
Erythropoietin (EPO) secretion from the kidney is stimulated
mRNA processing, and translation. The varying clinical sever-
by hemolysis and a decrease in hemoglobin. Tissue anoxia ity observed in β-thalassemia is directly correlated with the
also leads to EPO production. EPO causes excessive iron degree to which such mutations decrease β-globin gene
absorption and iron overload and increases erythroid expression.
hyperplasia in bone marrow and extramedullary sites. Clinically, β-thalassemias are classified as thalassemia
Marrow expansion leads to skeletal deformities by major, thalassemia intermediate, or thalassemia minor. The
invading bone and impairing proper growth. It also affects three differ in severity of disease and types of inter-
extramedullary sites—the liver and spleen. Extreme cases ventions. As suggested, thalassemia major is the most
involve extra-osseous masses in the thorax, abdomen, and severe and requires transfusions. Thalassemia minor is often
pelvis.
asymptomatic.

TABLE 6-11. Comparison between α- and β-Thalassemias

CLINICAL CONDITION GENOTYPE DISEASE MOLECULAR GENETICS

α-Thalassemias

Silent carrier -α/αα Asymptomatic; no RBC

abnormality
α-Thalassemia trait –/αα (Asian) Asymptomatic, like β-thalassemia
-α/-α (Black African) minor; microcytosis
Hb H disease –/-α Severe, resembles β-thalassemia Gene deletions usually
intermediate; moderately
severe hemolytic anemia
Hydrops fetalis –/– Lethal in utero, Hb Barts

β-Thalassemias

Thalassemia major Homozygous β0-thalassemia Severe, requires blood

(β0/β0) transfusion regularly
Thalassemia intermediate β /β
0
Severe, but does not require Rare gene deletions in β0/β0.
regular blood transfusions Defects in transcription,
processing, or translation of
β-globin mRNA
Thalassemia minor β0/β Asymptomatic with mild or
β+/β absent anemia; RBC
abnormalities seen

Deleted genes are indicated by hyphens (-) in α-thalassemias. The absence of chain production in β-thalassemia is indicated by (0), whereas mutations resulting in
decreased β-globin chains are indicated by (+).
 110 Hematologic Genetics and Disorders

The majority of individuals with minor and intermediate B, and von Willebrand disease. Among these, the hemophilias
thalassemia do not need regular transfusions, although some affect 1 in 10,000 individuals per year and are best known
individuals do require either occasional or regular transfu- because of their historic association with European royal
sions. The most effective therapy, if needed, is the use of families. However, von Willebrand disease is the most common
transfusions with prophylactic antibiotics. Unfortunately, just coagulation disorder, affecting 1% to 2% of the U.S. popula-
as with thalassemia major, repeated transfusions, especially tion (Table 6-12).
in children, can create a state of iron overload, which damages Hemophilia A and B are characterized by defects in key
the tissues in which it is deposited, such as the heart and liver. components of the clotting cascade—factors VIII and IX,
Chelation with an iron-binding resin is administered to respectively—that render the patient incapable of normal
prevent this overload. The intensive use of transfusions and coagulation processes. Clinical expression can range from
chelation can extend the life expectancy of a patient for 20 mild to excessive bleeding due to major insult to frequent
to 30 years. Bone marrow transplantation is available but has spontaneous internal bleeding without insult. As exhibited by
been successful in only a small percentage of patients. a number of recessive disorders, the degree of clinical mani-
To summarize, α- and β-thalassemia are caused by a molec- festation depends on the amount of gene products available.
ular defect in the α- or β-globin genes that prevents normal Accordingly, the amount of available clotting factor is deter-
expression of these genes. Because the α-globin gene is essen- mined by the severity of the genetic mutation. Finally, both
tial both to fetal life and to postpartum life, α-thalassemia hemophilias are sex-linked diseases and serve as a paradigm
generally is either fatal in utero or compatible with a normal for X-linked recessive disorders. Females only exhibit bleed-
lifestyle. On the other hand, the β-globin gene is not impera- ing problems via unfortunate lyonization, or the co-occurrence
tive in fetal life and is not even fully expressed until after of two independent allelic mutations.
birth. Hence, β-thalassemia in its full expression is the crip- In von Willebrand disease, both platelet aggregation and
pling disease of childhood. α-Thalassemias and β-thalassemias clot formation fail to occur properly. The von Willebrand
are compared in Table 6-11. protein, also known as the von Willebrand factor or vWF,
normally promotes platelet adhesion to endothelium and
is a carrier for factor VIII in the clotting cascade. There-
●●● BLEEDING DISORDERS fore, von Willebrand disease has an association with hemo-
The most common hereditary deficiencies of coagulation, philia A that is caused by a mutation in the factor VIII
resulting in excessive bleeding, are hemophilia A, hemophilia gene.

TABLE 6-12. Comparison between Hemophilia A, Hemophilia B, and von Willebrand Disease

VON WILLEBRAND
FEATURE HEMOPHILIA A HEMOPHILIA B DISEASE

Inheritance X-linked X-linked Dominant with variable

expressivity;
chromosome 12
Mutations Flip inversion, deletions, Severe form—frameshifts, splicing Missense, deletion
rearrangements, frameshift errors, nonsense and missense
mutations, splicing errors, mutations
and nonsense mutations Mild to moderate form—missense
Mild form—missense mutations mutations
Primary sites of Muscle, joints; posttrauma, Muscle, joints; posttrauma, Mucous membranes;
hemorrhage postoperative postoperative skin cuts; posttrauma,
postoperative
Platelet count Normal Normal Normal
Bleeding time Normal Normal Prolonged
Prothrombin time Normal Normal Normal
Partial thromoboplastin Prolonged Prolonged Prolonged or normal
time
Factor VIII Low Normal Normal
Factor IX Normal Low Normal
vWF Normal Normal Low
Ristocetin-induced Normal Normal Impaired
platelet aggregation

Data from Hoffbrand AV, Pettit JE, Moss PAH. Essential Haematology, 4th ed. Oxford, England, Blackwell, 2001, p 265.
 Bleeding Disorders 111

Hemophilia B
PHYSIOLOGY
Hemophilia B, sometimes referred to as Christmas disease,
Coagulation results from a reduction in the amount of factor IX, a serine
Abnormal coagulation results from the failure to clot or the protease, available for thrombin generation by the clotting
failure to prevent excessive clotting. Coagulation comprises cascade. The incidence of hemophilia B is roughly one-seventh
an intrinsic and an extrinsic pathway. that of hemophilia A. This is in part attributable to the much
The intrinsic pathway is initiated by a negatively charged smaller size of the factor IX gene—8 exons comprising 34
surface, which may occur with damaged endothelium or by
kb—at the tip of the X chromosome (Xp27) and very close
surface contact with certain foreign substances. Partial
to the factor VIII gene. Still, hundreds of different missense,
thromboplastin time (PTT) detects intrinsic factor
abnormalities reflected by increased PTT.
nonsense, frameshift, and deletion mutations have been
The extrinsic pathway is activated by tissue found with hemophilia B. The most common cause of mild to
thromboplastin (factor III), which is released after cell injury moderate hemophilia B results from missense mutations.
of endothelium or other cells. Prothrombin time (PT) mainly There have been occasional reports of large deletions associ-
detects abnormalities in extrinsic factors (prothrombin; ated with severe disease, but usually cases are associated with
factors V, VII, and X), although prothrombin is commonly frameshifts, splicing errors, nonsense, and missense muta-
considered the major factor measured by PT. Most factors tions. For both hemophilia A and B, nearly one third of cases
adversely affecting the extrinsic coagulation pathway, are due to new, spontaneous mutations.
including clotting factors, result in an increased PT.
However, a few situations, such as vitamin K
supplementation, thrombophlebitis, and use of certain
drugs, will decrease PT.
BIOCHEMISTRY
Serine Proteases
Serine proteases are a family of enzymes that cleave
Hemophilia A between specific amino acids. They are grouped according
to structural homology and play important roles in
Hemophilia A occurs with an incidence of approximately coagulation, inflammation and immunity, and digestion.
1 in 5000 live male births. A deficiency or absence of Generally, there is an enzyme-specific preference for
clotting factor VIII ultimately results in impaired thrombin cleaving adjacent to a specific type of amino acid. For
production. The gene for factor VIII is large, encoding 26 example, trypsin cleaves after the basic amino acids arginine
exons that span 186 kb on the tip of the long arm (Xp28) and lysine. The coagulation factors, except for factors VIII
of the X chromosome. Base pair changes, deletions, frame- and V, which are glycoproteins, are all serine proteases.
shift mutations, and protein-truncating mutations have been Serine proteases are synthesized in an inactive form
found in the factor VIII gene, and clinical severity is pro- (zymogen) and require proteolysis for activation. Those
portional to the loss of factor VIII activity conferred by participating in the coagulation cascade are synthesized in
the liver, secreted as zymogens, and activated following
the mutation. Hence, it is possible for a female to exhibit
vascular injury. The zymogen, or proenzyme, form generally
a modest reduction in factor VIII due to X inactivation.
has an “-ogen” suffix.
About 45% of the most severe cases of hemophilia A are
caused by the so-called “flip” inversion in intron 22. Here,
recombination with nearly homologous X chromosome
sequences located near the chromosome-terminating telo-
mere disrupts the normal reading frame. Approximately 50% The presentation of hemophilia B is quite similar to hemo-
of remaining cases of severe hemophilia have deletions, philia A. For both disorders, there may be prolonged bleeding,
rearrangements, frameshift mutations, splicing errors, or spontaneous bleeding, hemarthrosis, deep muscle bruising,
nonsense mutations. Mild to moderate cases typically harbor intracranial bleeding at birth, unexplained GI bleeding, and
missense mutations. excessive bruising. Both hemophilias have mild to severe
In severe cases, the diagnosis of hemophilia may be made forms. Only by determining the deficient factor can a proper
during the first year of life. If the diagnosis is not made, an diagnosis be made.
affected child may have large bruises from minor injuries,
which may even suggest a “battered” child. The child or adult
with severe forms of the disease may have five or more spon-
Von Willebrand Disease
taneous bleedings per month. Often, these are in joints and Von Willebrand disease differs from the hemophilias in its
deep muscles and can be painful. This is in contrast to indi- mode of inheritance. It is transmitted in an autosomal domi-
viduals with mild disease who may not have spontaneous nant manner with variable expression. In hemophilia, bleed-
bleeding and may experience abnormal bleeding once a year ing is generally in joints and muscles, whereas in von
to once every 10 years. Willebrand disease, bleeding is more common in mucous
 112 Hematologic Genetics and Disorders

membranes and after routine operations. As with hemophilia,

Activated Protein C Resistance and
there are mild to severe forms. Missense mutations or large
Factor V Leiden
deletions cause either a reduced amount of vWF or an abnor-
mal function of the protein. The most common is the mild In families suspected of having a familial thrombotic disorder,
form, type I, in which vWF and perhaps factor VIII are 20% to 65% of these disorders have been attributed to acti-
reduced. Type II results from a structural defect in vWF, and vated protein C resistance. Known as factor V Leiden, this
the presentation reflects the severity of the defect. In type III, defect is present in 2% to 5% of the asymptomatic white
there may be a complete absence of vWF and factor VIII population and 1.2% of the black population. The factor V
levels are often less than 10%. Leiden mutation is relatively uncommon in the native popula-
tions of Asia, Africa, and North America. In contrast, in
Greece and southern Sweden, rates above 10% have been
●●● THROMBOPHILIA reported.
A final group of disorders to be considered are those abnor- Risk of venous thrombosis is increased 3- to 8-fold for
malities that cause excessive clotting; in other words, this heterozygous individuals and 30- to 140-fold for homozy-
group could be considered the antithesis of those coagulation gous individuals (Table 6-13). Factor V Leiden accounts for
disorders discussed above that result in excessive bleeding. about 40% of idiopathic venous thromboembolic disease. It
Their occurrence is sometimes not recognized as a coagula- has been associated with recurrent venous thromboembolism
tion disorder. Eighty percent of all strokes result from isch- and thrombosis following pregnancy and the use of oral
emic events—blood clots blocking a vessel. One in 1000 contraceptives.
individuals in the United States is at risk for venous throm- The primary factor V Leiden mutation is an A-to-G mis-
bosis, most commonly occurring in the lower extremities. sense mutation in the factor V coagulation factor, leading
There are 2 million cases per year in the United States, with to an arginine-to-glutamine substitution at position 506 of
mortality estimated at 60,000 from pulmonary emboli. the protein, which represents the proteolytic site of the
Several genetic conditions contribute to these clotting disor- protein. This mutation occurs in over 95% of cases and
ders. Antithrombin III (AT3) deficiency, protein C deficiency, is the most common genetic risk factor for venous
and protein S deficiency account for 5% to 15% of these thrombosis.
inherited thrombophilias. The function of protein C in the clotting cascade is to
inactivate factor V and factor VIII. The arginine-to-glutamine
substitution prevents factor V from being cleaved by acti-
vated protein C and thus it remains active. The reality is
that factor V Leiden is inactivated by activated protein C
ANATOMY but at a much slower rate. The factor V Leiden mutation
has been associated with venous thrombotic clots, pulmo-
Thrombosis of the Leg
nary emboli, and arterial clots. The probability of thrombosis
Three major veins drain the lower leg, so thrombosis in one before age 33 is 44% and 20% in homozygous and het-
does not obstruct venous return. Deep vein thrombosis erozygous individuals, respectively. In addition, it may play
involving the popliteal, femoral, and iliac veins may be a role in stillbirths or recurrent miscarriages, preeclampsia,
tender and palpable over the involved vein. With iliofemoral
and eclampsia.
venous thrombosis, dilated superficial collateral veins may
appear over the leg, hip, and lower abdomen.

PATHOLOGY
Venous Thrombosis TABLE 6-13. Risk of Deep Vein Thrombosis with
■ Superficial thrombophlebitis affects superficial veins. Factor V Leiden Mutation
■ Deep vein thrombosis affects deep veins.
■ Prolonged thrombosis can lead to chronic venous RISK AVERAGE FACTOR V LEIDEN
insufficiency with edema, pain, stasis pigmentation, (AGE) POPULATION MUTATION
dermatitis, and ulceration.
■ Because thrombosis is almost always associated with <40 years 1 in 10,000 1 in 1750
phlebitis, “thrombosis” and “thrombophlebitis” are used 40–50 years 1 in 1250 1 in 1100
interchangeably. 50–60 years 1 in 1100 1 in 476
■ Venous thrombosis may occur as a result of a coagulation 60–70 years 1 in 833 1 in 250
disorder or related to an underlying malignancy. 70–80 years 1 in 625 1 in 120
 Questions 113

allele and that during gamete formation the sperm receives

KEY CONCEPTS the B allele that is inherited by the daughter. Because the
■ Red blood cells have antigens on the cell surface that determine mother expresses the A blood type, it is known that she is
blood groups and other compatibilities. either Hh or HH at the H locus. It is also known that the child
■ Anemia results from insufficient red blood cells and hemoglobin. inherits the H allele from the mother in order to express A
and B antigens.
■ Causes of anemia are membrane defects that change the integ-
rity, metabolic defects within the cell, or defects in hemoglobin. 2. Laboratory testing of a blood sample reveals H anti-
■ Membrane defects cause changes in the shapes of cells, such gens on the surface of red blood cells. Which of the
as spherocytes or elliptocytes. following blood types is expected to demonstrate
the most H antigen?
■ Glucose-6-phosphate dehydrogenase is the only RBC enzyme
that produces NADPH. Mutations decrease the amount of NADP+ A. Type A, Rh(D) negative
available for glutathione reductase and conditions of oxidative B. Type A, Rh(D) positive
stress. C. Type AB, Rh(D) negative
■ Hemoglobinopathies cause abnormal structures or insufficient D. Type B, Rh(D) negative
amounts of normal hemoglobin needed to provide O2 to cells. E. Type O, Rh(D) positive
■ A mutation in the same triplet can cause either sickle cell disease Answer. E
(Hb S) or hemoglobin C disease. Explanation: The presence of H antigen on the surface of
■ Thalassemias result from an imbalance of α and β chains in cells conveys that the person is either Hh or HH for this
hemoglobin, which requires two of each chain. protein. For type A, B, or AB blood, the A and/or B antigens
■ Bleeding disorders include hemophilia and von Willebrand must be expressed and attached to the H antigen. In the
disease. absence of either antigen, the H antigen is exposed and the
blood type is type O. The Rh allele is expressed by the RHD
■ Excessive coagulation is seen in thrombophilias such as anti-
gene separately and thus could be either positive or nega-
thrombin III deficiency, protein C deficiency, and protein S
tive. Rh D antigens are present or absent and designated D
deficiency.
or d, respectively; in this case they are just designated as
■ A factor V Leiden mutation leads to venous thrombosis. negative (d) or positive (D).

3. A 2-day-old infant is admitted to the neonatal inten-

sive care unit with mild jaundice and mild edema of
the extremities. The parents have one other child
●●● QUESTIONS and no significant medical history for newborns on
either side of the family. Laboratory tests reveal
1. A female with type AB blood learns that her mother anemia and a positive direct Coombs test. Which of
has type A blood and her father has type O blood. the following best explains the diagnosis for this
Testing at additional loci demonstrates a 99.9% child?
probability that the man is indeed the father of this
female. Which of the following best explains his A. Rhd mother, Rhd father, RhD child
blood type? B. Rhd mother, RhD father, RhD child
C. RhD mother, Rhd father, Rhd child
A. Bombay phenotype D. RhD mother, RhD father, RhD child
B. GPA phenotype
C. Hh genotype Answer. B
D. Null alleles Explanation: Hemolytic disease of the newborn can be
E. RhdCE genotype caused by an ABO incompatibility or an Rh incompatibility.
Answer. A The former is generally mild, but the latter is potentially life
threatening and can have severe consequences if not rec-
Explanation: ABO blood types occur because of antigens ognized and treated quickly. This infant has hemolytic
expressed on the surface of erythrocytes. These antigens anemia due to Rh incompatibility, also called erythroblasto-
result from different alleles of one gene. Once produced, the sis fetalis. The mother has Rh-negative blood and the father
proteins must attach to an H antigen at the membrane. This is Rh-positive. The first child had no antibodies directed
antigen is expressed by a different gene. In the case pre- toward its Rh factor. An immune response was mounted at
sented, the mother is either AA or AO and contributes the birth, and memory cells were generated toward the father’s
A allele to the child. The father seemingly has two O alleles, Rh antigen. The direct Coombs test is positive for antibodies
but the child inherits a B allele from the father. For this to bound to erythrocytes.
occur, the father must be hh at the H locus and have no H
antigen expressed on the surface of erythrocytes for the B
antigen to attach. This explains his O blood type. What is Additional Self-assessment Questions can be Accessed
important to note is that his genotype indicates he has a B at www.StudentConsult.com
Musculoskeletal Disorders 7
CONTENTS shape, plasticity, and partitioning. The ECM is composed of
three associated macromolecules: (1) fibrous structural pro-
teins such as collagen and elastin, (2) glycoproteins, and (3)
CONNECTIVE TISSUE AND BONE DISEASES
proteoglycans and hyaluronic acid. Typically, the ECM forms
Extracellular Matrix and Connective Tissue either basement membrane or interstitial matrix and, in doing
Collagen so, performs several functions, including retaining water,
Collagen Genes minerals, and nutrients as well as acting as the substrate for
Disorders of Connective Tissues cell-cell contact, migration, and adherence.
Differential Considerations Connective tissues have an extensive ECM that serves to
MUSCULOSKELETAL DISEASE DUE TO GROWTH bridge, interconnect, and support a variety of cellular and
FACTOR RECEPTOR DEFECT organ structures. These structures are typically composed of
cells, blood vessels, and a particular type of ECM. For
Achondroplasia
example, skin, fibroblasts and blood vessels are interwoven
MUSCLE CELL DISEASES within an extracellular matrix that is an amalgam of struc-
tural proteins, proteoglycans, and adhesion molecules. Other
Muscular Dystrophies
types of connective tissue include tendon and cartilage. Here,
Molecular Basis and Genetics of Duchenne and Becker
the discussion of connective tissues focuses on skin con-
Types of Muscular Dystrophy
Mitochondrial Myopathies
nective tissue, since much is known about the structure
Myoclonic Epilepsy with Ragged Red Fiber Syndrome and function of this anatomic element and many well-
Chronic Progressive External Ophthalmoplegia and characterized connective tissue diseases are localized to the
Kearns-Sayre Syndrome skin. Central to any discussion of skin connective tissue is
collagen.

In this chapter, the most common forms of a heterogeneous

group of inherited musculoskeletal diseases are highlighted. BIOCHEMISTRY
Musculoskeletal disorders have many etiologic origins, includ-
Extracellular Matrix (ECM)
ing connective tissue/extracellular matrix deficiencies such as
osteogenesis imperfecta, Ehlers-Danlos syndrome, and Marfan The extracellular matrix occupies the intercellular spaces. It
syndrome; faulty growth factor biology as seen in achondro- is most abundant in connective tissues such as the
basement membrane, bone, tendon, and cartilage, where
plasia; and both structural and metabolic muscle cell abnor-
definition is given to the ECM by the proportions and
malities represented by Becker and Duchenne muscular
organization of various components. The elastin of skin and
dystrophies and mitochondrial myopathies, respectively. blood cells provides resiliency, collagen provides strength to
Although individually these may be somewhat rare, collec- tendons, and the calcified collagen matrix of bone provides
tively the musculoskeletal diseases constitute a significant strength and incompressibility.
proportion of human disease. Integrins are a family of heterodimeric proteins composed
of α and β subunits that are the main cellular receptors for
the ECM. Integrins have several distinctive features from
●●● CONNECTIVE TISSUE AND other adhesion proteins. They interact with an arginine-
BONE DISEASES glycine–aspartic acid (RGD) motif of ECM proteins. Integrins
link the intracellular cytoskeleton with the ECM through this
Extracellular Matrix and RGD motif. Without this attachment, cells normally undergo
Connective Tissue apoptosis. Integrins can bind to more than one ligand and
many ligands can bind to more than one integrin. Examples
The extracellular matrix (ECM) is found in the spaces between of integrins include fibronectin receptors and laminin
cells, forming a large proportion of tissue volume. It is also receptors.
found between organs and as such contributes to the body’s
 Connective Tissue and Bone Diseases 115

HISTOLOGY HISTOLOGY
Types of Connective Tissue Skin
Connective tissues are classified by the cells and fibers Skin is composed of the epidermis and the dermis. The
present in the tissue as well as the characteristics of the epidermis is composed of two main zones of cells:
ground substance. ■ Stratum corneum: outer layer of cells without nuclei

Connective tissue (CT) proper consists of loose ■ Stratum germinativum: composed of three strata (basal,

connective tissue (areolar tissue) and dense connective spinous, granular)

tissue, which has more and larger fibers than loose CT. The dermis consists of a three-dimensional matrix of
Dense connective tissue can be either irregular, in which the loose connective tissue, including fibrous proteins such as
fibers are usually arranged more or less haphazardly, or collagen and elastin as well as proteins embedded in
regular, in which the fibers are arranged in parallel sheets or ground substance (glycosaminoglycans). Skin collagen
bundles. (type I) is rich in glycine, proline, and hydroxyproline.
Specialized CT is distinct in either structure or function Hydroxyproline is unique to collagens, and synthesis
from CT proper. Examples are adipose tissue, blood, bone, requires vitamin C.
cartilage, hematopoietic tissues, and lymphatic tissues.
Embryonic CT encompasses mesenchymal and
mucoid CT. Collagens have a distinctive primary amino acid sequence
featuring a repeated motif of (glycine-X-Y)n, where Y is often
proline or hydroxyproline and X can be any amino acid. Typi-
cally, a fibrillar collagen is synthesized in the endoplasmic
reticulum as a precursor molecule—procollagen—that is com-
posed of a short signal peptide, an amino-terminal and
carboxy-terminal propeptide, and a central α-chain segment
Collagen
(Fig. 7-1). The α-chain segment includes the repeated motif
A major component of skin connective tissue is the fibrous in which glycine represents every third amino acid, and it is
structural protein collagen. The collagens form a family of this chain that constitutes the biochemical core of collagen.
insoluble, extracellular proteins that are produced by a Three separate chains coalesce in the Golgi complex to form
number of cell types but primarily by fibroblasts. Collagen a triple helix, or tropocollagen, which is characterized by
is the most abundant protein found in the human body and numerous disulfide bonds. The formation of a stable triple
is a key structural component of bone, cartilage, tendons, helix requires the presence of glycine in the restricted space
ligaments, and fascia in addition to skin. Nineteen types of where the three chains come together. Collagen triple helices
collagen have been characterized, and each localizes to a are either heterotrimers or homotrimers, depending on the
specific part of the body. The major collagens are type I, of collagen type. The heterotrimeric type I collagen molecule has
skin, tendons, bone, and ligaments; type II, found in cartilage; two identical polypeptide chains called α1(I) and one slightly
type III, found in skin and hollow tubular structures such as different chain called α2(I). The homotrimeric type II and type
arteries, intestines, and uterus; and type IV, represented in III collagen molecules are composed of three identical α1(II)
all basal laminae (Table 7-1). Types I through III form strong chains and three identical α1(III) chains, respectively. Upon
fibers and thus are called fibrillar collagens, while type IV secretion from the originating cell, tropocollagen is processed
is associated with a multibranched network. Fifteen addi- into individual fibrils that, in turn, assemble into large, linear,
tional types of collagen perform essential functions but are insoluble fibers that are strengthened by lysine-mediated
less abundant. covalent cross-links between individual fibrils.

TABLE 7-1. Characteristics of the Major Collagens

COLLAGEN TYPE CHAIN GENE LOCATION DISORDER

I α1(I) COL1A1 17q21-22 Osteogenesis

α2(I) COL1A2 7q21-22 Ehlers-Danlos syndrome
II α1(II) COL2A1 12q13-q14 Chondrodysplasias
III α1(III) COL3A1 2q31-q32 Ehlers-Danlos syndrome
IV α3(IV) COL4A3 2q35-q36 Alport syndrome
α4(IV) COL4A4 2q36-q37
α5(IV) COL4A5 Xq22.3
V α1(V) COL5A1 9q34.2-34.3 Ehlers-Danlos syndrome
α2(V) COL5A2
α3(V) COL5A3
 116 Musculoskeletal Disorders

Collagen triple helix

Tropocollagen

Microfibril

Figure 7-1. Structure of collagen. The

triple helical structure of collagen—
tropocollagen—is the basic unit of
microfibrils. Many microfibrils bundle
together to form a macrofibril. (Redrawn
Subfibril Fibril with permission from Dr. J. P. Cartailler at
Symmation LLC [www.symmation.com].)

Collagen Genes
The model exon in all collagen loci is a 54-bp unit that
Collagen genes are named starting with the prefix COL, fol- codes for 18 amino acid residues, or six triplets of Gly-X-Y
lowed by an Arabic numeral indicating the collagen type, the repeats. This model led to the hypothesis that the varied col-
letter “A,” and finally a second Arabic number denoting the lagen genes, although now widely scattered in the human
particular α chain. Four distinct genetic loci (COL1A1, genome, were derived from a single ancestral gene more than
COL1A2, COL2A1, and COL3A1) collectively encode the 50 million years ago. It is surmised that the numerous genes
unique chains of the three classic fibrillar collagens—types I, evolved by successive duplications of a primitive procollagen
II, and III—and these genes are dispersed throughout the gene consisting of 54 bp with six Gly-X-Y repeats that under-
genome. COL1A1 is found on chromosome 17, COL1A2 on went subsequent chromosomal rearrangements and sequence
chromosome 7, COL2A1 on chromosome 12, and COL3A1 divergence. These genes have remained highly conserved
on chromosome 2. Type IV collagen is a nonfibrillar, or amor- during evolution.
phous, form coded for by the COL4A3 gene on chromosome
2. Overall, there are more than 34 different collagen genes
dispersed on at least 15 chromosomes.
Collagen genes have several interesting features. Genes
encoding fibrillar collagen are quite similar in structure; the BIOCHEMISTRY
triple helical domain regions consist of greater than 40 exons
all of which are multiples of 9 nucleotides. Exons are typically Glycine
54 nucleotides in length, but multiples of 54 or combinations Glycine is the smallest amino acid and is a nonessential
of 45 and 54 base exons are not uncommon. Consistent with amino acid. Its size is convenient for “small places” in
the (Gly-X-Y)n primary amino acid sequence motif, each proteins such as the turns of helices. These are
exon begins with a glycine codon. The gene COL1A1 that evolutionarily very stable. Substituting the glycine with a
larger amino acid can dramatically change the shape of the
encodes α1(I) will serve as an example. The gene is large,
protein.
spanning 18 kb of genomic DNA and encoding 52 exons that
Glycine can also function as an inhibitory neurotransmitter
are 45, 54, 99, 108, or 162 base pairs in length. Hydroxypro- and serve as a co-agonist with glutamate to activate NMDA
line occupies the Y position in about a third of the exon (N-methyl-D-aspartate) receptors.
triplets and is often preceded by proline.
 Connective Tissue and Bone Diseases 117

that progresses to sensorineural loss occurs in roughly 50% of

Disorders of Connective Tissues
adult patients. Type I OI is most often associated with protein-
The large number of collagen genes, the widespread distribu- shortening nonsense, frameshift, or splice site mutations in
tion of collagen within the body, and the high degree of evolu- the COL1A1 gene leading to decreased α1 chains. Because
tionarily conserved primary amino acid sequences all strongly type I collagen is a heterotrimer consisting of two α1 chains
suggest that functional collagen is critical for health. Accord- and one α2 chain, triple helix formation is greatly reduced.
ingly, it follows that defects in collagen synthesis, processing
and maturation, and structure would result in tissue dysfunc-
tion and human disease. This is the case, as more than 1000
mutations have been described in the collagen genes. Here, we NEUROSCIENCE
discuss three such diseases: osteogenesis imperfecta and
Ehlers-Danlos syndrome as paradigms of collagen disorders Hearing Loss
and Marfan syndrome as an important noncollagen disorder. Hearing loss is classified as conductive, sensorineural,
central hearing disorder, and presbycusis. Lesions involving
Osteogenesis Imperfecta the external or middle ear characterize conductive hearing
Osteogenesis imperfecta (OI) represents a collection of type loss. It is most commonly caused by cerumen impaction,
although otitis media is the most common serious cause.
I collagen disorders due to mutations in the COL1A1 or
A lesion of the cochlea or auditory parts of cranial nerve VIII
COL1A2 genes that are generally characterized by weak
indicates sensorineural hearing loss, which includes
bones, dentinogenesis imperfecta, short stature, and adult- hereditary deafness. Intrauterine factors causing sensorineural
onset hearing loss. Classically, four types of OI have been hearing loss include infection, metabolic and endocrine
recognized (Table 7-2). However, with the availability of disorders, and anoxia. When hearing loss is unilateral, there is
DNA-based diagnostics, there are now seven subtypes of OI usually a cochlear basis, whereas bilateral loss is often due to
that represent distinct clinical entities. Considering all forms drug use. Aminoglycoside antibiotics are toxic; salicylates,
of OI, the overall prevalence is roughly 7 per 100,000, and furosemide, and ethacrynic acid can cause transient loss.
the disease crosses all racial and ethnic boundaries. Types I Central hearing disorders occur with lesions of the central
to IV illustrate the more common forms of OI. auditory pathways. The loss is unilateral if there is unilateral
Type I OI is the most common form of OI. It is relatively pontine cochlear nuclei damage in the brainstem owing to
ischemic infarction of the lateral brainstem, multiple sclerosis
mild and exhibits autosomal dominant inheritance with vari-
plaque, neoplasia, or hematoma. Bilateral degeneration of
able expressivity. Clinically, type I individuals exhibit skeletal
nuclei is seen in rare childhood disorders.
osteopenia and fractures, dentinogenesis imperfecta (Fig. Presbycusis is gradual age-related loss, which may be
7-2), joint hypermobility, and blue-colored sclerae. Stature is conductive or central.
usually within the normal range, but conductive hearing loss

TABLE 7-2. Summary of Osteogenesis Imperfecta (OI) Types

OI BIOCHEMICAL
TYPE CLINICAL FEATURES INHERITANCE ABNORMALITY

I Normal stature, little or no deformity, blue sclerae, hearing loss AD 50% reduction in type I
collagen synthesis
II Lethal in perinatal period, very few survive to 1 year; minimal AD Structural alteration in
calvarial mineralization, beaded ribs, compressed femurs, long type I collagen
bone deformity, platyspondyly chains—
overmodification
III Progressively deforming bones, moderate deformity at birth, scleral AD/AR (rare) Increased collagen
hue varies, dentinogenesis imperfecta, hearing loss, very short Structural alteration turnover
stature in type I collagen
chains—
overmodification
IV Normal to gray sclerae, mild to moderate deformity, variable short AD Excessive
stature, dentinogenesis imperfecta, some hearing loss posttranslational
modification to one
type I collagen chain
V Similar to OI type IV plus calcification of interosseous membrane of AD None identified
forearm, anterior radial head dislocation, hyperplastic callus
formation, normal sclerae
VI Similar to OI type IV with early-onset vertebral compression fractures, Unknown None identified
mineralization defect
VII Mild symptoms, short stature, shortened limbs, normal sclerae AR A non-collagen type I
mutation

Data from Sillence DO, Senn A, Danks DM. Genetic heterogeneity in osteogenesis imperfecta. J Med Genet 1979;16:101–116.
AD, autosomal dominant; AR, autosomal recessive.
 118 Musculoskeletal Disorders

side-chain amino acids or in the C-terminal two thirds of the

ANATOMY gene usually predict a severe clinical outcome. Some splice
site mutations have also been found. There is a rare form of
Dentinogenesis Imperfecta OI III that has autosomal recessive transmission. This form is
Dentinogenesis imperfecta results from a failure of associated with consanguinity and is the most common form
odontoblasts to differentiate normally. Odontoblasts produce of OI observed in Africa.
dentin; ameloblasts produce enamel. Dentinogenesis Types II and III are severe forms of OI. Type II is character-
imperfecta affects deciduous and permanent teeth, giving ized by multiple fractures and perinatal lethality. The mean
them a brown to grayish-blue appearance with an
birth weight and length is below the 50th percentile. Twenty
opalescent sheen. The enamel wears down quickly,
percent of these infants are stillborn and 90% die within 4
exposing the dentin. It occurs because of autosomal
recessive inheritance, drug toxicity (tetracycline), and
weeks after birth.
syndromic association. Type III features progressive skeletal abnormalities, includ-
ing frequent early-onset fractures and progressive kyphosco-
liosis. Fractures are frequently present at birth. Generalized
Mutations in either COL1A1 or COL1A2 can cause OI osteopenia leads to poor longitudinal growth that is well
types II to IV. Each of these forms of OI is transmitted in an below the third percentile in height. Blue sclerae are another
autosomal dominant fashion and most often results from mis- frequent clinical sign. Infants who survive the first months
sense mutations that alter the important glycine codon in the of life generally live reasonably long lives, and approximately
triple helical domain. Interestingly, the phenotypical conse- one third survive long term. In one example, a child was
quence depends on the nature and position of the glycine born with 132 fractures at birth and missing bones of the
amino acid substitution. For example, substitutions with large skull. This person attained a maximum height of 36 inches
and weight of 50 pounds by adulthood. This young woman
was above average in intelligence, completing college and
mastering five languages before her death at age 32.
Type IV OI is the most clinically variable of the four para-
digm types. Presentation may be severe to mild. Clinical signs
may include somewhat reduced stature, dentinogenesis
imperfecta, adult-onset hearing loss, and variable degrees of
skeletal osteopenia.
A particularly interesting skeletal anomaly found in osteo-
genesis imperfecta is the presence of wormian bones. These
are irregularly shaped bones within the sutures of the skull;
they are found most often within the lambdoid suture and
arranged in a mosaic pattern (Fig. 7-3). These intrasutural
bones have been associated with several congenital disorders
A but most commonly with OI. Other disorders associated with
wormian bones include cleidocranial dysplasia, hypophos-
phatasia, hypothyroidism, and pycnodysostosis. They may
also occur with no anomaly. In this last case, these extra
bones tend to be smaller and fewer in number. Wormian
bones tend to represent a pathologic condition when greater
than 4 to 6 mm and when more than 10 are present. Interest-
ingly, the name “wormian” has nothing to do with the appear-
ance of the bone but honors Olaus Worm, the Danish
anatomist who first described them in 1643.
It is important to emphasize that those children with
undiagnosed osteogenesis imperfecta or another type of
B bone disease may have the same symptoms as an abused
child. Bruising, unexplained fractures, and evidence of old
Figure 7-2. Dentinogenesis imperfecta is characterized by fractures in various stages of healing on radiography can
translucent gray to yellow-brown teeth and involves both lead a physician to consider abuse when a genetic defect
deciduous (baby) and permanent teeth. The enamel fractures has not been eliminated. A thorough family history may
easily. This condition occurs in osteogenesis imperfecta, or it reveal other minor or variable phenotypes not previously
can be caused by a separate inherited autosomal dominant recognized in family members. A careful physical examina-
trait. A, Panoramic radiographic view of permanent dentition
with bulb-shaped crowns and large pulpal chambers.
tion of the child may reveal additional features associated
B, Frontal view showing irregularly formed, opalescent teeth. with OI such as blue sclerae, opalescent and undermineral-
(Courtesy of Rebecca Slayton, DDS, PhD, University of ized teeth, bruising, a triangular face, a barrel-shaped chest,
Washington School of Dentistry.) and scoliosis.
 Connective Tissue and Bone Diseases 119

Figure 7-3. Wormian bones are

intrasutural cranial bones. They are
often associated with the lambdoid
suture and are generally only
considered pathologically significant
when greater than 6 × 4 mm in size and
10 or more are arranged in a mosaic
pattern. Pathologic associations of
wormian bones include osteogenesis
imperfecta, cleidocranial dysplasia,
pycnodysostosis, hypophosphatasia,
hypothyroidism, and acro-osteolysis.
The majority of observations represent
normal variants. (Courtesy of Owen
Lovejoy, PhD, Kent State University, and
Melanie McCollum, PhD, University of
Virginia School of Medicine.)

Figure 7-4. Ehlers-Danlos syndrome,

characterized by hyperelastic skin and
hypermobile joints, may also be
characterized by easy bruising, poor
healing, and “cigarette paper” scarring.
(Courtesy of Joshua Lane, MD, Mercer
University School of Medicine.)

Ehlers-Danlos Syndrome patients have not been identified. Of patients with COL5A
Ehlers-Danlos syndrome (EDS) is a group of connective tissue mutations, one half have inherited the mutation from a parent
disorders featuring joint hypermobility, hyperelasticity of the and the other half harbor a new mutation that occurred in a
skin, and abnormal wound healing (Fig. 7-4). Historically, parental gamete or during their own early embryonic develop-
EDS was classified into two subtypes, EDS type I and type ment. Both protein-truncating mutations and glycine substitu-
II, the discriminator being clinical severity. However, it is now tion mutations have been found in COL5A-associated EDS.
recognized that EDS represents a continuum of clinical mani-
festations. Today, most of EDS types I and II have been reclas- Marfan Syndrome
sified as classic EDS on the basis of diagnostic criteria. Three Marfan syndrome is a systemic connective tissue disorder that
of four major criteria must be met for diagnosis of EDS: skin typically manifests as skeletal, ocular, and cardiovascular
hyperextensibility; wide, atrophic scars; joint hypermobility; defects. Individuals are typically tall with arachnodactyly
and a positive family history for EDS. There are six major (Fig. 7-5). Ectopia lentis, mitral valve prolapse, and dilation
types of EDS (Table 7-3). Newer terminology has replaced of the ascending aorta are also common.
the use of Roman numerals to designate types. Unlike osteogenesis imperfecta and Ehlers-Danlos syn-
Classic EDS is an autosomal dominant disease caused by drome, Marfan syndrome is caused by mutations in the FBN1
mutations in genes encoding the α chains of type V collagen. gene that encodes fibrillin, a glycoprotein that is the major
Specifically, approximately 50% of patients presenting with structural component of extracellular microfibrils. Microfi-
classic EDS have mutations in either COL5A1 or COL5A2. brils are part of the ECM and form a network for elastin
The genetic defects responsible for the remaining half of the deposition in the formation of elastic fibers. Microfibrils are
 120 Musculoskeletal Disorders

ANATOMY ANATOMY
Ciliary Body of the Eye Craniosynostosis
The ciliary body lies behind the iris and is attached to the Craniosynostosis is the premature closure of sutures in the
lens by ciliary zonules. It produces aqueous humor and skull that leads to a change in the shape of the skull. Major
controls accommodation—the changing of lens shape. sutures include coronal, sagittal, metopic, and lambdoid.
There are several types of craniosynostosis:
■ Brachycephaly: premature closure of coronal sutures
■ Scaphocephaly, also called dolichocephaly: premature

closure of the sagittal suture

■ Trigonocephaly: premature fusion of the metopic suture
■ Plagiocephaly: premature fusion of one of the coronal or
widely distributed in the body, but they are most abundant lambdoid sutures
in ligaments, the aorta, and the ciliary zonules of the lens—all ■ Acrocephaly, oxycephaly, and turricephaly: premature
tissues prominently affected in Marfan syndrome. closure of the coronal and lambdoid sutures; creates
Several skeletal phenotypes are associated with Marfan pyramidal shape
syndrome. These individuals are noted for tall stature, where
the mean height is greater than the 97th percentile, along
with a decrease in the upper body segment to lower body
segment ratio, designated as US:LS. Normally, US:LS is 0.93,
but in individuals with Marfan syndrome it is 0.85 or at least Coronal Metopic
2 standard deviations below the mean for age, sex, and race.
Often the arm span exceeds height, an advantageous charac-
teristic for some sports such as basketball. It is not unusual
for normal males and females to also meet this criterion; Sagittal
however, arm span exceeds height by more than 8 cm in only
5% to 6% of normal individuals. The greater the arm span
exceeds height, the less likely that normal individuals will be
identified. Other skeletal features frequently found in
Lambdoid

TABLE 7-3. Summary of Ehlers-Danlos Syndrome Types

TYPE FORMER TYPE CLINICAL FEATURES PREVALENCE

Classic I and II Skin hyperextensibility, velvety skin 1 in 20,000–40,000

Fragile skin—bruises and tears easily
Poor wound healing leading to widened, atrophic scarring
Molluscoid pseudotumors on elbows and knees; spheroid
bodies on shins and forearms
Hypermobile joints
Mitral valve prolapse
Hypermobility III Hypermobile, unstable joints Most common form:
Chronic joint pain 1 in 10,000–15,000
Mitral valve prolapse
Fragile blood vessels and organs—at risk for rupture, aneurysm,
dissection
Thin, fragile skin—bruises easily
Vascular IV Veins visible beneath skin One of the most
Characteristic facies—protruding eyes, thin nose and lips, malar serious forms: 1 in
flattening, hypoplastic mandible 100,000–200,000
Kyphoscoliosis VI Progressive scoliosis Rare
Fragile eyes
Progressive muscle weakness
Arthrochalasis VIIA Hypermobile joints prone to dislocations, especially hips Rare
VIIB Skin hyperextensibility—prone to bruising
Early-onset arthritis
Increased risk of bone loss and fracture
Dermatosparaxis VIIC Extremely fragile, sagging skin Rare
Hypermobile joints—may delay development of motor skills

In 2002, Steinmann et al questioned the existence of EDS V as a distinct entity. The phenotype, described in 1975, was poorly defined and may have represented
another disorder.
 Connective Tissue and Bone Diseases 121

A B

C D

Figure 7-5. Individuals with Marfan syndrome have characteristic arachnodactyly with joint hypermobility. A, Long fingers.
B, Positive wrist sign (Walker sign). C, Positive thumb sign (Steinberg sign). D and E, Hypermobile joints.

individuals with Marfan syndrome are dolichocephaly (Fig. fibrillin within the aorta, dilatation of the aortic root, where
7-6), prominent brow, hypognathic or retrognathic mandible, maximum stress occurs, is a serious concern (Fig. 7-7).
and high-arched and narrow palate. Vertebral and pectus Dilatation is seen in approximately 25% of children and
deformities are present in 30% to 60% of individuals. 70% to 80% of adults. These individuals carry a significant
Although the skeletal features may be the most readily risk of aortic dissection that may begin as a gradual dilata-
recognized phenotype of Marfan syndrome, the earliest tion at the aortic root that progresses into the ascending
manifestation may be mitral valve disease. Eighty percent aorta. Marfan syndrome does not preclude childbirth by
of individuals will show evidence of prolapse; in more than females, but these women must be monitored regularly by
25% of these individuals, prolapse will progress to regur- echocardiography.
gitation by adulthood. Aortic regurgitation is also common Marfan syndrome is an autosomal dominant disease;
and progressive in 70% of individuals. Because of the altered approximately 75% of the cases are inherited and 25%
 122 Musculoskeletal Disorders

represent de novo mutations. Over 500 independent FBN1 throughout this text as well as conditions not discussed in
gene mutations are associated with Marfan syndrome, nearly this text. Careful consideration of all physical and clinical
70% of which are missense mutations. This suggests that findings is important in establishing a presumptive diagnosis.
the production of normal microfibrils is altered by the A definitive diagnosis is easily attainable with molecular
presence of mutant fibrillin. In the heterozygous state, this analysis for each of these disorders with a specific known
defines autosomal dominant disorders—the interaction family mutation or in situations in which limited mutations
between mutant and normal fibrillin is sufficient for disease are associated with a disorder, such as for achondroplasia
expression. (see next section). An equally definitive diagnosis is also
Allelic heterogeneity at the FNB1 locus accounts for the possible with other tests such as muscle biopsy used for
overall symptom variability observed in individuals with muscular dystrophies. Less secure diagnoses are described
Marfan syndrome and between different affected families. in Table 7-4.
Some clinical variability can also be seen within a family
sharing the same mutation, suggesting that other genetic or
epigenetic factors play a role in disease expression. ●●● MUSCULOSKELETAL DISEASE
DUE TO GROWTH FACTOR
Differential Considerations RECEPTOR DEFECT
Achondroplasia
Osteogenesis imperfecta, Ehlers-Danlos syndrome, and
Marfan syndrome all share variably expressed phenotypic Achondroplasia, also known as short-limb dwarfism, is the
features. This is also true for other conditions discussed most common form of dwarfism and occurs in roughly 1
in 20,000 live births. In short-limbed dwarfism, affected
individuals have short stature and particularly short arms
and legs; the average height of adult men is 132 cm and
that of adult women is 125 cm. Although all bones formed
from cartilage are involved in achondroplasia, the prolif-
eration of cartilage is greatly retarded in the metaphyses
of long bones. In essence, the maturation of the chon-
Cephalic index (CI) ≤75.9 drocytes in the growth plate of the cartilage is affected.
Head width¥100 Other skeletal abnormalities include macrocephaly with
CI =
Head length frontal bossing, midface hypoplasia, and genu varum (Fig.
7-8). The spine and ribs are also affected, as is the car-
tilaginous base of the skull. Life span and intelligence are
typically normal, although 5% to 7% of infants with
Figure 7-6. Dolichocephaly. The head is long and narrow. achondroplasia die within the first year of life from central
The cephalic index can be calculated to determine whether a or obstructive apnea due to brainstem compression or
shape is dolichocephalic or within normal limits. midface hypoplasia.

Normal aorta Enlarged aorta

(aneurysm)

Figure 7-7. Aortic aneurysm. The

aortic root is the site of greatest
pressure. Mutations in fibrillin weaken
the connective tissue of the aorta,
causing bulging, tearing, and
dissection.
 Musculoskeletal Disease Due to Growth Factor Receptor Defect 123

TABLE 7-4. Security of Diagnosis

SECURITY LEVEL OF SECURITY EXPLANATION

Definite 100% The diagnosis is absolutely not in question.

Full genetic counseling is appropriate relative to prognosis, treatment, and recurrence
risk.
Probable 80–99% Absolute diagnosis is not able to be made.
A remote possibility of other diagnoses exists.
Reevaluation of diagnosis should occur with each follow-up visit.
Full genetic counseling is given just as for “definite diagnosis.”
Possible 50–79% Diagnosis is considered likely, but the presence or absence of some historical, clinical,
or laboratory findings leaves some question about the diagnosis.
No specific genetic counseling can be offered, but the family should be informed of the
diagnostic possibilities and of any opportunities for helping arrive at a diagnosis.
Rule out 1–49% The diagnosis being considered is among a list of possibilities, none being particularly
likely.
Specific genetic counseling cannot be given.
Follow-up is very important to monitor growth and development and to check for new
findings that may make the diagnosis more likely.
Unknown 0% There is no real clue to the diagnosis.
Continued follow-up is essential, and new diagnostic tests should be pursued.
No specific genetic counseling can be offered except that a 4–6% empirical risk of
recurrence can be given.

ANATOMY & EMBRYOLOGY

Appendicular Skeleton
The appendicular skeleton consists of the pectoral and
pelvic girdles with the limbs. Ossification of long bones
begins by the eighth week of development, although all
primary centers and most secondary centers of ossification
are present at birth. For the diaphysis of long bones, bone
is ossified from a primary center, whereas for the epiphysis,
ossification occurs from a secondary center. The epiphyseal
plate consists of cartilage formed between diaphysis and
epiphysis, and it is eventually replaced by bone when bone
growth ceases. Flat and irregular bones have no diaphysis
or epiphysis.

Achondroplasia results from mutations in the fibroblast

growth factor receptor 3 (FGFR3) gene. Four FGFR genes have
been identified that interact with at least 23 different fibroblast
growth factors; the receptors are all transmembrane tyrosine
kinases involved in binding fibroblast growth factor and sub-
sequent cell signaling. The FGFRs share a common protein
structure, characterized by three extracellular immunoglobulin-
like domains, an acidic box, a lipophilic transmembrane
domain, and intracellular tyrosine kinase domains (Fig. 7-9).
FGFR3 is expressed at high levels in the prebone cartilage
rudiments of all bones and in the central nervous system.
Defective FGFR proteins lead to altered interaction with fibro-
blast growth factor, which affects signal transduction. The most
common mutations, G1138A and G1138C (discussed below),
introduce a charged amino acid into the hydrophobic domain
of the receptor and activate dimerization. This region of the
Figure 7-8. Achondroplasia. Note the short limbs relative to
receptor regulates the kinase activity.The mutations, therefore, the length of the trunk. Also note the prominent forehead, low
cause constitutive activation in a ligand-independent manner. nasal root, and redundant skinfolds in the arms and legs.
Since FGFRs are widely expressed in bone, consequences of (From Jorde LB, Carey JC, Bamshad MJ, White RL. Medical
mutations are more profound in bone development. Genetics, 3rd ed. Philadelphia, Elsevier, 2006, p 67.)
 124 Musculoskeletal Disorders

nucleotide. Hence, nearly all achondroplasia worldwide is

BIOCHEMISTRY due to the alteration of a single amino acid (glycine 380) in
FGFR3, thus demonstrating once again the importance of
Receptors glycine in protein function. A very rare mutation that substi-
Transmembrane receptors have three parts: intracellular tutes lysine for methionine at position 650 results in severe
domain, transmembrane domain, and extracellular domain. achondroplasia with developmental delay and acanthosis
The extracellular domain recognizes and responds to nigricans (SADDAN).
specific ligands, such as hormones, neurotransmitters, and
antigens. The transmembrane domain may form a channel
or pore when the extracellular domain is activated. The
intracellular domain interacts with the inside of the cell to
PATHOLOGY
relay a signal through effector proteins or enzymatic activity.
Acanthosis Nigricans
Receptor tyrosine kinases are single-membrane-spanning
receptors that can autophosphorylate as well as Acanthosis nigricans are dark, thick, velvety areas of skin
phosphorylate other proteins, such as epidermal growth in body folds and creases that are most commonly seen
factor, platelet-derived growth factor, insulin, insulin-like in individuals of African descent. They can occur with
growth factor type 1, fibroblast growth factor, and nerve growth some drugs—particularly growth hormone and oral
factor. Binding of a ligand causes a conformational change contraceptives—and with obesity and diabetes, GI or
that facilitates dimerization of two cytoplasmic domains and genitourinary cancers, lymphoma, and certain genetic
phosphorylation of tyrosines to activate the complex. The conditions.
active complex then signals effectors downstream. Therefore,
the phosphorylation of the intracellular domain is a regulatory
Achondroplasia is a congenital defect, a defect present at
mechanism for effector function.
birth. A significant number of achondroplastic infants are
stillborn or die in infancy; those surviving to adulthood
produce fewer offspring than normal. The mortality and low
Achondroplasia-associated FGFR3 mutations exhibit auto- fecundity generate a strong force of selection against affected
somal dominant transmission with complete penetrance. individuals. About 80% of the children born with this condi-
Over 200 independent FGFR3 mutations have been identi- tion do not produce offspring. If this selective force were the
fied. Remarkably, 99% of individuals with achondroplasia only one operating, the frequency of the disorder would
have one of two missense mutations in the gene. Approxi- steadily decrease from one generation to the next. But this
mately 98% of individuals harbor a G-to-A mutation at force is opposed by mutation. Greater than 80% of individu-
nucleotide 1138 (G1138A) of the FGFR3 gene that causes als with achondroplasia have normal parents, and thus de
the replacement of a glycine with an arginine at amino acid novo mutation occurs within a parental germ cell or very
residue 380. One percent of patients have a G1138C muta- early in embryologic development. The former is strongly
tion that substitutes a cysteine for glycine at the same suspected, since FGFR3 de novo mutations are associated

Ig-l

Ig-l
Extracellular domain

Ig-l

TM

Mutation introduces a charged G1138A and G1138C mutations

amino acid into a hydrophobic K Constitutive receptor activation in a
domain and activates dimerization i ligand-independent manner.
(i.e., TM regulates kinase activity). n
a
s
e

K
i
n
a Figure 7-9. Typical FGFR structure.
s
Ig, immunoglobulin domain; TM,
e
transmembrane domain; kinase,
tyrosine kinase domain.
 Muscle Cell Diseases 125

example, the G-to-A transition produces a new restriction

site for the enzyme SfcI, which recognizes the sequence
CTACAG (Fig. 7-10) (see Chapter 13). As a result, prenatal
diagnosis of heterozygous achondroplasia, homozygous
achondroplasia, and the homozygous unaffected state is
rapid, accurate, and unequivocal.

— 164 bp ●●● MUSCLE CELL DISEASES

— 109 bp Muscular Dystrophies
The muscular dystrophies are a large and heterogeneous
Figure 7-10. Prenatal analysis of FGFR mutation. Paternal group of disorders distinguished by the progressive loss of
DNA demonstrates the ScfI restriction fragments created with muscular strength and morphologic integrity. Individual
the G1138A mutation. Maternal DNA does not have the
muscle fibers show variation in size, metabolic oxidative
mutation. Fetal DNA isolated from maternal serum has the
G1138A mutation. The normal amplified fragment is 164 bp. stress, sarcolemmal fragility, and ultimately fiber loss due to
Following digestion, one allele produces a 109-bp allele and necrosis and replacement by fat or connective tissue. As a
a 55-bp allele that is not shown on the gel. Solid-colored group, the muscular dystrophies are represented by greater
symbols indicate affected individuals. (Gel from Li Y, than 30 independent disorders with etiologic links to nearly
Holzgreve W, Page-Christiaens GC, et al. Improved prenatal 30 different genetic loci. Here, we focus on the most common
detection of a fetal point mutation for achondroplasia by the and severe form of muscular dystrophy, Duchenne muscular
use of size-fractionated circulatory DNA in maternal plasma—
dystrophy (DMD), and its associated milder form, Becker
case report. Prenat Diagn 2004;24:896–898. Copyright, John
Wiley and Sons Ltd. Reproduced with permission.) muscular dystrophy (BMD), as illustrative muscle diseases.
DMD is an X-linked disease of childhood. The incidence
is approximately 1 in 3600 live male births. Unfortunately,
for those males who inherit or harbor the DMD mutation,
penetrance is essentially complete and their muscles rapidly
with advanced paternal age. The magnitude of recurrent deteriorate during early childhood. The earliest symptom
mutations at FGFR3 nucleotide 1138 represents one of the is usually clumsiness in walking with a tendency to fall.
highest new mutation frequencies observed for autosomal At about age 3, the child experiences difficulty in climbing
dominant diseases. Obviously, the remaining 20% of patients stairs and rising from the floor (Fig. 7-11). One of the
have at least one parent with achondroplasia. Couples in most obvious physical features in the early stages of the
whom both individuals are heterozygous for achondroplasia disease is pseudohypertrophy of the calf muscles due to
are not uncommon, and their offspring have a 25% risk of the replacement of muscle with adipose and fibrous
being homozygous for the achondroplasia mutation. The connective tissue.
homozygous state of achondroplasia is not compatible with As the profound wasting of muscles progresses, the major-
survival, and affected infants inevitably die of respiratory ity of affected individuals are unable to walk by 12 years of
failure in the first or second year of life. age (Fig. 7-12). As the disease progresses, loss of strength in
Prenatal diagnosis of homozygous achondroplasia can be shoulders and proximal arms is often noted and cognitive
accomplished by ultrasonography in the mid to late second abilities decline. Respiratory muscles weaken, and cardiomy-
trimester. However, women who are heterozygous for achon- opathy is essentially ubiquitous in patients over 18 years.
droplasia face maternal obstetric risks, several of which Most patients die before the age of 20 years of chronic respi-
increase during gestation and may complicate a late preg- ratory insufficiency or pneumonia and, occasionally, of heart
nancy termination. Moreover, prenatal diagnosis in the failure.
second trimester creates anxiety for the couple and increases BMD is a milder variant of DMD and is distinguished from
the emotional burden of terminating a homozygous fetus DMD by the later onset of skeletal muscle weakness and the
at a relatively late stage. Identification of the key mutations ability to walk into the third decade of life. The disease usually
in the FGFR3 gene enables first-trimester DNA-based pre- progresses to death due to cardiomyopathy-related heart
natal diagnosis. In the simplest case, a disease mutation failure in mid to late adulthood.
either creates or destroys a restriction endonuclease site.
Thus, PCR amplification of the relevant portion of the
FGFR3 gene, followed by mutation-specific restriction endo- Molecular Basis and Genetics
nuclease digestion and gel electrophoresis, enables the visu-
of Duchenne and Becker Types
alization of the heterozygous and homozygous states.
Accordingly, both the G1138A transition and the G1138C
of Muscular Dystrophy
transversion create new recognition sites for restriction DMD and BMD are caused by mutations in the dystrophin
enzymes, rendering it straightforward to test for the pres- gene. Dystrophin is a 427-kDa intracellular protein, found
ence or absence of the mutation in genomic DNA. For predominantly in skeletal, smooth, and cardiac muscle. Low
 126 Musculoskeletal Disorders

A B

C D

E F

Figure 7-11. Gowers maneuver. The photographs show the characteristic movements by which a child with muscular
dystrophy uses his hands to walk up his legs to a standing position. (Used with permission from the MDA Foundation.)

levels are also found in the central nervous system. Dystro- actin-binding portion of spectrin in the cytoskeleton of the
phin is localized to the sarcolemmal membrane, where it red blood cell (see Chapter 6). This is followed by a rod-like
functions in a mechanical fashion by linking the ECM with domain consisting of 24 repeats of similar sequences of nearly
actin components of the cytoskeleton, thereby helping to 109 amino acids. The rod-like domain terminates at a cysteine-
resist stresses associated with muscle contraction. rich region of about 150 amino acids. The C terminus com-
The N-terminal region of the dystrophin molecule (Fig. prises a 420-amino-acid region that is interactive with other
7-13) is an actin-binding domain, homologous to the membrane proteins.
 Muscle Cell Diseases 127

Laminin 2

Sarcoglycan
Extracelluar matrix complex aDG

Sarcolemma Dystroglycan
a b g d bDG complex

SYN

Dystrophin aDB

Actin SYN
Hinge Cytoplasmic
regions complex

Figure 7-13. Skeletal muscle membrane organization. DB,

dystrobrevin; DG, dystroglycan; SYN, syntrophin. (Used with
Figure 7-12. As muscle wasting progresses, postural permission and adapted from Blake DJ, Weir A, Newey SE,
changes occur in Duchenne muscular dystrophy (DMD). Davies KE. Function and genetics of dystrophin and
(Used with permission from the MDA Foundation.) dystrophin-related proteins in muscle. Physiol Rev
2002;82:291–329.)

Dystrophin Organization

Dp427
Dp427 Dp260 Dp140 Dp116 Dp71
Dp427
EXON
2 30 44 56 63
0 500 1000 1500 2000 2500kb

Deletion cluster II Deletion cluster I

Figure 7-14. Exon of the dystrophin gene showing the seven different promoter sites for each of the isoforms: three full-length
and four shortened forms. The 427-kDa forms differ only at the NH2-terminal sequences. Two “hot spots” for deletions are
shown: cluster I between exons 45 and 53 affects the rod domain of the protein, and cluster II between exons 2 and 20 affects
the actin-binding domain. Dp, dystrophin protein. (Used with permission and adapted from Blake DJ, Weir A, Newey SE, Davies
KE. Function and genetics of dystrophin and dystrophin-related proteins in muscle. Physiol Rev 2002;82:291–329.)

There is tissue-specific expression of the 427-kDa protein are correlated with abnormal electroretinograms and electro-
that is controlled by different promoters. Brain dystrophin is encephalograms in individuals with abnormal dystrophins.
expressed in the cortex and hippocampus; muscle dystrophin DMD and BMD are caused by many mutations within the
is expressed in skeletal muscle, cardiomyocytes, and some dystrophin gene that are located in the middle of the short
glial cells; and Purkinje dystrophin is expressed in cerebellar arm of the X chromosome (Xp21). It is the largest known
Purkinje cells and skeletal muscle. These three dystrophins human gene, spanning about 2000 kb and containing more
have a unique first exon followed by 78 common exons. Four than 70 exons. The gene is approximately 0.001% of the total
internal promoters give rise to shorter forms of dystrophin human genome, representing about one third the amount of
isoforms. Figure 7-14 shows the seven different promoters the entire Escherichia coli genome. Although the gene is 2
within the dystrophin gene. In addition to the three 427-kDa million base pairs in size, the mature dystrophin mRNA is 14
proteins already mentioned, the four shorter dystrophins are kb, which encodes a very large polypeptide of 3685 amino
present in the retina (260 kDa); the brain and fetal kidney acids in muscle.
(140 kDa); Schwann cells and nodes of Ranvier (116 kDa); DMD is allelic with BMD in that both are caused by dif-
and the glia, kidney, liver, and lung (71 kDa). These findings ferent mutations within the same gene locus. BMD affects
 128 Musculoskeletal Disorders

about 1 in 30,000 male newborns. Both diseases usually

Mitochondrial Myopathies
result from deletions—65% of DMD cases and 85% of BMD
cases—with duplications (6% of DMD cases) and point, small Mitochondrial myopathy is a muscle disease caused by mito-
insertion, or deletion mutations (30% of DMD cases) account- chondrial dysfunction. Mitochondria provide several func-
ing for the remainder of cases. The deletions are widely scat- tions to the cell, but the primary function is producing cellular
tered over the length of the gene, with two areas representing energy in the form of adenosine triphosphate (ATP). This is
clusters of deletions (see Fig. 7-14). No clustering of deletions accomplished by the electron transport chain (ETC) and oxi-
differentiates DMD from BMD. The deletions are not uniform dative phosphorylation (OXPHOS). The ETC comprises four
in size, ranging from 6 to greater than 1000 kb. There is no enzyme complexes (complexes I to IV) that systematically
apparent correlation between the size and location of the oxidize NADH and FADH2 molecules and ultimately reduce
deletion and the severity and progression of the disorder. molecular O2 to form water. Concurrent with the flow of
Large deletions have been found in some patients with the electrons down the ETC is the pumping of protons from the
milder BMD, whereas some of the patients with severe DMD inner membrane space to the matrix, forming an electro-
have small deletions. chemical gradient across the mitochondrial inner membrane.
The enigma regarding deletion size and disease severity is This gradient, known as a membrane potential, is utilized by
resolved by the understanding of reading frames. The DNA ATP synthase (complex V) to condense inorganic phosphate
deletions resulting in the clinically less severe BMD bring with ADP to form ATP, which is then utilized by nearly all
together exons that maintain the translational reading frame cells for function and maintenance. Hence, mitochondria
(“in-frame” deletions) of the messenger RNA. If two “com- serve as the “powerhouses” of the cell and are essential for
patible” exons (multiples of three bases) are juxtaposed in aerobic respiration. However, mitochondria are also central
the sliced message, then the reading frame is preserved, and players for two other relevant cellular metabolic processes:
a functional, although shorter than normal, transcript is pro- oxidative stress and programmed cell death, or apoptosis. In
duced. Conversely, deletions associated with the more severe fact, mitochondrial myopathy, and mitochondrial diseases in
DMD bring together exons that disrupt the translational general, can be envisioned as resulting from the interplay
reading frame and inevitably lead to a stop codon. The between bioenergetic deficiency, increased cellular oxidative
outcome is the production of a severely truncated and non- stress, and the provocation of apoptosis.
functional protein. Likewise, duplications and point muta-
tions associated with DMD typically render little or no
dystrophin.
The integrity of the dystrophin protein is the key to distin- PATHOLOGY
guishing between the two diseases. Most DMD patients have
between 0 and 5% detectable dystrophin while most BMD Creatine Kinase
patients have between 20% and 80% of functional dystro- Creatine kinase (CK), also known as creatine phosphokinase,
phin. Based on dystrophin quantification, an intermediate or is an important indicator of muscle damage. Both the muscle
“outlier” form (IMD) can be recognized with a cognate inter- and the brain have high ATP demands, and CK functions to
mediate phenotype. Thus, the presence or absence of dystro- regenerate ATP rapidly. Muscle breakdown causes elevated
serum CK (>10 times normal in DMD; >5 times normal in
phin is of diagnostic and prognostic significance.
BMD) even before clinical symptoms appear. CK is also
In its typical form, DMD is expressed in affected males with
elevated in many muscle diseases and is a dimer of muscle-
complete penetrance. Hence, affected males experience specific (M) or brain-specific (B) monomers.
reduced reproductive fitness, since they cannot transmit the ■ Muscle: MM form
deleterious gene. Roughly one third of the mutant alleles are ■ Brain: BB form
“lost” each generation. Because the incidence of DMD is ■ Heart: MB form
maintained at 1 in 3600 live male births, de novo mutations
must occur in a significant number of cases either in parental
gametes or very early in zygotic cleavage, just as seen with
achondroplasia. Indeed, the extremely large size of the dys- Complexes I to V (Fig. 7-15) are generated from two
trophin gene makes it an excellent target for mutation, and different genetic systems. Eighty percent of the ETC and
the DMD locus accordingly has a high new mutation fre- OXPHOS proteins are provided by nuclear-encoded (nDNA)
quency. As a result, the affected male is often the solitary genes. The remaining proteins are provided by the only
member of the family to be afflicted. extranuclear DNA found in cells, the mitochondrial DNA
Although heterozygous females generally are normal, (mtDNA). mtDNA is a closed, circular molecule 16,569
about 8% have muscle weakness, fatigability, and mild respi- bases in length that encodes 37 different gene products,
ratory and cardiac problems. The overt muscle weakness in including 13 mRNAs that contribute polypeptides to com-
female carriers is explicable by the chance predominance of plexes I to V (see Fig. 7-15). Specifically, mtDNA provides
mutant-expressing X chromosomes during lyonization. seven subunits to complex I, none to complex II, one subunit
Indeed, monozygotic females have been identified with one to complex III, three subunits to complex IV, and two sub-
normal twin and one DMD-affected twin resulting from units to complex V. The remaining mtDNA genes code for
skewed X chromosome inactivation. 22 tRNAs and 2 rRNAs. Transcription and translation of
 Muscle Cell Diseases 129

OH
PH T
12s F
rRNA D-loop Cyt b
V
A P
0/16569
16s
PL
rRNA
DEAF 1555G E
ND6

L LHON 14484C
MELAS 3243G LHON 14459A
ND5
ND1 LHON 3460A

I Q
M L
S
H

ND2 LHON 11778A

A

ND4
OL
N
C Y

NARP 8993G/C
ND4L
MERRF 8344G
R
S ND3
CO I G
COIII

D K ATPase6
CO II
5 kb deletion
ATPase8
KSS

Complex I genes (NADH dehydrogenase) Complex III genes (ubiquinol:cytochrome-c oxidoreductase) Transfer RNA genes
Complex IV genes (cytochrome c oxidase) Complex V genes (ATP synthase) Ribosomal RNA genes

Figure 7-15. Human mitochondrial DNA (mtDNA) chromosome. The human mitochondrial genome is 16,569 bp in length and
encodes 37 gene products, including 13 mRNAs, 22 tRNAs, and 2 rRNAs. The chromosome is present in multiple copies per
cell. DNA replication is initiated at the origins of DNA replication (OH and OL), and polycistronic RNAs are produced by the two
major promoters (PH and PL). The large RNAs are then processed to yield the mRNAs, tRNAs, and rRNAs. tRNA genes are
identified by a single-letter code that refers to the cognate amino acid in the charged tRNA. CO, cytochrome oxidase genes;
Cyt b, cytochrome b gene; D-loop, noncoding displacement loop; ND, NADH dehydrogenase genes. (From MITOMAP:
A human mitochondrial genome database. Available at: http://www.mitomap.org.)

mtDNA gene products take place within the mitochondrial principles. First, mitochondria are present in multiple copies
matrix. Thus, the mtDNA-encoded polypeptides are essential per organelle, and thus a cell harbors thousands of mtDNAs.
for aerobic respiration and, therefore, life. Because most of Second, mitochondria are strictly maternally inherited.
the well-characterized inherited mitochondrial diseases are Defects in mtDNA that result in disease show maternal
due to mtDNA mutations, these will be discussed further. (and no paternal) transmission. Third, mtDNA has an
However, it should be noted that, because most mitochon- extremely high mutation rate due to lack of an efficient
drial proteins are provided by the nucleus, the number of DNA repair mechanism. Fourth, when a new mutation occurs
nDNA-based mitochondrial diseases might be significantly within a mitochondrion of a cell, a mixture of mutant and
underrecognized. normal mtDNA is produced. This mixture is called hetero-
mtDNA has a number of features distinguishing “mito- plasmy and, unlike the homozygous/heterozygous situation
chondrial genetics” from the better known nuclear genetic found for nDNA mutations, a heteroplasmic proportion can
 130 Musculoskeletal Disorders

be anywhere from 1% to 99%. When heteroplasmic mito-

chondria divide, heteroplasmy can be propagated to an entire
tissue, organ system, or organism, depending on when and
where a mutation originated. Fifth, different organ systems
and tissues rely on OXPHOS for energy to varying extents
and thus display varying “threshold expression” of mito-
chondrial products. For example, the central nervous system
and skeletal/cardiac muscle depend most strongly on
mitochondrial ATP production. Thus, deleterious mtDNA
mutations tend to feature neurologic and neuromuscular
clinical signs, since these tissues are the least tolerant of
perturbations in mitochondrial function.
As noted, mtDNA-encoded genes are essential for cellular
energy in multiple cell types and these genes suffer a very
high mutation rate. Therefore, it is reasonable that a number
of mutations in mtDNA will be deleterious and result in
mitochondrial dysfunction and human disease. In practice— Figure 7-16. Ragged red fibers in skeletal muscle section.
again owing to the ubiquity of the mitochondrion— Muscle cells in mitochondrial myopathy contain a
mitochondrial diseases that originate from both the mtDNA mitochondrial defect and exhibit a classical subsarcolemmal
accumulation of abnormal mitochondria, which stain red with
and the nDNA include a large spectrum of disorders ranging
Gomori trichrome stain.
from lethal perinatal disease to late-onset, progressive, aging-
related disorders. Typically, mitochondrial diseases progress
and involve more than one organ system. Exceptions to this
generality demonstrate the tremendous clinical variability Hence, vomiting, seizures, dementia, movement disorder,
found in bioenergetic disease. For example, Leber hereditary stroke-like episodes, ptosis, ophthalmoplegia, blindness, and
optic neuropathy (LHON), a late-adolescence blindness, does cardiomyopathy/cardiac conduction abnormalities often
not progress after rapid-onset degeneration of only the retinal occur in patients. Neurologic signs are common in mito-
ganglion cell and its axon, the optic nerve. Below, mtDNA- chondrial myopathy, and when present, this group of dis-
based mitochondrial myopathy is presented as the paradigm orders can be termed mitochondrial encephalomyopathies.
for mitochondrial muscle disease. Classic examples of mitochondrial myopathies include
Mitochondrial myopathy is characterized by the degenera- Kearns-Sayre syndrome (KSS); chronic progressive external
tion of individual muscle fibers associated with an accumula- ophthalmoplegia (CPEO); myoclonic epilepsy with ragged
tion of abnormal mitochondria in the subsarcolemma of the red fiber syndrome (MERRF); and mitochondrial encepha-
fiber. The proliferation of abnormal mitochondria represents lomyopathy, lactic acidosis, and stroke-like symptoms
a compensatory response by the cell to bioenergetic defi- (MELAS).
ciency and is perhaps the most reliable pathologic sign of a
mitochondrial myopathy. Histochemical studies consistently
demonstrate that such aggregates of abnormal mitochondria
form red subsarcolemmal blotches, providing the pathogno- NEUROSCIENCE & ANATOMY
monic name ragged red fibers (RRFs) (Fig. 7-16). Likewise,
staining for mitochondrial electron transport chain enzymes Ocular Mitochondrial Myopathies
(typically complex IV or cytochrome c oxidase [COX]) sug- Kearns-Sayre syndrome (KSS) and chronic progressive
gests COX-deficient fibers. Electron microscopic examination external ophthalmoplegia (CPEO) are known as ocular
of RRF mitochondria reveals disordered cristae, and they mitochondrial myopathies characterized by blepharoptosis
often have paracrystalline inclusions (“parking lot” inclusion and ophthalmoparesis. Extraocular muscles are affected
bodies) consisting of precipitated mitochondrial creatine preferentially because the fraction of mitochondrial volume is
several times greater in these muscles than in other skeletal
phosphokinase. Thus, from a histochemical and ultrastruc-
muscles. The levator palpebrae superioris (CN III), one of
tural point of view, mitochondrial myopathy is characterized
eight extraocular muscles, is weakened. As ptosis
by RRFs, an absence or diminution of mitochondrial respira- progresses, the individual may use the frontalis muscle to
tory chain enzyme signals, and morphologically abnormal elevate the eyelids. Ophthalmoplegia occurs when a general
mitochondria. weakness of extraocular muscles progresses to paralysis.
Clinically, mitochondrial myopathies encompass a fairly
wide range of symptoms of varying severity. Most often,
however, these are characterized by onset prior to age 20,
muscle weakness, and exercise intolerance with lactic aci- Most cases of mitochondrial myopathy arise from either
dosis. Although mitochondrial myopathy can stand alone point mutations in mtDNA tRNA (MERRF, MELAS) or dele-
as the sole clinical presentation of mitochondrial disease, tions or rearrangements (KSS, CPEO). MERRF and KSS illus-
it frequently is part of a more complex clinical picture. trate the general principles behind mitochondrial myopathy.
 Muscle Cell Diseases 131

Myoclonic Epilepsy with Ragged Red similarly aged individuals harboring 85% mutant tRNALys can
appear healthy and unaffected, exhibiting normal muscle bio-
Fiber Syndrome
energetic capacity, OXPHOS enzyme activity, and phenotype.
MERRF typically presents with mitochondrial myopathy and Interestingly, maternally related individuals age 60 years and
myoclonic epilepsy—a periodic, uncontrolled jerking that greater, with the same 85% mutant tRNALys, can be severely
often begins focally but progresses to generalized cyclic mus- affected with mitochondrial myopathy, little muscle energy
cular contractions. Roughly 85% of the cases are due to an capacity, and dramatically reduced respiratory chain enzyme
A-to-G mutation in the mtDNA tRNALys gene at nucleotide activities. Hence, for mitochondrial diseases due to tRNA
position 8344, and many of the remaining cases result from mutations, age of onset often reflects the proportion of mutant
a G-to-C mutation at position 8356 in the same tRNA gene mtDNA inherited, and symptoms tend to progress with age
(Fig. 7-17). Both of these mutations disrupt the TψC loop of in terms of the number and severity of clinical signs. This
tRNALys. These mutations have consistently been found het- suggests that at least two factors—an inborn error mutation
eroplasmic in patients and have never been found in normal and an age-related factor—collaborate to precipitate many
mtDNA. forms of mitochondrial disease.
As indicated above, clinical variability and age-related pro-
gression of symptoms are common in the mitochondrial dis-
orders. Upon diagnosis of MERRF, examination of the clinical Chronic Progressive External
status of maternally related individuals within the proband’s
Ophthalmoplegia and
family often reveals affected individuals exhibiting a spec-
trum of clinical signs, including mitochondrial myopathy,
Kearns-Sayre Syndrome
ataxia, cardiomyopathy, sensorineural hearing loss, diabetes, Frequently, a mild to moderate mitochondrial myopathy
and dementia. The wide range of clinical signs and the vari- features ophthalmoplegia and ptosis in addition to frank
able expression found within families is in part due to the mitochondrial myopathy. This combination of symptoms is
varying heteroplasmic proportioning found in affected indi- termed chronic progressive external ophthalmoplegia
viduals. MERRF pedigrees show a strong association between (CPEO). Some patients manifest CPEO before age 20, with
phenotype and genotype (heteroplasmic proportion) in rela- retinitis pigmentosa and at least one of the following:
tion to aging. For example, a young (15- to 25-year-old) cardiac conduction abnormality, cerebellar ataxia, or cerebral
patient with 95% mutant tRNALys often will have a complex spinal protein level above 100 mg/dL. These patients have
and severe clinical picture, including mitochondrial myopa- the more severe Kearns-Sayre syndrome (KSS). Occasion-
thy, reduced muscle oxidative capacity, and diminished mito- ally, other signs manifest with KSS or CPEO, including
chondrial respiratory chain enzyme activities. In contrast, optic atrophy, sensorineural hearing loss, dementia, seizures,
cardiomyopathy, diabetes, and lactic acidosis. Like the
mtDNA tRNA mutations that result in MERRF and MELAS,
KSS and CPEO patients typically feature RRFs and dimin-
ished histochemical staining of respiratory chain enzymes
in skeletal muscle.
The np8344 MERRF Mutation in tRNALys Nearly 85% of KSS patients harbor rearrangements of the
mtDNA that take the form of a group of interrelated, complex
OH 3' molecules including normal, duplicated, and deleted mtDNA.
J

A Occasionally, insertions are found. Such rearrangements are

5' P J C J G almost always heteroplasmic in patients, and the vast major-
AJT ity of patients are singleton cases representing a new muta-
C JG
T JA tional event that occurred either in the maternal oocyte or
GJC early in embryonic development.
T JA TYC Loop In practice, deleted mtDNA is easiest to detect, charac-
AJT C A CA
DHU Loop A TTCTC terize, and quantitate; hence, this form of mitochondrial
A A
J
J
J
J
J

AATCG C myopathy has historically been erroneously associated

J
J
J
J
J

C A A G A GA C strictly with deletions. Nevertheless, a consideration of

T A
TTAGC
A T mtDNA deletions associated with KSS and CPEO illustrates
T JA GA G
T JA the magnitude and mechanism of the genomic rearrange-
AJT ments. Nearly all deletions, for example, are associated
AJT with direct repeats 4 to 16 nucleotides in length, suggest-
C JG
C A ing that a specific DNA sequence motif is necessary for
T A mtDNA rearrangement. Nearly 100 different rearrangement
TTT
breakpoints and 200 different deletions have been charac-
Anticodon
terized in KSS and CPEO patients, suggesting that this is
Figure 7-17. Human tRNALys showing the MERRF-associated a relatively common pathogenic mutational mechanism in
A-to-G transition at mtDNA nucleotide position 8344. mitochondrial diseases.
 132 Musculoskeletal Disorders

Currently there is no effective treatment for mitochondrial A. Ehlers-Danlos syndrome

diseases. Administration of vitamins, O2 radical scavengers, B. Homocystinuria
artificial electron acceptors (in an attempt to bypass a block- C. Marfan syndrome
age in the electron transport chain), and the dimunition of D. Osteogenesis imperfecta
harmful metabolites have been tried with anecdotal success. E. Kearns-Sayre syndrome
Frequently, a combination of the above is formulated as Answer. A
a “cocktail,” indicating the dearth of knowledge regarding
Explanation: From the options presented, Ehlers-Danlos
the underlying pathophysiologic basis of mitochondrial
syndrome is the best option, with types I, II, and III being
disorders.
the best candidates because they are the most common
and because of the hypermobility, murmur, and atrophic
scars. Option E is a mitochondrial myopathy with optic and
neurologic involvement. Options B, C, and D have similari-
ties with EDS, but additional information is needed, and
KEY CONCEPTS
generally provided, to make one of these the best option.
■ Collagens are involved in diseases affecting skin and bone. The
nomenclature specifies the origin of each chain in the triple helix
Individuals with Marfan syndrome and homocystinuria may
collagen structure. have normal height at this age but later are taller than the
average male.
■ Osteogenesis imperfecta and Ehlers-Danlos syndrome have
mutations in collagens I and V, respectively. EDS may also occur
with mutations in collagen II and III genes. 2. A 3-year-old male is evaluated for short stature. At
birth, he was 47.2 cm (18.5″) with shortened limbs.
■ Marfan syndrome has many phenotypic similarities to EDS but At age 3, he has an enlarged head with mild frontal
is a fibrillin mutation; fibrillin is found in the aorta, which can
bossing, a low nasal bridge, and rhizomelic short
dissect in affected individuals.
stature. Both parents are normal. Achondroplasia
■ Achondroplasia is the most common of the dwarfism syndromes is suggested pending DNA testing. Which of the
and is caused by mutations in the fibroblast growth factor following is the etiology of this disorder?
receptor.
A. Collagen mutation
■ Common muscle cell diseases are Duchenne and Becker mus- B. Fibrillin mutation
cular dystrophies. These are allelic conditions—mutations in the C. Mitochondrial mutation
dystrophin gene cause both diseases.
D. Receptor mutation
■ Dystrophin has multiple promoters that demonstrate tissue- E. tRNA mutation
specific expression.
Answer. D
■ Mitochondrial mutations can cause myopathies. Mitochondrial
disease is caused by either mitochondrial mutations or mutations Explanation: Achondroplasia is the most common form
in nuclear-encoded genes. Complex II genes are nuclear of dwarfism and is caused by a mutation in the fibroblast
encoded, but there are many other nuclear-encoded proteins growth factor receptor 3 gene. Almost all cases result
needed for oxidative phosphorylation. from mutations in one DNA triplet. The mutation occurs
in the transmembrane segment of the receptor, resulting
■ Mitochondria demonstrate maternal inheritance.
in dimerization of receptors that leads to constitutive
■ Ragged red fibers are associated with many mitochondrial activation of the kinase activity. FGFR3 proteins are
diseases. expressed in bone, where the consequences are seen.
While it is an autosomal dominant disorder, most muta-
tions are spontaneous with no family history. Collagen
mutations are typically associated with osteogenesis
imperfecta and Ehlers-Danlos syndrome. Fibrillin is
●●● QUESTIONS mutated in individuals with Marfan syndrome. tRNA muta-
tions are common in most mitochondrial mutations, and
1. A 10-year-old boy was evaluated for a heart murmur.
mitochondrial mutations are not commonly associated
Physical examination revealed a healthy male of
with short stature.
normal height and weight with hypermobile joints
and a history of recurrent joint dislocations. Also
present were several wide, atrophic scars remaining
from a prior accident. Which of the following is most Additional Self-assessment Questions can be Accessed
consistent with this patient’s presentation? at www.StudentConsult.com
Neurologic Diseases 8
CONTENTS metabolism. However, both have a significant neurologic
impact. Lysosomal storage disorders are also single-gene dis-
orders and underscore the importance of lysosomes and
SINGLE-GENE DISORDERS
proper degradation of substrates. A discussion of two triplet
Neurofibromatosis repeat disorders—fragile X syndrome and Huntington
Lesch-Nyhan Syndrome disease—serves as a bridge between the easier-to-understand
Lysosomal Storage Disorders classic single-gene disorders and complex, multifactorial dis-
Triplet Repeats orders. Triplet repeat amplification occurs at or near a specific
COMPLEX DISEASES OF THE BRAIN gene and affects that specific gene. These regions of amplifica-
tion are also inherited in the mendelian manner but may not
Tools for Behavioral and Psychiatric Studies represent a parental complement. Expression of complex dis-
Alzheimer Disease orders is more intricate than noted in the earlier discussed
Parkinson Disease
examples. The amplifications in these disorders demonstrate
Schizophrenia
effects in brain development and disease. The second group
Bipolar Disorder
of disorders represents more complex diseases that have both
a genetic and an environmental component affecting expres-
sion. The effects of Alzheimer disease, Parkinson disease,
schizophrenia, and bipolar disorder on families and society
are profound. Though pathphysiologies and molecular mech-
The advent of molecular techniques initiated a remarkable anisms are not fully understood for any of these, they are
period of achievement in the understanding of inherited neu- areas of major research and interest.
rologic diseases. Linkage analysis, combined with molecular
analysis, provided powerful tools for mapping genes for which
no gene product was previously known. Investigators were ●●● SINGLE-GENE DISORDERS
then able to proceed with isolating, cloning, and sequencing
mutant alleles. The determination of the normal and disease
Neurofibromatosis
mutant allele products permitted an explanation for the Until the 1970s, different forms of neurofibromatosis were
molecular pathogenesis of disease. This isolation and charac- not distinguished. Today several forms are recognized, with
terization process of a gene before the gene product is known the most common being neurofibromatosis type 1 (NF1) and
as reverse genetics and has been responsible for better under- type 2 (NF2).
standing of many diseases. NF1 is the most common form of the disease. It was origi-
The classification of genetic neurologic disorders is confus- nally called von Recklinghausen disease or peripheral neuro-
ing because there are an immense number that may be clas- fibromatosis. NF1 is a common disease with an incidence of
sified in several different ways. For example, disorders 1 in 4500 and complete penetrance by age 5. It is inherited
classified by gene function may not represent the clinical in an autosomal dominant manner, and most patients have
manifestation of the disease. In some disorders, more than café-au-lait spots, peripheral neurofibromas, and Lisch
one gene may be involved, such as in neurofibromatosis—a nodules (Table 8-1). Two thirds of individuals also have freck-
disease that results from two different genes. In Duchenne ling in the axilla, base of the neck, groin, and submammary
and Becker muscular dystrophies, mutations in the same gene regions.
yield different clinical presentations. Café-au-lait spots, also found in other disorders, invariably
The disorders discussed in this chapter represent two broad develop by 2 years of age. These pigmented regions demon-
categories: single-gene disorders and complex disorders. strate variable expressivity in size, number, and coloration,
Among the single-gene disorders, neurofibromatosis repre- and while six or more are required for diagnosis, the absolute
sents a disorder characterized by tumors, and Lesch-Nyhan number is not linked to the severity of the disease (Fig. 8-1).
syndrome is a metabolic disorder of purine and pyrimidine Peripheral neurofibromas begin to appear during the teen
metabolism. Thus, the former could be discussed along with years and are found in nearly all adults with NF1. These
cancer disorders and the latter as an inborn error of neurofibromas are discrete nodules within the dermis and
 134 Neurologic Diseases

TABLE 8-1. Comparison of Neurofibromatosis 1 and Neurofibromatosis 2

NEUROFIBROMATOSIS 1 NEUROFIBROMATOSIS 2

Gene symbol NF1 NF2

Gene location 17q11.2 22q12.2
Protein Neurofibromin-1 Merlin (neurofibromin-2)
Incidence 1 in 4500 1 in 40,000–50,000
Mode of inheritance AD with variable expression, complete penetrance
Age of onset Before age 5 years Between ages 15 and 25 years

Major Clinical Features

Café-au-lait spots >99% Generally fewer than NF1

Freckling 67% Generally absent
Peripheral neurofibromas >99% Yes
Lisch nodules 90–95% Absent
Plexiform neurofibroma 25–30% Yes
Vestibular schwannomas
Bilateral 85%
Unilateral 6%
Hearing loss (bilateral) 35%
Cranial or spinal tumors
Meningiomas 45%
Spinal meningiomas 26%
Peripheral schwannomas 68%
Ocular abnormalities 90%

Minor Clinical Features

Macrocephaly 45%
Short stature (below third percentile) 31.5%

A B C

Figure 8-1. Isolated café-au-lait spots are found in many people without neurofibromatosis; however, individuals with more
than five or six of these should be investigated further for NF, particularly if the spots appear within the first 5 years of life. These
dizygotic twins are age 7.
 Single-Gene Disorders 135

epidermis, found mainly on the trunk of the body, and may mutations produce a truncated protein. This protein has a
range in size from less than a centimeter to several centime- domain homologous to the GTPase-activating family. The
ters in diameter. Although cosmetically unappealing, they are protein also has a GAP-related domain (GRD) that interacts
rarely painful (Fig. 8-2). One of the most common causes of with the RAS proto-oncogene. Mutations that truncate neu-
morbidity in individuals with NF1 is the development of rofibromin in the GRD region inactivate the tumor suppressor
plexiform neurofibromas along large nerves. These may function of neurofibromin. The large size of the protein and
invade adjacent structures and impinge on organs. the large number of mutations identified indicate that many
Lisch nodules are asymptomatic hamartomas of the iris mutations are family specific and that carrier screening can
associated with NF1 in 90% to 95% of individuals with NF1 yield many false negatives in a large population.
(Fig. 8-3). These nodules generally develop before peripheral
neurofibromas.
The gene responsible for NF1 is neurofibromin. It is a large BIOCHEMISTRY
gene, and more than 500 mutations have been described that
disrupt normal neurofibromin expression, but most germline GTPases, Components of the G
Protein Complexes
G proteins exist in an active or inactive state and participate
in signal transduction. In the inactive state, GDP is bound to
the G protein, which is composed of α, β, and γ subunits.
Ligand binding to a receptor allows interaction with a G
protein, causing GDP to be replaced by GTP in the α
subunit of the G protein. This α-GTP complex dissociates
from the β and γ subunits as well as from the receptor and
interacts with an appropriate effector, such as adenylate
cyclase, to produce cAMP.
To inactivate the active G protein, GTPase activity that is
intrinsic to the complex converts GTP to GDP and the α
subunit recombines with the β and γ subunits. The rate-
limiting step in this cycle is the GDP dissociation, since GTP
cannot bind until GDP dissociates.

Neurofibromatosis type 2 (NF2) was previously referred to

as bilateral acoustic or central neurofibromatosis. It shares
Figure 8-2. Cutaneous neurofibromas. There are four types
of neurofibromas: cutaneous, subcutaneous, nodular
several phenotypic similarities with NF1, such as café-au-lait
plexiform, and diffuse flexiform. These cutaneous spots and peripheral nerve tumors, but it is a distinct disorder.
neurofibromas have no malignant potential. (From Feit J, Morbidity and mortality are predominantly due to vestibular
et al. Hypertext Atlas of Dermatology. Available at: http:// schwannomas (Fig. 8-4) and other cranial or spinal tumors (see
www.muni.cz/atlases.) Table 8-1). Because many schwannomas may be asymptomatic
and symptoms leading to diagnosis, such as tinnitus or vertigo,
may begin as vague complaints, diagnosis usually occurs much
later than for NF1. Nearly all patients develop symptoms that
lead to deafness, but this progression can take years.
Also inherited in an autosomal dominant manner, NF2
results from mutations in the merlin gene and has strong
homology to protein 4.1, important in membrane cytoskele-
tons (see Fig. 6-4). There are differences in the presentation of
mild and severe forms of the disease. Mild forms usually have
an earlier onset (15–25 years vs. 25–30 years) and slower
progression of tumor development. Mild forms usually dem-
onstrate vestibular schwannomas, whereas the more severe
forms generally have meningiomas and other spinal tumors.
Unlike NF1, which has an early age of onset with complete
penetrance, onset of symptoms due to NF2 is generally in the
late teens with almost complete penetrance by age 60.

Figure 8-3. Lisch nodules (arrow) are associated with Lesch-Nyhan Syndrome
neurofibromatosis type 1. (From Digre K, Corbett JJ. Practical
Viewing of the Optic Disc. Philadelphia, Butterworth Lesch-Nyhan syndrome is a metabolic disorder of purine and
Heinemann, 2003, p 223.) pyrimidine metabolism. It is an X-linked deficiency of the
 136 Neurologic Diseases

Hypoxanthine IMP

Xanthine PRPP HPRT PPi

Xanthine
oxidase Guanine GMP

Uric acid

Figure 8-5. Hypoxanthine and guanine pathway. A deficiency

in hypoxanthine-guanine phosphoribosyltransferase (HPRT)
leads to the overproduction of uric acid and the symptoms
associated with Lesch-Nyhan syndrome. GMP, guanosine
5′-monophosphate; IMP, inosine 5′-monophosphate;
PPi, inorganic pyrophosphate; PRPP,
5-phospho-α-D-ribosyl-1-pyrophosphate.

PHARMACOLOGY
Allopurinol
Allopurinol is a xanthine oxidase inhibitor used to treat
chronic gout. It is also administered with colchicine to
prevent gouty arthritis. Xanthine oxidase oxidizes allopurinol
Figure 8-4. Bilateral vestibular schwannomas (arrows) shown to alloxanthine, which also inhibits xanthine oxidase and de
by magnetic resonance imaging (MRI) with contrast. The novo purine synthesis.
arrowhead shows extension of the right schwannomas into the
internal auditory canal. (Courtesy of Simin Dadparvar, MD.)

The first symptom of this disease may be the observation

enzyme hypoxanthine-guanine phosphoribosyltransferase of orange-colored crystals in the infant’s diaper. Symptoms
(HPRT), encoded by the HPRT1 gene. More than 210 HPRT1 progress, however, and the child becomes hypotonic and
mutations are associated with Lesch-Nyhan syndrome, and hyperreflexic as spasticity of the limbs develops. Self-
its severity correlates with the severity of the genetic lesion. mutilation behavior develops and is demonstrated in children
Infants born with this disorder appear normal at birth, but who bite and chew away fingertips, lips, and tongue. A mild
symptoms begin to appear between 3 and 6 months of age. form in which the enzyme has some activity is rare but, when
Enzyme deficiencies block the ability of hypoxanthine and present, lacks neurologic symptoms.
guanine to form inosine 5′-monophosphate and guanosine
5′-monophosphate, respectively (Fig. 8-5), leading to increased
de novo synthesis. This metabolic block results in the conver-
Lysosomal Storage Disorders
sion of hypoxanthine and guanine to uric acid crystals that Lysosomes are cytoplasmic organelles that enzymatically
accumulate in joints, tissues, and the central nervous system degrade glycoproteins, glycolipids, and mucopolysaccharides.
(CNS). These crystals ultimately lead to kidney stones and Deficiencies in the enzymes responsible for degradation of
impaired kidney function, blood in the urine, and swollen and these substrates result in substrate accumulation. The sub-
painful joints. strate accumulates in the cytoplasm or is taken up by phago-
cytes, leading to clinical features. Lysosomal storage diseases
represent a continuum of disease severity characterized by
BIOCHEMISTRY age of onset, clinical course, and whether the CNS is involved.
Clinical symptoms in lysosomal storage disorders are variable
HPRT with the disorder but all are progressive. The major sites
Hypoxanthine-guanine phosphoribosyltransferase (HPRT) is a affected by the accumulation of substrate are the brain, skel-
ubiquitous enzyme found in the cytoplasm. Its highest activity eton, and organs such as the heart, liver, and spleen. There
is in the brain and testes. HPRT catalyzes the transfer of the are two major classes of lysosomal storage disorders: sphin-
phosphoribosyl group of phosphoribosylpyrophosphate golipidoses and mucopolysaccharidoses.
to hypoxanthine and guanine; this forms inosine
monophosphate and guanosine monophosphate. In
situations in which hypoxanthine and guanine cannot be
Sphingolipidoses
recycled, there is a lack of feedback control of synthesis, Sphingolipids are complex lipids and a major component of
resulting in rapid catabolism of these bases to uric acid. cell membranes. Sphingolipids include sphingomyelins and
glycosphingolipids. This latter group includes cerebrosides
 Single-Gene Disorders 137

N-acetylneuraminic acid (NANA)

GM1
Cer-Glu-Gal-GalNAc-Gal

b-Galactosidase

N-acetylneuraminic acid (NANA)

GM2
Cer-Glu-Gal-GalNAc
Tay-Sachs disease — hypotonia,
b-Hexosaminidase A seizures, spasticity, blindness,
cherry red spot of macula
Cer-Glu-Gal-NANA

Neuraminidase

Cer-Glu-Gal Cer-Glu-Gal-Gal
Fabry disease —pain in lower Sandhoff disease—
extremities, renal failure, optic atrophy,
a-Galactosidase b-Hexosaminidase B seizures, spasticity
hypertension, cardiomyopathy

Cer-Glu Cer-Glu-Gal-Gal-GalNAc
Gaucher disease (type IA) —
hepatosplenomegaly,
b-Glucocerebrosidase
osteonecrosis, anemia,
thrombocytopenia, Gaucher cells

Sphingomyelinase Galactocerebrosidase

Cer-phosphocholine Ceramide Cer-Gal

Niemann-Pick disease—liver Krabbe disease —demyelination

disease, pulmonary infiltrates, and atrophy of brainstem and
ataxia, VSG palsy, dementia Sphingosine+fatty acid cerebellum, hypertonicity, blindness,
progressive peripheral neuropathies

Figure 8-6. Degradation of sphingolipids by lysosomal enzymes. Fabry disease has X-linked inheritance. Other examples
demonstrate autosomal recessive inheritance. VSG, vertical supranuclear gaze.

(glucocerebrosides and galactocerebrosides) and gangliosides.

PATHOLOGY & HISTOLOGY
Normally, phagocytic cells, particularly histocytes or macro-
phages, degrade membranes. In the brain, there is normally Histiocytes
rapid turnover of gangliosides, the most common sphingo-
Histiocytes are fixed cells of the immune system found in
lipid, during development. Any accumulation of sphingolipid many organs and connective tissue. They are phagocytic
degradation products results in a sphingolipid disorder (Fig. cells of the reticuloendothelial system and may also be
8-6). The entire degradation pathway occurs in the lysosome, known as macrophages or mononuclear phagocytes.
where the pH of 3.5 to 5.5 is optimal for enzymatic Examples include:
activity. ■ Gaucher cells: uniformly vacuolated mononuclear,

kerasin-containing cells present in the bone marrow,

Tay-Sachs Disease and Sandhoff Disease spleen, liver, and lymph nodes in patients afflicted with
Tay-Sachs disease occurs with a deficiency of the lysosomal Gaucher disease
■ Kupffer cells: phagocytes located within the sinusoids of
enzyme hexosaminidase A and results in the accumulation of
the normal liver
GM2 gangliosides. These gangliosides accumulate in all tissues,
■ Dust cells: macrophages located in the alveoli and
but the clinical symptoms are seen in those cells with the
interalveolar spaces of the lung
greatest accumulation. As expected from the discussion above, ■ Langerhans cells: dendrite-shaped cells found in the
these cells are neurons and specifically those of the central stratum spinosum of skin
and autonomic nervous systems as well as retinal cells.
 138 Neurologic Diseases

BIOCHEMISTRY
Gangliosides
Gangliosides are the most complex group of
glycosphingolipids. These ceramide (a family of lipids
composed of sphingosine and fatty acid) oligosaccharides
are composed of a sugar and at least one sialic acid
residue. They are the primary component of cell membranes
and make up 6% of brain lipids.

Three gene products are required to form a complex

resulting in the degradation of GM2 gangliosides. Hexosa-
minidase A is composed of an α and a β subunit and an
activator—each expressed from different genes. Proper
binding of this complex to the ganglioside causes hydrolysis
between N-acetylgalactosamine and galactose (Fig. 8-7). A Figure 8-8. A cherry-red spot (arrow) in Tay-Sachs disease
mutation of the α subunit leads to a deficiency of hexosa- is due to glycolipid deposits in ganglion cells everywhere
minidase A activity. The mutation has an autosomal recessive except in the macula, where there are no ganglion cells.
mode of inheritance. (From Digre K, Corbett JJ. Practical Viewing of the Optic
Disc. Philadelphia, Butterworth Heinemann, 2003, p 518.)
Normal at birth, these children develop mental and physical
deterioration, blindness, deafness, and muscle atrophy fol-
lowed by paralysis. A cherry-red spot of the macula is present have similar presentations; however, individuals with Sand-
in Tay-Sachs just as in other lipid storage diseases (Fig. 8-8). In hoff disease have hepatosplenomegaly and the disease is not
this classic presentation that begins around the fifth or sixth predominant in any particular population.
month, death usually occurs by age 5. Tay-Sachs disease is
common in the Ashkenazi Jewish population and French Cana- Fabry Disease
dians. Ninety-eight percent of Tay-Sachs cases result from one Among the enzymes in the sphingolipid degradation pathway,
of three mutations in the Ashkenazi Jewish population, thus only α-galactosidase A is inherited in an X-linked manner,
providing a basis for carrier screening within this population. and mutations in the cognate GLA gene are specific for Fabry
Sandhoff disease results from a mutation in the β subunit disease (see Fig. 8-6). Over 300 GLA mutations have been
and thus causes a deficiency in hexosaminidase A and hex- characterized in affected males. The substrate for this enzyme
osaminidase B complexes, the latter being composed of two is globotriaosylceramide, a cerebroside containing glucose
β subunits and an activator. Tay-Sachs and Sandhoff diseases and galactose. Mutations in the enzyme cause substrate accu-
mulation within endothelial cells, pericytes, smooth muscle
cells, renal epithelial cells, myocardial cells, peripheral nerves,
and dorsal ganglia. The most conspicuous features of Fabry
disease are angiokeratomas (Fig. 8-9) and acroparesthesia
with recurrent episodes of severe neuropathic pain. Burning
pain occurs in the distal extremities, particularly in the palms
αβ
GM2 activator protein and soles of the feet. These episodes of pain may last from a
few minutes to days and usually occur during exercise, emo-
tional stress, fatigue, or rapid changes in temperature or
humidity. Pain can be excruciating, and it is presumed that
the acroparesthesia results from deposition of sphingolipid in
GM2 ganglioside small vessels supplying blood to the peripheral nerves.

PATHOLOGY
GalNAC-Gal-Glu-Cer
Angiokeratoma Corporis Diffusum
NANA Angiokeratoma corporis diffusum is the hallmark of Fabry
Cleavage site
disease, causing red to blue-black cutaneous vascular
lesions. Early lesions may be small and non-hyperkeratotic,
Figure 8-7. Hexosaminidase A complex is composed of but they tend to increase in number and size with age and
three proteins: the α subunit, the β subunit, and an activator. are nonblanching with pressure. Lesions may occur
A mutation in the α subunit results in Tay-Sachs disease. Cer, anywhere but tend to concentrate between the umbilicus
ceremide; Gal, galactose; GalNAC, N-acetylgalactosamine; and the thighs; they rarely occur on the face, scalp, or ears.
Glu, glucose; NANA, N-acetylneuraminic acid.
 Single-Gene Disorders 139

Figure 8-9. A, Angiokeratomas are

characteristic dark red to blue-black
angiectases. In Fabry disease, they are
often found between the umbilicus and
thigh. B, They become larger and more
numerous with age. C, The whorled
corneal opacity is seen with a slit lamp
and does not affect vision. It is found in
almost all males and 70% to 90% of
female carriers of Fabry disease. (From
Desnick RJ, Brady R, Barranger J, et al.
Fabry disease, an under-recognized
multisystemic disorder: expert
recommendations for diagnosis,
management, and enzyme replacement
therapy. Ann Intern Med
2003;138:338–346.)

reduces or stabilizes general symptoms as well as stabilizing

NEUROSCIENCE renal deterioration and improving cardiac function. Treatment
should begin as early as possible in all males with Fabry
Pain disease, even those with end-stage renal disease, as well as in
Pain receptors, called nociceptors, are free nerve endings in female carriers with significant symptoms.
the periphery that initiate a pain sensation. Pain fibers enter
the spinal cord in the lateral region of the dorsal root and Gaucher Disease
divide into Lissauer’s tract. Synapses occur in the substantia Gaucher disease occurs from an inability to degrade a sulfa-
gelatinosa, located in the apical region of the posterior horn
tide to ceramide (see Fig. 8-6). A defect in glucocerebrosidase
of the spinal cord gray matter and extending the entire
causes an accumulation of glucocerebroside in the spleen,
length into the medulla oblongata. Pain sensation is
transmitted via a second-order neuron in the lateral
liver, lung, and bone marrow, and occasionally in the brain.
spinothalamic tract and ascends in the contralateral Glucocerebrosides are a component of cell membranes, as are
quadrant of the spinal cord. From there it travels to the sphingolipids in general, and are released when cells are
ventral posterolateral nucleus of the thalamus. A third-order degraded; in the brain, glucocerebrosides arise from complex
neuron travels to the postcentral gyrus of the cortex for lipid turnover during brain development and the formation
conscious awareness of pain. of myelin sheaths. When this substrate is not degraded, it
accumulates in Gaucher cells, a type of histiocyte (Fig. 8-10).
The accumulation of Gaucher cells in bone marrow can cause
pain and even fractures. Hepatosplenomegaly occurs from
There are three forms of Fabry disease: classic, a cardiac accumulation in the spleen and liver, contributing to anemia,
variant, and a renal variant. In the classic form of the disease, bruising, and impaired clotting. Neurologic damage occurs
less than 1% of the enzyme is present. Cardiac and renal when Gaucher cells accumulate in the nervous system.
variants have greater than 1% activity. As expected, the most
severe clinical presentation occurs with the classic form, and
the life expectancy is 41 years. Onset of symptoms in the
classic form is during childhood (4–8 years) rather than adult-
hood, as seen with the cardiac and renal variants, where onset PATHOLOGY & HISTOLOGY
is generally after ages 25 and 40 years, respectively. The varia-
tions in the later-onset presentations often lead to delayed Gaucher Disease
diagnosis and missed diagnosis. As a result, a correct diagnosis Gaucher disease is a group of autosomal recessive
may not be made until more than a decade after the first disorders with mutations in the gene encoding
symptoms appear. It has been estimated that 4% of individu- glucocerebrosidase. This defect causes glucocerebroside to
als with hypertrophic cardiac disease may have the cardiac accumulate in massive amounts within phagocytic cells
variant of Fabry disease. Similarly, 1.2% of all dialysis patients throughout the body. These malfunctioning phagocytic cells
with end-stage renal failure have been shown to have the (Gaucher cells) are distended and found in the spleen, liver,
bone marrow, lymph nodes, tonsils, thymus, and Peyer
renal variant of Fabry disease.
patches. In severe cases, Gaucher cells may displace bone
The development of recombinant α-galactosidase A pro-
marrow, causing bone to become demineralized and
vides a treatment for Fabry disease worldwide. This enzyme weakened, a situation that can lead to fractures. In the lung,
effectively degrades the substrate and prevents accumulations these cells can lead to hypoxia, cyanosis, and clubbing.
leading to symptoms. Studies demonstrate that treatment
 140 Neurologic Diseases

There are three clinical forms: adult, infantile, and juvenile platelets, hepatosplenomegaly, bone pain, and aseptic necro-
(Table 8-2), also characterized as types 1, 2, and 3. The most sis (Fig. 8-11). Unlike in other lipidoses, there is no brain
common form of Gaucher disease is type 1. This is a chronic involvement.
non-neuropathic form and is responsible for 85% of cases. Type 2 Gaucher disease is an acute neuropathic disease.
The onset of symptoms may be early or delayed until adult- The clinical onset of hepatosplenomegaly is apparent by 3
hood. These include easy bruising and fatigue, anemia, low months of age. There is extensive and progressive brain
damage. Death occurs by age 2.
Type 3 Gaucher disease is a subacute neuropathic form
with variable hepatosplenomegaly. Brain involvement, such
as seizures, is gradual. Other than the neurologic symptoms,
symptoms in type 3 may be similar to those in type 1.
Life expectancy is generally three to four decades without
treatment.

B Figure 8-11. Children with type 1 Gaucher disease will

develop hepatosplenomegaly early in childhood. Many will
Figure 8-10. Gaucher cells in bone marrow (A) and spleen develop a characteristic “Erlenmeyer flask deformity” of the
(B). Note the fibrillar cytoplasm that looks like parchment or distal femur, reflecting thinned cortices and dilatation of the
crumpled tissue paper. (Courtesy of Dr. Jerome Tift, Mercer medullary cavity. (From GE Healthcare. Available at: http://
University School of Medicine.) www.medcyclopaedia.com.)

TABLE 8-2. Comparison of the Three Forms of Gaucher Disease*

TYPE SIGNS AND SYMPTOMS INCIDENCE LIFE EXPECTANCY

1, Adult form Anemia, bruising, fatigue, low platelets, 1 in 450 in Ashkenazi Jewish Variable; in general, the later
(most common) hepatosplenomegaly, bone pain and population in life symptoms appear,
aseptic necrosis, growth retardation the less severe the disease
2, Acute or Rapid, progressive development of 1 in 100,000—no ethnic focus 2 years
infantile form neurologic symptoms
3, Subacute or Slowly developing neurologic symptoms 1 in 100,000—no ethnic focus 3 to 4 decades
juvenile form beginning in childhood; other
symptoms similar to type 1

*There is also a perinatal form that is lethal and associated with skin abnormalities or with nonimmune hydrops fetalis. A cardiovascular form is characterized by
calcifications of the aortic and mitral valves, supranuclear ophthalmoplegia, corneal opacities, and splenomegaly.
 Single-Gene Disorders 141

Gaucher disease is an autosomal recessive disorder caused (see Fig. 8-6). Type A is the most severe form and is seen
by mutations in the gene for glucocerebrosidase (GBA). in the majority of individuals with Niemann-Pick disease.
Although approximately 200 Gaucher-associated mutations There is less than 1% of the normal enzyme in type A,
have been characterized within the GBA gene, only 4 variants resulting in feeding difficulties, an enlarged abdomen within
account for 90% of disease in the Ashkenazi Jewish popula- the first 3 to 6 months, a cherry-red spot, and progressive
tion. These same mutations account for 50% to 60% of muta- loss of motor skills. These individuals usually die by 2 to 3
tions in other populations. Type 1 affects 1 in 450 Ashkenazi years of age.
Jewish individuals and is the most common genetic disorder Individuals with type B have more enzymatic activity, gen-
affecting this population. Types 2 and 3 occur in 1 in 100,000 erally between 10% and 60%, and thus experience less
individuals and have no predilection for any particular severe symptoms. These symptoms may include hepato-
ethnic group. splenomegaly and respiratory problems that can lead to car-
Treatment has dramatically reduced the symptoms of diovascular stress, but there is little or no neurologic
Gaucher disease since the introduction of enzyme replace- involvement. These individuals may survive into late child-
ment therapies for types 1 and 3 Gaucher disease in hood or adulthood. In addition to types A and B, some indi-
1991. The administration of a recombinant analog for β- viduals may have an intermediate form of disease with more
glucocerebrosidase eliminates the symptoms but does not neurologic problems than type B but milder symptoms than
cure the disease. This therapy reduces the size of the liver and type A.
spleen, reduces skeletal anomalies, and normalizes blood cell Type C Niemann-Pick disease has a different genetic defect
counts. Therapy must be maintained for the life of the indi- that may lead to a secondary deficiency of sphingomyelinase
vidual or the symptoms will recur as substrate once again due to the large accumulation of lipids. The total sphingomy-
begins to accumulate. Bone marrow transplantation has been elinase may be normal but inadequate. Two genes are respon-
used in some patients to replace hematopoietic cells; however, sible for type C—NPC1 and NPC2. NPC1 is a member of a
the risks of graft-versus-host rejection may be high. Enzyme gene family of membrane-bound proteins with a sterol-
replacement therapy is a better choice of treatment. There is sensing domain. Mutations in NPC1, which occur in 95% of
no treatment for the severe brain involvement seen with type C disease, disrupt transport of cholesterol and other
types 2 and 3. Treatment can increase the life expectancy of lysosomal products, leading to neuronal degeneration.
individuals significantly, and it may even approach normal. The second gene, NPC2, produces a cholesterol-binding
However, while Gaucher disease is the most common lyso- protein. Mutations in this protein decrease cholesterol binding,
somal storage disorder in the general population, lifelong causing it to accumulate in the cell. NPC1 and NPC2 there-
enzyme replacement therapy is expensive and not an option fore work together to transport lipids. Mutations in NPC1
for every family. Yet without this therapy, the options are prevent proper transport of lipids out of the cell, whereas
limited. mutations in NPC2 affect lipid movement within the lyso-
some and perhaps between lysosomes and other organelles
such as the endoplasmic reticulum.
Cholesterol enters the cell normally, but in Niemann-Pick
IMMUNOLOGY disease cholesterol and other unmetabolized lipids accumu-
late in cells of the liver, spleen, and brain. Infants often
Complications of Bone Marrow Transplantation have jaundice at or shortly after birth, followed by the
Graft-versus-host (GVH) reaction is the major complication progressive development of hepatosplenomegaly, ataxia, dys-
of bone marrow transplantation. The reaction occurs when tonia, dysarthria, and dementia. Vertical supranuclear gaze
immunocompetent cells contained in the bone marrow graft palsy is highly suggestive of type C. Individuals with type
recognize the host tissue as foreign and initiate rejection. C disease generally die before age 20.
During the afferent phase of the disease, alloreactive
donor T cells recognize major and minor histocompatibility
antigens of the host. The efferent phase follows, and
inflammatory effector cells are activated. Cytopathic
molecules, including cytokines, are also secreted and
induce pathology in the skin, gastrointestinal tract, liver,
NEUROSCIENCE
lung, and immune system. GVH reaction symptoms include
Vertical Supranuclear Gaze Palsy
rash, jaundice, hepatosplenomegaly, diarrhea, and infection.
Cyclosporine may be used to decrease the GVH reaction. Supranuclear and internuclear pathways are involved with
voluntary conjugate eye movement. “Nuclear” refers to
cranial nerve nuclei. The pathways originate in the middle
frontal gyrus and require bilateral activation of nuclei for
movement. The principal pathway controlling coordinated
Niemann-Pick Disease
eye movement is the internuclear pathway, which is linked to
Niemann-Pick disease is an autosomal recessive group of
the third, fourth, and sixth cranial nerve nuclei via the medial
related neurologic disorders designated as A, B, and C. Both longitudinal fasciculus. The supranuclear pathway feeds into
A and B have mutations in sphingomyelinase, causing sphin- the medial longitudinal fasciculus.
gomyelin to accumulate rather than be degraded to ceramide
 142 Neurologic Diseases

Some populations are more affected than others with a In the infantile form of Krabbe disease, a 30-kb deletion is
specific type of Niemann-Pick disease. For example, types A responsible for 45% and 35% of cases occurring in individu-
and B occur more frequently in the Ashkenazi Jewish popula- als of European and Mexican ancestry, respectively. This
tion. Type B is also prevalent among North African popula- differs from late-onset disease, which often has a G809A
tions. Type C is more likely to occur among Hispanic mutation. Compound heterozygotes with both mutations
Americans in southern New Mexico and Colorado. demonstrate late-onset disease.

Krabbe Disease Mucopolysaccharidoses

Krabbe disease results from a mutation in galactocerebro- Mucopolysaccharidoses (MPSs) are caused by excessive intra-
sidase (see Fig. 8-6). This enzyme is responsible for the lysosomal accumulation of glycosaminoglycans, specifically
degradation of galactocerebroside to ceramide and of galac- heparan sulfate and dermatan sulfate (Table 8-3). Mucopoly-
tosylsphingosine, also known as psychosine, to sphingosine. saccharides, more appropriately called glycosaminoglycans
Galactocerebroside is a major component of myelin. Myelin (GAGs), are unbranched polysaccharides made up of repeat-
is not abnormal in these individuals, but the enzyme defi- ing disaccharide units that may be sulfated. Glycosaminogly-
ciency causes an accumulation of substrates. This is par- cans linked to protein are called proteoglycans, the major
ticularly critical during the first 18 months of life when component of ground substance in connective tissues (see
myelin formation and turnover are high. It is the increased Chapter 7). Similar to the sphingolipidoses, defects in lyso-
galactosylsphingosine levels that are toxic and lead to the somal enzyme activity result in the accumulation of partially
destruction of oligodendroglia and impaired Schwann cell degraded molecules; however, rather than sphingolipids,
function in the CNS and to demyelination. The undegraded GAGs accumulate and are secreted in urine.
substrates accumulate in multinucleated macrophages in
demyelinated regions of the brain. These “globoid” cells
characterize this disease, which is also known as globoid
cell leukodystrophy. BIOCHEMISTRY
Proteoglycans
Proteoglycans are found in the extracellular matrix and on
cell surfaces. These large and complex molecules consist of
BIOCHEMISTRY
a protein core with covalently bound glycosaminoglycans
Psychosine (GAGs) or mucopolysaccharides. Several types of GAGs
are dermatan sulfate, heparan sulfate, and chondroitin
Galactosphingosine, also called psychosine, is a constituent sulfate. These GAGs are removed from the protein core in
of cerebrosides. It is synthesized by direct galactosylation of lysosomes by specific enzymes. Defects in any of these
sphingosine. The accumulation of psychosine leads to the enzymes lead to an accumulation of GAG metabolites, with
formation of “globoid cells” that may lead to cytotoxity of clinical consequences.
oligodendrocytes. These multinuclear, globular giant cells
are derived from macrophages and microglia. Although the
complete mechanism is still unclear, it has been shown that
psychosine acts through a G protein–coupled receptor to
block cytokinesis but not mitosis. The observation of There are at least 14 known types of lysosomal storage
cytoplasmic filamentous structures suggests that psychosine diseases affecting glycosaminoglycan degradation. It is the
affects vesicle transport, actin reorganization, or both, accumulation of GAGs, or mucopolysaccharides, that leads
resulting in abnormal cytokinesis. to the commonly associated clinical features of coarse facies,
thick skin, corneal clouding, and organomegaly. The accumu-
lation also results in defective cell function represented by
mental retardation, growth deficiencies, and skeletal dyspla-
Symptoms begin at 3 to 6 months of age in the classic sias. Because GAGs are such important constituents in the
infantile form (85% to 90% of cases) with irritability, unex- extracellular matrix, hernias and joint contractures may also
plained crying, fever, limb stiffness, seizures, feeding difficul- be seen. As noted, the most commonly known disorders of
ties, vomiting, and decreased mental and motor development. GAG degradation involve heparan sulfate and dermatan
Unlike in the other sphingolipidoses, lipid storage clinically sulfate (Fig. 8-12). Among these Hurler syndrome, Hunter
differs in that the galactosylceramide and total brain lipids syndrome, Scheie syndrome, and Sanfilippo syndrome will be
are actually reduced because myelinogenesis fails following described (Table 8-4). Each of these, with the exception of
the loss of myelin-producing cells. As noted above, individual Hunter syndrome, is inherited in an autosomal recessive
globoid cells, however, accumulate galactosylceramide. As manner; Hunter syndrome is an X-linked recessive disorder.
may be anticipated from this description, Krabbe disease is a
disease of white matter because of the presence of myelin; MPS-I: Hurler, Scheie, Hurler-Scheie Syndromes
gray matter is relatively unaffected. Many mutations have The most severe of the mucopolysaccharidoses is Hurler syn-
been defined in the galactocerebrosidase gene. Some are drome, also known as MPS-IH. A mutation in iduronidase,
associated with a later onset and milder adult form. encoded by the IDUA gene, leads to the accumulation of
 Single-Gene Disorders 143

heparan and dermatan sulfate (see Fig. 8-12). The Hurler large hernias, respiratory infections, and thickening of the
phenotype—coarse facies, enlarged skull, corneal clouding, meninges, leading to decreased cerebrospinal fluid circulation
hepatosplenomegaly, thickened skin, hernias, and and increased cranial pressure.
contractures—becomes evident by the age of 1. Mental dete- Scheie syndrome is a milder form of MPS, designated
rioration is progressive. Heparan sulfate and dermatan sulfate as MPS-IS. Intermediate to Hurler and Scheie is MPS-IH/S.
accumulate in the coronary artery, leading to ischemia and MPS-IH and MPS-IS forms are allelic. The intermediate form,
cardiac insufficiency. Other problems are the potential for MPS-IH/S, is a compound heterozygote. The milder form has
no CNS involvement (Table 8-5).
Both enzyme replacement and bone marrow transplanta-
tion have been used to treat MPS-I. Recombinant α-L-
TABLE 8-3. Comparison of Heparan, Heparan iduronidase is available for treatment. Individuals with severe
Sulfate, and Dermatan Sulfate disease may be treated with bone marrow transplantation
(BMT) or umbilical cord blood transplantation to modify
GLYCOSAMINOGLYCAN* DISTRIBUTION disease progression and improve survival. This treatment may
improve some of the physical features and decrease the CNS
Heparan Component of intracellular deterioration. Because of the morbidity and mortality risks
granules of mast cells; associated with transplants, BMT is generally not used in
lining of arteries of the milder forms of the disease.
lungs, liver, and skin
Heparan sulfate Basement membranes,
components of cell MPS-II: Hunter Syndrome
surfaces MPS-II, also known as Hunter syndrome, is an X-linked dis-
Dermatan sulfate Skin, blood vessels, heart order. The wide spectrum of clinical severity correlates with
valves the amount of iduronate sulfatase activity present. Like
*Heparan sulfate contains more acetylated glucosamine than heparan, and
MPS-I, this is a progressive disorder. Typically, symptoms
heparan is more sulfated than heparan sulfate. begin between ages 2 and 4 years. Clinical features are similar

Figure 8-12. Structures of dermatan

Hurler-Scheie sulfate and heparan sulfate. Disease
occurs when these glycosaminoglycans
(2)
Dermatan sulfate IdUA GalNAc GlcUA GalNAc are not degraded and accumulate.
(1)
Hurler and Scheie syndromes are
Hunter allelic. Shown here are the resulting
OSO3H OSO3H OSO3H mucopolysaccharidoses that occur
when specific enzymes are absent or
have reduced activity. The numbers in
Hurler-Scheie Sanfilippo C Sanfilippo B parentheses refer to enzymes that
(2) (4) (6) hydrolyze the bonds: (1) iduronate
Heparan sulfate IdUA GlcN GlcUA GalNAc sulfatase; (2) α-L-iduronidase; (3)
(1) (3) (5) heparan N-sulfatase; (4) acetyl-CoA:α-
Hunter Sanfilippo A Sanfilippo D glucosaminide N-acetyltransferase; (5)
OSO3H OSO3H OSO3H N-acetylglucosamine-6-sulfatase; (6)
α-N-acetylglucosaminidase.

TABLE 8-4. Summary of Mucopolysaccharidoses

MPS TYPE DISEASE LIFE EXPECTANCY ENZYME DEFICIENCY ACCUMULATED PRODUCT

I Hurler <10 years (teens to 20s) α-l-Iduronidase Heparan sulfate and dermatan
Hurler-Scheie Normal expectancy sulfate
II Hunter 15 years (severe) Iduronate sulfatase Heparan sulfate and dermatan
Normal expectancy sulfate

!
(mild)
IIIA Sanfilippo A Heparan N-sulfatase Heparan sulfate
IIIB Sanfilippo B α-N-acetylglucosaminidase Heparan sulfate
20 years
IIIC Sanfilippo C Acetyl-CoA:α-glucosaminide Heparan sulfate
N-acetyltransferase
IIID Sanfilippo D N-acetylglucosamine-6-sulfatase Heparan sulfate
 144 Neurologic Diseases

TABLE 8-5. Comparison of MPS-I Types

INTERMEDIATE
SEVERE FORM FORM MILD FORM

Hurler Hurler-Scheie Scheie

MPS-IH MPS-IH/S MPS-IS
• Severe • Normal or near- • Normal
developmental normal intelligence intelligence
and mental • Respiratory • Less
delay disease progressive
• Severe • Obstructive airway physical
respiratory disease problems
disease • Joint stiffness, • Corneal
• Obstructive contractures clouding
airway disease • Skeletal • Valvular
• Progressive abnormalities disease of
• Decreased vision the heart

to those in MPS-I. Atypical retinitis pigmentosa or retinal

degeneration may occur without the corneal clouding seen in
MPS-I. Progressive loss of hearing also occurs. Figure 8-13. Mucopolysaccharidosis is accompanied by
many skeletal abnormalities. Oval-shaped vertebrae and
hook-shaped spinous processes result from defective
MPS-III: Sanfilippo Syndromes development, leading to a gibbus deformity and lumbar
There are four types of MPS-III; each results from a defect kyphosis. (From GE Healthcare. Available at: http://
in a different enzyme (see Fig. 8-12). The four enzymes www.medcyclopaedia.com. Used with permission.)
affected are heparan N-sulfatase, α-N-acetylglucosaminidase,
acetyl-CoA:α-glucosaminide N-acetyltransferase, and N-
acetylglucosamine-6-sulfatase. These enzymes participate in
ANATOMY
the degradation of heparan sulfate but not of dermatan
sulfate. As expected, heparan sulfate accumulates in tissues Dysostosis Multiplex
and is found in the urine of these individuals (see Table
Mucopolysaccharidoses have clinical and radiologic features
8-4). Sanfilippo type A, MPS-IIIA, has the most severe known as dysostosis multiplex. These features include the
presentation; types IIIB to IIID have decreasing severity. following:
The physical changes of Sanfilippo syndromes tend to be ■ Thickened calvaria
mild when compared with MPS-I and MPS-II. Infants are ■ J-shaped sella turcica

normal at birth. The first changes may appear in a child ■ Dolichocephalic skull due to premature sagittal suture

between the ages of 2 and 6 years, presenting as develop- fusion

■ Malformed teeth and flattened mandibular condyles
mental delay, particularly of speech, and behavioral changes
■ Oval vertebral bodies
such as sleep disturbances, short attention span, and impul-
■ Pelvic abnormalities such as an underdeveloped
siveness. The facies are coarse with thickened eyebrows that
acetabular region leading to coxa valga
meet, also called synophrys. Individuals may also have
■ Wide ribs
coarse, thick hair that is often blond or light brown. Diarrhea ■ Short, wide clavicles
is common. ■ Delayed epiphyseal ossification and cortical thinning
Skeletal changes are similar to those seen in MPS-I. Typi- ■ Abnormal carpal and metacarpal development
cally, the calvarium is thickened, vertebral bodies are oval ■ Abnormal taper of distal radius and ulna
shaped (Fig. 8-13), and hypoplasia of the pelvis may occur. It
is not uncommon for individuals with MPS-III to be shorter
than the mean. Joint stiffness is not uncommon, and in some
individuals contractures may occur.
Triplet Repeats
Intellectual changes become evident, followed by mental The mechanism of triplet repeat expansion and inheritance
retardation, progressive neurologic disease, and severe CNS was discussed in Chapter 3. As noted, the expansions are
degeneration. Eventually the individual is unable to speak parent-of-origin specific and occur within or near particular
and progressively loses all motor skills. Life expectancy is genes. Two examples will be discussed to better illustrate
about two decades, although BMT treatment may extend these features in neurologic disease: fragile X syndrome and
this. Huntington disease.
 Single-Gene Disorders 145

gene results in the expression of fragile X syndrome. This

Fragile X Syndrome protein has been called FMRP for “fragile-X mental retar-
Fragile X syndrome is the most common type of inherited dation protein.”
mental retardation and has been called the second most
common chromosomal cause of mental retardation after
Down syndrome; however, it is questionable whether this
disorder should be classified as having a chromosomal etiol- BIOCHEMISTRY
ogy in the usual sense. Fragile X is not caused by nondisjunc- Methylation
tion, chromosomal translocations, or deletions. Rather, the
Methylation of DNA occurs primarily at cytosines of CpG
DNA is greatly expanded at one particular region of the DNA
dinucleotides, called CpG islands. About 70% of CpG
on the X chromosome. It is inherited just as other genes are sequences are methylated to form 5-methylcytosine, and
by independent assortment. However, the expansion, or this occurs on both strands. Methylation of CpG islands in
amplification, resulting in fragile X occurs during maternal promoter regions correlates with a lack of transcription. It
meiosis. No change occurs to the expanded region in males. also correlates with deacetylation of histones and decreased
Expression of the gene affected by amplification is dependent transcription.
upon the extent of amplification. Methylation plays an important role in imprinting.
DNA analysis revealed an unstable region of DNA char-
acterized by a long, repeating sequence of three nucleotides:
CGG. The CGG repeat localizes with the brain-expressed
fragile-X mental retardation gene, or FMR1 gene. Four BIOCHEMISTRY
categories of CGG repeats are recognized: normal, inter-
mediate, premutation, and full mutation. Normal alleles FMRP Protein
range from approximately 5 to 44 and are stably transmit- FMRP protein transports mRNAs to the cytoplasm for
ted without a change in number. There is a threshold of translation. It is very abundant in the brain, and contains two
CGG repeat copies (about 44–58 copies, or about 50 on receptor-binding sites known as the nuclear localization
average) at which the DNA segment becomes prone to signal (NLS) and the nuclear export signal (NES).
DNA expansion. Repeats within the intermediate range Once FMRP mRNA is translated in the cytoplasm, an NLS
(approximately 45–58 repeats) are usually transmitted stably receptor binds to the protein and allows it to return to the
by females with occasional minor increases or decreases in nucleus. This FMRP-NLS complex then binds an NES
the repeat number. Premutation alleles range from approxi- protein. The complex interacts with specific mRNAs, which
bind to the NES to become a messenger ribonuclear protein
mately 59 to 200 repeats and are not associated with mental
(mRNP). The mRNP is transported to cytoplasm, where it
retardation. Premutations may become full mutations during
binds to the 80S ribosome for translation.
oogenesis, but remain stable during spermatogenesis. Women
with alleles in this range are at risk for having affected
children owing to the instability of the alleles. The upper
limit of the premutation range is sometimes noted as approxi- Males affected with fragile X syndrome have moderate to
mately 230 repeats. Both 200 and 230 are approximations severe mental retardation and show definitive facial features
from laboratory data. Full mutations have more than 250 (Table 8-6). The classic physical phenotype of a fragile X male
repeats and often expand to several hundred to several includes a long narrow face, large and protruding ears, and a
thousand repeats. Once expansion extends beyond 200, prominent jaw (Fig. 8-14). Additional features include velvet-
hypermethylation of deoxycytidylate residues usually occurs. like skin, hyperextensible finger joints, and double-jointed
Any person with a full mutation—100% of males and as thumbs. There are other problems, which may be briefly men-
high as 50% of female carriers—will be mentally impaired, tioned, such as eye disturbances, otitis, orthopedic conditions,
whereas the risk is very low (about 3%) in carriers of a skin problems, and cardiac involvement (mitral valve pro-
premutation. lapse). As one might suspect, these phenotypic features are
The CGG repeat is located within the 5′-untranslated generally not observed until maturity.
region of the FMR1 gene. Loss of expression of the FMR1 A striking feature of most adult fragile X males is the
gene is associated with methylation. One region of particular enlarged testicular volume (macro-orchidism). Normal adult
note is characterized by a high density of cytosine phos- males have a mean testicular volume of 17 mL, whereas the
phate guanine (CpG) dinucleotides, belonging to a class testicular volume in fragile X patients ranges from 40 to
of regulatory sequences called CpG islands. These CpG 140 mL. Fragile X men are fertile and offspring have been
islands frequently identify the promoter regions of eukary- documented, but those with significant mental retardation
otic genes. DNA methylation in such regions generally rarely reproduce. The hormonal basis for the oversized testis
down-regulates expression of adjacent genes. It is likely is unclear; the levels of testosterone are normal, but there
that expression of the FMR1 gene is repressed by methyla- may be excessive gonadotropin stimulation.
tion of the CpG islands, since amplification of the CGG Despite being X-linked, fragile X syndrome can manifest in
sequences can down-regulate the production of FMR1 mRNA females. About 50% of heterozygous females show some
transcripts. The inability to produce a protein from the degree of mental impairment, and as many as 30% are
 146 Neurologic Diseases

mentally retarded (IQ range is 55–75). The prevalence of in which the fragile X allele is active. Hence, the fragile X
mentally impaired females is 1 in every 2000 to 2500. The mutation predisposes affected females to a characteristic cog-
high rate of heterozygous expression is unprecedented for nitive profile deficit. In addition to the neurologic features,
X-linked inheritance. The fact that carrier females may be recall that heterozygous females also have a different clinical
affected is consistent with random inactivation of one of the profile that includes premature ovarian failure and features of
two X chromosomes. If the active chromosome is the fragile fragile X–associated tremor/ataxia syndrome (FXTAS; see
one in the majority of the affected female’s brain cells, then Chapter 3).
brain function is affected, with results comparable to that of
fragile X males. It has been surmised that the degree of Huntington Disease
mental retardation is correlated with the percentage of cells Huntington disease (HD) is a progressive disorder of motor,
psychiatric, and cognitive functions resulting from selective
neuronal death. It is a particularly devastating disease for the
patient and family because there is no cure and because
TABLE 8-6. Predominant Clinical Features of symptoms of HD usually begin in midlife between the ages
Fragile X Syndrome in Males of 35 and 45. This later age of onset is significant, since the
mutated allele may have already been passed on to offspring
Prepubertal Postpubertal before symptoms begin to appear. It is inherited as an auto-
Delayed developmental Mental retardation somal dominant disease with complete penetrance. Mean
milestones Pronounced craniofacies
survival time after onset of symptoms is 15 to 18 years.
Sit alone, 10 months Macro-orchidism
Walk, 20.6 months Like fragile X syndrome, HD results from the expansion of
First clear words, 20 months Additional Features a triplet repeat. Fragile X and Huntington disease differ by
Developmental delay Strabismus the triplet that amplifies—CGG for fragile X and CAG for
Abnormal behavior Joint hyperextensibility Huntington—and the number of repeats necessary for patho-
Tantrums Mitral valve prolapse
Soft, smooth skin genesis. The number of CAG triplets normally present in the
Hyperactivity
Autism HTT (Huntington) gene is 10 to 26. Unlike the fragile X
Mental retardation: IQ 30–50 repeat that expands during maternal gametogenesis and lies
Abnormal craniofacies outside the gene, the HD repeat is found in the first exon of
Long face the gene and is inherited from the father. The disease displays
Prominent forehead
anticipation, in which a larger expansion is associated with
Large ears
Prominent jaw an earlier onset of the disease and more severe symptoms.
De novo mutation of this gene has never been reported.

Rights were not granted to include this figure in electronic media. Please refer to the printed publication.

Figure 8-14. Fragile X syndrome. A, Two-year-old male with a full mutation exhibiting a relatively normal appearance with an
elongated face and prominent ears; also note tapering fingers, a minor anomaly. B, At age 5 years, his head is large with large
ears and a prominent jaw. C, At age 22 years. (From Scriver CR, Beaudet AL, Sly WS, et al. Metabolic and Molecular Bases of
Inherited Disease, 8th ed, Vol 1. New York, McGraw-Hill, 2000, p 1269, Fig. 64-7.)
 Complex Diseases of the Brain 147

The initial effects of the disorder are subtle, with minor

difficulties of coordination, slight adventitious movements
of the fingers, and abnormal darting eye movements. The
motor disturbances are generally accompanied by behavioral
changes, including chronic depression, impulsiveness, and
irritability. Until the diagnosis is made, individuals with
this disease can be mistakenly thought to abuse alcohol
or drugs. The movement disorders gradually worsen, with
more frequent and exaggerated chorea and abnormal pos-
turing. Speech becomes disturbed, and memory is disrupted.
Depression and apathy are heightened. Extreme weight loss
occurs. Eventually, the victim of HD is totally incapacitated
and the choreic movements give way to dystonia and
rigidity.
The expansion of CAG-encoded polyglutamines is a gain-
of-function toxic mutation. This is better understood with
Wolf-Hirschhorn syndrome, which results from a terminal
deletion of chromosome 4; HTT can be deleted on one chro- Figure 8-15. Huntington disease brain showing reduced
mosome but without progression to the development of HD. caudate nucleus (arrow) and abnormal lateral ventricular
angle (arrowheads). (Courtesy of Dr. Jerome Tift, Mercer
There is strong support suggesting that cleavage differences
University School of Medicine.)
between normal and mutant proteins are important in the
pathogenesis of the disease. The formation of intranuclear and
intracytoplasmic inclusions are hallmarks of the disease.
Tools for Behavioral and
These huntingtin aggregates are seen in brains of HD patients,
Psychiatric Studies
and the density of the aggregates is associated with increased
length of repeats. For many years the study of behavior has been very much a
Normal HTT has been associated with many functions. quantitative science, and the classic methods employed by sci-
Found predominantly in the cytoplasm, some is also found entists are twin and family studies to associate the degree of
in the nucleus; it is associated with the cell membrane relatedness with behavioral expression. These studies consis-
endocytic and autophagic vesicles, endosomes, endoplasmic tently demonstrate that complex disorders, such as Alzheimer
reticulum, Golgi apparatus, mitochondria, and microtubules. disease, Parkinson disease, schizophrenia, and bipolar affec-
As an example of these actions, HTT normally stimulates tive disorder, run in families. They remain excellent tools for
the production of brain-derived neurotrophic factor (BDNF), studying complex diseases caused by a constellation of predis-
important in striatal neuron survival. BDNF gene transcrip- posing variant alleles acting in concert with environmental
tion occurs in the cerebral cortex, and BDNF is then trans- influences to convert a vulnerability to a personality disorder.
ported to striatal targets. In the presence of mutant protein,
BDNF transcription is decreased and less is available in the Twin Studies
striatum. This mechanism may explain increased neuronal Twin studies are based on the genetic identity that occurs in
toxicity and progressive death of medium spiny GABAergic monozygotic (MZ), or identical, twins and the dissimilarity
neurons of the striatum and in the deep layers of the that occurs between dizygotic (DZ) twins, also called non-
cortex. In later stages, degeneration extends to a variety of identical twins. In the latter, the genetic similarity is similar
brain regions, including the hypothalamus and hippocampus to that of siblings. The basic assumption in these studies is
(Fig. 8-15). that differences in gene expression result in phenotypic
behavioral differences between the twins, since the prenatal
and postnatal environments are either identical or very
●●● COMPLEX DISEASES similar. Comparisons provide estimates of genetic variability
OF THE BRAIN for a particular trait.
The earliest twin studies focused on intelligence, mental
Single-gene disorders follow the rules of mendelian seg- illness, and personality. Perhaps the most startling finding
regation and inheritance. The special characteristics of triplet is that MZ twins are more than twice as similar as DZ
repeat amplification provide better explanations for inheri- twins. This finding led to three laws of behavioral genetics,
tance and the pathogenesis of gene action. For many presented by Irving Gottosman and Eric Turkheimer:
common disorders, however, inheritance patterns are more First Law: Behavioral traits are heritable.
complex, and the genes affecting susceptibility and disease Second Law: The effect of being raised in the same
conditions are more slowly identified. It is expected that, family is smaller than the effect of genes.
for diseases such as Parkinson disease and Alzheimer disease, Third Law: A substantial portion of variation in complex
multiple and quite possibly a combination of genetic and behavioral traits is not accounted for by the effects of
environmental factors may be involved. genes or families.
 148 Neurologic Diseases

Although seemingly straightforward, each of these laws actu- siblings, then they should have 50% of their genes in common.
ally interacts with each other in ways that are not fully MZ twins have 100% of their genes in common. The findings
understood. What is becoming clearer is that even minor of twin studies demonstrate that DZ twins have less than
variations in allele expression, both temporally and physi- 50% of their genes in common, since MZ twins cannot have
cally, may affect function and alter the cascade of following more than 100% of their genes in common. An explanation
events in profound ways. Overall, however, studies demon- for this and similar observations has been less than satisfac-
strate the robustness of genetics among twins, and that twins tory, but certainly discussions about the role of the cellular
reared apart are about as similar as twins reared together. environment during chromosome assortment and gamete for-
mation were prompted.
Adoption Studies Epigenetics is the study of changes in gene activity that
Adoption studies are important to understanding the effects do not involve alterations to the genetic code but that are
of environment on inherited traits because they address the still passed to successive generations. It is through epigenetics
role of genes from biological parents along with differences that environmental factors such as diet, stress, and prenatal
encountered in growth and development resulting from the nutrition imprint genes passed from one generation to
environment the child is adopted into. Such studies have another. The most frequently studied epigenetic mechanisms
involved large cohorts and have demonstrated that adopted are DNA methylation and histone modifications. Studies of
children share a greater similarity with their biological parents human postmortem brain demonstrate that aging and
than with the siblings of their adoptive families. Alzheimer disease are associated with epigenetic dysregula-
tion. Other studies show that early exposure to toxins can
Association Studies alter methylation patterns of genes involved in Alzheimer
Association studies are a common tool for genetic epidemiol- disease that affect brain development, leading to observa-
ogy studies and employ both case-control studies and case- tions seen later during the disease. Twin studies also suggest
parent trio studies. More recently, they have also been used that epigenetic mechanisms mediate the risk for Alzheimer
to determine the frequency of a particular trait in a popula- disease.
tion occurring with a particular polymorphism; in other
words, they have been used as a mapping tool. It is possible
to use a population-based association study, and many of
Alzheimer Disease
these have been reported; however, there are far fewer prob- Alzheimer disease (AD) is typically a late-onset disorder with
lems with family studies, where variables that may alter symptoms appearing after the age of 65. The predominant
results can be minimized. The greatest difficulty with these clinical feature of Alzheimer disease is progressive dementia,
studies is ascertainment bias, in which there is a difference but the disorder is most readily identified by its neuropatho-
in the likelihood that affected relatives of the case individual logic effects evident in postmortem brain tissue—the forma-
will be reported compared with affected relatives of the con- tion of neuritic plaques containing insoluble spherical amyloid
trols. Family members of an affected individual are also more deposits and of intracellular neurofibrillary tangles composed
likely to know of other affected relatives than control fami- of the cytoskeletal protein termed tau (τ). At the protein level,
lies. Family members of an affected individual are also more protein phosphatase 2A is methylated normally; its hypo-
keenly aware of symptoms or presentations related to a methylation, which is an epigenetic effect, results in tau
disease in other family members than are those individuals hyperphosphorylation and the neurofibrillary tangles. The
with no affected family member. The relatives of affected degeneration and death of neurons in the hippocampus and
individuals are also more likely to respond to questionnaires portions of the cerebral cortex shackle the minds of victims
than relatives of controls. in confusion. Studies of the epidemiology of Alzheimer
The advent of molecular tools affected the study of behav- disease indicate that the disease is present in 2% of the
ior just as it did other areas of science. Now the focus has over-65 population and at least 8% of the over-85 population
turned more to the effect of gene expression on behavior. The worldwide. The prevalence of Alzheimer disease is lower than
greatest area of interest is the area of psychiatric genetics. expected because of the competing causes of death. That is
Tremendous efforts are being made in the areas of schizo- to say, many individuals destined to express geriatric genetic
phrenia and bipolar disorder—the two most intensely studied disease will not express symptoms because they fail to survive
psychiatric illnesses. However, the task of identifying suscep- to the age of onset.
tibility genes and gene interactions is extremely challenging Attention was initially focused on a small subset of clinical
and perhaps even daunting. cases in which the patients showed an early onset of symp-
toms in the fourth and fifth decades. Although appearing
Epigenetic Studies relatively early in life, the symptoms are clinically and neu-
In recent years, more attention has been directed toward rologically indistinguishable from the later-onset Alzheimer
environmental factors to explain situations that fail to comply disease. Since the early-onset patients are clustered in large
with mendelian and mathematical expectations for inheri- pedigrees, the early-onset form is generally referred to as
tance. Revisiting the finding mentioned above that MZ twins familial Alzheimer disease (FAD). In FAD families, the disor-
are more than twice as similar as DZ twins helps illustrate der segregates as an autosomal dominant trait over multiple
this. If DZ twins share the same percentage of genes as generations.
 Complex Diseases of the Brain 149

There is genetic heterogeneity in the etiology of FAD,

which means early-onset disease can be caused by mutations PHYSIOLOGY & BIOCHEMISTRY
in different genes. Four known genes—APP, PSEN1, PSEN2,
and APOE—contribute to Alzheimer disease, which is the APOE and Cardiovascular Disease
most commonly occurring dementia, affecting almost half of In general, lipoproteins are complex molecules that transport
all individuals with dementia. nonpolar lipids in an aqueous environment, and each
lipoprotein contains one or more apolipoproteins on its
APP Gene surface that have a variety of functional or structural roles.
Apolipoprotein E is the main apoprotein of intermediate-
The major proteinaceous constituent of neuritic plaques is
density lipoprotein (IDL) and remnants to low-density
amyloid β-peptide. This is a proteolytic fragment of the larger
lipoprotein (LDL) receptors and LDL receptor–related
transmembrane protein precursor β-amyloid precursor proteins. It is synthesized in the brain, spleen, lung,
protein (APP). This cell surface protein is expressed in many adrenals, ovary, kidney, and liver. The presence of APOE
cell types, with the highest expression in the brain. The cere- mediates the uptake of IDL by the liver and conversion of
bral cortex and limbic structures, which are particularly vul- IDL to LDL. Individuals who either lack APOE or are
nerable to senile plaque formation in Alzheimer disease and homozygous for variants that bind less efficiently to
Down syndrome, exhibit disproportionately large amounts of receptors will have elevated IDL and chylomicron remnants.
mRNA encoding APP. APP gene mutations are a rare cause These situations result in hypercholesterolemia and
of early-onset FAD, accounting for no more than 10% to 15% hypertriglyceridemia.
of cases.
The clinical hallmarks of FAD—amyloid plaques and neu-
rofibrillary tangles—also occur in Down syndrome (trisomy
21). Plaques and tangles appear as early as the third decade There are three variant alleles at the APOE locus—APOE
of life in Down syndrome patients and affect virtually all ε2, APOE ε3, and APOE ε4. These alleles result in different
patients by the fourth decade of life. However, in spite of protein isoforms and therefore can occur in different allelic
these similarities, there is no evidence of association of combinations, such as ε2/ε3 or ε3/ε3, reflecting the inheritance
chromosome 21 markers with late-onset AD. of one allele from each parent. Different allelic combinations
demonstrate differences in age-specific predisposition to AD
PSEN1 Gene (Table 8-7). Generally speaking, the ε4 allele increases the risk
The presenilin 1 (PSEN1) gene at chromosome 14q24.3 and decreases the age of onset of AD. In a study of 42 families
has been associated with FAD by linkage studies. More with late-onset AD, 91% of the AD individuals had ε4/ε4
than 70% of FAD pedigrees show linkage to this locus alleles; this combination normally occurs in only 2% of the
and not to the APP locus. This gene codes for a protein population. Table 8-7 also reveals that individuals with this
localizing mainly in the endoplasmic reticulum. PSEN1 genotype demonstrate symptoms earlier than those with a
mutations apparently disrupt Ca++ regulation in the endo- single ε4 allele. The other two alleles provide a protective
plasmic reticulum and result in impaired mitochondrial effect by apparently slowing the rate of the process leading
function, the formation of reactive O2 species, and an to AD.
increased vulnerability to apoptosis. This suggests that aber- The ε4 allele is not a reliable predictor that a person will
rant APP processing in cells with mutant presenilins may develop AD. In the United States, 64% of individuals with
be secondary to disturbed Ca++ homeostasis and increased AD have at least one ε4 allele. This is twice the frequency
oxidative stress. observed in the general population, and the remaining 36%
have no ε4 allele.
PSEN2 Gene
A second presenilin gene (PSEN2) is located at chromosome
1q31-242. It has a greater than 80% homology to PSEN1
and is localized to the same regions of the brain as PSEN1. TABLE 8-7. Five Common APOE Genotypes
Mutations in this gene are associated with less than 5% of and Mean Age of Onset of
FAD cases. Alzheimer Disease

APOE Gene AGE OF ONSET (YEARS)

PERCENTAGE
There is compelling evidence that the apolipoprotein E OF U.S.
(APOE) gene predisposes individuals to late-onset Alzheimer GENOTYPE Mean Range POPULATION
disease. This “susceptibility” locus is on chromosome 19. A
susceptibility gene is one that affects the risk of developing ε2/ε2 Unknown Unknown <1
a disease; it is not causal. The APOE protein product plays a ε2/ε3 >90 50 to 140 11
critical role in triglyceride-rich lipoprotein metabolism and ε2/ε4 80–90 50 to >100 5
ε3/ε3 80–90 50 to >100 60
cholesterol homeostasis. APOE is a known risk factor for
ε3/ε4 70–80 50 to >100 21
cardiovascular disease and was not initially considered as a ε4/ε4 <70 50 to >100 2
risk factor for AD.
 150 Neurologic Diseases

Oxidative insults associated with an age-related increased

frequency of mutations in mitochondrial DNA apparently
represent a risk factor in the pathogenesis of AD. Excess
O2 radicals result in a cascade of neuronal dysfunction,
including membrane alterations, perturbed calcium homeo-
stasis, vascular damage, and apoptosis. By reducing the
level of oxidative stress, the development and progression
of AD may slow.

BIOCHEMISTRY & PHYSIOLOGY

Adverse Effects of Oxygen
Figure 8-16. Parkinson disease midbrain showing
Oxygen is a biradical that can form toxic reactive oxygen decreased substantia nigra neurons (arrows) compared with
species (ROS), such as superoxide (O2−), hydroxyl radical normal (right image). (Courtesy of Dr. Jerome Tift, Mercer
(OH), and hydrogen peroxide (H2O2). Oxidative stress occurs University School of Medicine.)
when ROS are produced faster than they are removed,
particularly for the respiratory, cardiovascular, nervous,
and gastrointestinal systems. Damage arises from the
mitochondria, where O2− is the final acceptor of electrons
in oxidative metabolism. These ROS leak out of the PATHOLOGY
mitochondria into the cytoplasm and initiate oxidative
damage, or oxidative stress to tissue and cells in DNA, Lewy Bodies
proteins, and polyunsaturated fats. Lewy bodies are characteristically found within the
Oxidative stress caused by O2 toxicity may have adverse pigmented neurons of the substantia nigra. They are
effects on virtually every organ of the body. This has been abnormal aggregates of protein in nerve cells. The major
most extensively demonstrated in neonates, in whom O2 component of Lewy bodies is α-synuclein, which has an
toxicity has long been linked to retrolental fibroplasia and altered shape: it is changed from a β structure into
bronchopulmonary dysplasia. In adults, prolonged exposure filamentous sheets. The cells stain for α-synuclein and for
to hyperbaric O2 can cause CNS toxicity, atelectasis, ubiquitin, which conjugates with proteins prior to
pulmonary edema, and seizures. degradation. Lewy bodies are also found in dementia with
Lewy bodies, Alzheimer disease (Lewy body variant), and
Hallervorden-Spatz syndrome.

Parkinson Disease
One of the most common progressive degenerative diseases Underlying the complexity of Parkinson disease are seven
affecting individuals over the age of 60 is Parkinson genes implicated in its etiology. Four have an autosomal dom-
disease. Most of the causes of this disease are unknown; inant mode of expression, and three have an autosomal reces-
however, oxidative damage, environmental toxins, and sive mode (Table 8-8). Originally, these genes had other
accelerated aging processes have been discussed as possible abbreviations, but as they became more closely associated
contributors. As expected, the complex nature of Parkinson with Parkinson disease, the gene abbreviation was changed to
disease etiology makes diagnosis difficult and often impre- PARK. In addition to these loci, there are at least three sus-
cise until the disease has advanced. Diagnosis is based ceptibility genes. Most twin studies across a broad spectrum
on the clinical presentation of bradykinesia, tremor, and of ages and focusing on late-onset disease have been ambiva-
rigidity. lent in distinguishing a genetic influence. However, the best
Although most commonly associated with aging, Parkinson association indicates that disease onset before age 50 may be
has several forms: juvenile-onset disease occurring before better related to genetic factors, suggesting that environmen-
age 20 years, early-onset disease occurring before age 50 tal influences in association with other genes have a stronger
years, and late-onset disease occurring after age 50 years. association with later-onset disease.
Each of these is expected to have a different etiology, but It is important to note that clinical signs of bradykinesia,
they all share the cardinal feature of the disease: dopami- tremor, and rigidity may be associated with other neurologic
nergic neurons are lost in the substantia nigra and Lewy diseases that mimic true Parkinson disease. Included among
bodies are present in the remaining, intact nigral neurons these are Alzheimer disease, Lewy body dementia, progres-
(Fig. 8-16). As many as 80% of the dopaminergic neurons sive supranuclear palsy, and others. Parkinson disease may
may be lost before clinical symptoms are apparent in affected also accompany diseases such as Huntington disease, spino-
individuals. cerebellar ataxia, and Wilson disease. These co-occurrences
 Complex Diseases of the Brain 151

TABLE 8-8. Comparison of Several Genes Implicated in Parkinson Disease

MODE OF
GENE PROTEIN LOCATION INHERITANCE PATHOGENESIS

PARK1 α-Synuclein 4q21 AD Neurotoxic aggregates of α-synuclein

PARK2 (juvenile form) Parkin 6q25 AR Impaired protein degradation
PARK3 Unknown 2p13 AD Unknown
PARK5 Ubiquitin carboxyl-terminal 4p14 AD Impaired C-terminal hydrolysis of
esterase L1 ubiquitin
PARK6 PTEN-induced putative 1p36 AR Mitochondrial dysfunction
kinase 1
PARK7 DJ-1 1p36 AR Impaired oxidative stress response
PARK8 Leucine-rich repeat 12q12 AD Impaired interaction with parkin for
aggregate ubiquitination

AD, autosomal dominant; AR, autosomal recessive.

underscore the need for critical assessment of family history, TABLE 8-9. Prevalence Rates for Schizophrenia
laboratory findings, and individual presentation of symptoms
for correct diagnosis. The correctness of the diagnosis can
COUNTRY AFFECTED INDIVIDUALS*
have familial consequences for diagnoses and management of
other members of the family and future generations.
Australia 285,000
Britain 250,000
Schizophrenia Canada 280,000
China 6–12 million (estimate)
Schizophrenia is a neurologic disorder afflicting about 2.2 India 4.3–8.7 million (estimate)
million people in the United States and more than 51 United States 2.2 million
Worldwide 51 million (estimate)
million people worldwide (Table 8-9). It is characterized
by delusions, hallucinations, reduced interest and drive, *Approximately 1.1% of the population over 18 years of age.
altered emotional reactivity, and disorganized behavior.
Structural brain abnormalities observed in individuals with
schizophrenia include decreased volume, loss of gray matter
(Fig. 8-17), and ventricular enlargement. The clinical features 13%. When both parents are schizophrenic, the risk to their
are often not recognized until late in the second decade offspring rises sharply to 46%.
or early in the third. Retrospectively, cognitive and behav- The risk of siblings of a proband (affected child) is influ-
ioral signs are present from early childhood in individuals enced by the status of the parents’ mental health. When the
with schizophrenia. parents are free of schizophrenia, the risk to a sibling of an
Twin studies, family studies, and adoption studies all dem- affected individual is 9%. When one parent is also affected,
onstrate a strong genetic component to schizophrenia. These the risk increases to 17%.
studies show conclusively that the risk of schizophrenia Both classic and modern studies concur that the concor-
increases among close relatives. Figure 8-18 reveals the dance rate in MZ twins—the proportion of co-twins who are
morbid risk for relatives of schizophrenics. The morbid risk is affected—is 48% (see Fig. 8-18). This concordance rate holds
the probability that a person who survives through the period even when MZ twins are reared apart. This rate differs appre-
of greatest manifestation, in this case 18 to 45 years of age, ciably from that of DZ twins, which is 17%. The concordance
will develop schizophrenia. The morbid risk in the general rate in DZ twins is in good agreement with family studies
population is approximately 1%. This serves as a benchmark showing a 9% to 17% rate of schizophrenia among nontwin
for comparing the risk of schizophrenia in relatives of schizo- siblings. Therefore, as stated earlier, twin data favor a signifi-
phrenics. As shown, the risk for relatives is considerably cant genetic component in the etiology of schizophrenia, but
higher than for the general population, and the risk varies as since 52% of MZ twins are discordant, environmental factors
a function of the degree of genetic relatedness to the affected are also strongly implicated.
individual. The risk declines sharply as one moves from near Numerous studies have associated environmental factors
to distant relatives. with schizophrenia. The most common study design for these
In samples of parents of a schizophrenic child, the risk of studies is the case-control study in which individuals with
a given parent developing schizophrenia is 6%. This value is the disorder are compared with individuals not experiencing
of dual importance: the majority of schizophrenics do not the disorder. These groups are matched for as many variables
have affected parents, and the lack of schizophrenia in as possible, such as age, sex, race, weight, background for
parents cannot be used to exclude the diagnosis in the child. different diseases, and geographic area. Statistical analysis
The risk to a child of a schizophrenic parent is, on the average, demonstrates the strength of proposed associations. For
 152 Neurologic Diseases

Rate of Gray Matter Loss

Normal Adolescents Schizophrenic Subjects

Average
annual loss
Figure 8-17. Three-dimensional maps
of brain changes in childhood-onset
schizophrenia derived from high-
resolution magnetic resonance images.
A, Average rates of gray matter loss in
normal adolescents and in
schizophrenia reveal profound,
progressive gray matter loss in
schizophrenia. Average rates of gray
matter loss from 13 to 18 years in the
same subjects show severe loss (red
and pink; up to 5% annually) in parietal,
motor, and temporal cortices, whereas
inferior frontal cortices remain stable
(blue; 0 to 1% loss). Dynamic loss is
also observed in the parietal cortices of
normal adolescents, but at a much
A
slower rate. B, Deficits occurring during
the development of schizophrenia are
detected by comparing average profiles Early and Late Gray Matter Deficits in Schizophrenia
of gray matter between patients and Earliest deficit
controls at their first scan (age 13;
upper) and their last scan 5 years later
(age 18; lower). Although severe
parietal, motor, and diffuse frontal loss
Average
has already occurred (upper) and deficit
subsequently continues, the temporal
and dorsolateral prefrontal loss
characteristic of adult schizophrenia is
not found until later in adolescence Five years later (same subjects)
(lower), where a process of fast attrition
occurs over the next 5 years. The color
code shows the significance of these
effects. (Reprinted with permission from
Thompson PM, Vidal C, Giedd JN, et al.
Mapping adolescent brain change
reveals dynamic wave of accelerated STG DLPFC
gray matter loss in very early-onset
schizophrenia. Proc Natl Acad Sci
U S A 2001;98:11650–11655.) B

schizophrenia, previous associations include illegal drug use, specifically implicated in the schizophrenic phenotype,
winter and spring birth, pregnancy and delivery complica- including the basal ganglia, areas of the frontal lobe, Wer-
tions, delayed development, low IQ, immigration status, and nicke’s area, the occipital lobe, the hippocampus, and the
urban birth and domicile. The biggest disadvantage of case- limbic system (Fig. 8-20). As might be expected, these are
control studies is that they provide little information about the areas of intense investigation for candidate genes involved
the absolute risk of the disorder. in the pathophysiology of the disease.
The overarching implication of twin and family studies The mechanism of inheritance for schizophrenia is
along with case-control studies is that genes and environ- complex. The number of specific susceptibility loci, the risks
mental factors involved in the etiology of schizophrenia associated with these, and their interaction with each other
most likely affect neurologic development (Fig. 8-19). Many and other genes is clearly unknown. It is even possible
studies have substantiated the role of genetics and heritabil- that schizophrenia is not a single disorder but a group of
ity in brain morphology. In general, brains of individuals disorders with overlapping presentations that remain to be
with schizophrenia are smaller than brains of controls and clearly delineated. Several susceptibility genes have strong
cerebral ventricles are larger. Several areas of the brain are associations with schizophrenia.
 Complex Diseases of the Brain 153

12.5% General population 1%

3rd-degree First cousins 2%
relatives Uncles/aunts 2%
25% Nephews/nieces 4%
2nd-degree Grandchildren 5%
relatives Half siblings 6%
50% Parents 6%
1st-degree Siblings 9%
relatives Children 13%
Fraternal twins 17%
Children with one 17%
100% affected parent
Children with 46%
two affected parents
Identical twins 48%

Genes shared Relationship to 0 10 20 30 40 50

person with Risk of developing
schizophrenia schizophrenia Figure 8-18. Risks of schizophrenia for relatives of individuals
with schizophrenia.

Environment Different genes

Birth order
Parenting
Variation Variation
Reinforcement
caused by caused by
Peers
environment genes

Developmental interactions

Figure 8-19. Behavior is dependent

Behaviors on both genes and environmental
factors.

Occipital lobe
Disturbances can lead to
Wernicke's area difficulties interpreting
Overactivity can create complex images, recognizing
hallucinations motion, and interpreting
Basal ganglia emotions in others
Disturbances
contribute to paranoia
and hallucinations

Frontal lobe
Disturbances contribute to
difficulty in planning
actions and organizing
thoughts

Limbic system Hippocampus

Disturbances are thought Learning and memory
to contribute to agitation functions are impaired
Figure 8-20. Regions of the brain
affected by schizophrenia.
 154 Neurologic Diseases

DTNBP1 Gene Other Candidate Genes

The dystrobrevin-binding protein 1 gene (DTNBP1) is local- Several chromosomal regions have strong linkage and asso-
ized on chromosome 6p22.3. This protein, also known as ciations with schizophrenia. At least nine regions associated
dysbindin, has a strong association with schizophrenia by with schizophrenia have been identified; these include chro-
single nucleotide polymorphism (SNP) and haplotype asso- mosomes 6p22-p24, 6q21-q25, 1q42, 1q21-q22, 13q32-q34,
ciation studies. It localizes primarily in axon bundles, particu- 8p21-p22, 22q11-q12, 5q21-q33, and 10p11-p15. Two of
larly at termini, of mossy fiber synaptic terminals in the these are supported in multiple studies: chromosomes 8p and
cerebellum and hippocampus. This protein is ubiquitously 22q. Chromosome 8p21-22 has been discussed above with
expressed and binds to dystrobrevins seen in the dystrophin- neuregulin. Deletions of chromosome 22q11 account for
associated protein complex (see Fig. 7-13). Dysbindin is also associations in a smaller set of individuals with schizophrenia.
expressed in the hippocampus in the presynaptic termini of Candidate genes in this region include those for catechol-O-
glutaminergic pathways. In individuals with schizophrenia, methyltransferase (COMT), proline dehydrogenase (PRODH),
protein expression in the hippocampus is reduced and the and a zinc finger–DHHC domain (ZDHHC8). COMT partici-
expression of vesicular glutamate transporter proteins is pates in catecholamine degradation. A V158M mutation
increased. Thus, glutamate release may be decreased and may affects the activity and stability of the enzyme and is seen in
be related to decreased cognitive function. decreased frontal lobe function tests for some individuals.
Data are mixed for schizophrenic individuals but continue to
NRG1 Gene support this finding in certain populations. SNP haplotype
A strong association between the neuregulin gene and schizo- analysis (see Chapter 13) demonstrates strong association
phrenia exists according to several population studies. within specific populations for mutations in this gene.
However, different studies have demonstrated different The focus of many hypotheses about the etiology of schi-
haplotype associations. This gene is located at chromosome zophrenia and bipolar disorder is that there is a defect
8p21-22. This region is also deleted in individuals with velo- in GABAergic signaling. Studies demonstrate that down-
cardiofacial syndrome (VCFS), a disorder highly associated regulation of two genes, reelin (RELN) and glutamic acid
with an increased risk for schizophrenia. Twenty to 30% of decarboxylase (GAD1) in telencephalic GABAergic neurons
individuals with this disorder develop schizophrenia. The of schizophrenic patients correlates with increased expression
NRG1 gene spans more than 1.1 Mb and contains 21 alter- of a DNA methyltransferase (DNMT1) responsible for meth-
natively spliced exons that produce at least 14 isoforms. Neu- ylating cytosines in promoter CpG islands. Decreased expres-
regulins and their receptors, the ERBB protein kinases, are sion of RELN and GAD1 leads to decreased conversion of
essential for neuronal development and are important in glutamic acid to GABA. However, it is unclear whether these
expression and activation of neurotransmitter receptors such results are contributory to schizophrenia or a consequence of
as the glutamate receptors. Neuregulin signaling from the the disease.
axon is essential for Schwann cell survival, and these cells are
essential for axon maintenance.

DAO and DAOA Genes NEUROLOGY

The D-amino acid oxidase gene (DAO) and D-amino acid
oxidase activator gene (DAOA) are located at different loci— γ-Aminobutyric Acid (GABA)
chromosome 12q24 and chromosome 13q34, respectively— GABA is a neurotransmitter. It acts at inhibitory synapses in
but interact together. The exact functions of these two genes the CNS. Inhibition results from hyperpolarization of the
are unknown, but they are involved in glutamate receptor transmembrane potential when GABA binds to receptors. It
activation, and four specific SNPs are associated with schizo- is highly concentrated in the substantia nigra and globus
phrenia in case-control studies. pallidus nuclei of the basal ganglia, the hypothalamus, the
periaqueductal gray matter, and the hippocampus.
GABAergic neurons use GABA as a neurotransmitter.

BIOCHEMISTRY
D-Amino Acid Oxidase (DAO) Is
Another area of focus is the metabolism of dopamine by
a Peroxisomal Enzyme
catechol-O-methyltransferase in schizophrenia and bipolar
The reaction disorder, since an imbalance of dopamine is considered key
D-Amino acid + H2O + O2 → a 2-oxo acid + NH3 + H2O2 in the pathogenesis of psychosis. As noted above, COMT is
requires a FAD coenzyme and catalyzes oxidative
located in a region deleted in VCFS, a syndrome with increased
deamination of histamine, putrescine, or both. It is
risk for schizophrenia. This enzyme catalyzes the transfer
ubiquitous and highly expressed, especially in the brain,
where it oxidizes D-serine—an activator of N-methyl-D- of a methyl group from S-adenosylmethionine to catechol-
aspartate–type glutamate receptor. amines, including the neurotransmitters dopamine, epineph-
rine, and norepinephrine (Fig. 8-21). Genetic variation
 Complex Diseases of the Brain 155

COMT
Tyrosine Epinephrine Metanephrine
BH4+O2 S-adenosylhomocysteine
Phenylethanolamine
Tyrosine hydroxylase
BH2+H2O S-adenosylmethionine N-methyltransferase

MAO COMT MAO

DOPA Norepinephrine Vanillylmandelic acid

CO2 H2O
DOPA decarboxylase O2 Dopamine β-hydroxylase

Dopamine
MAO
COMT

3-Methoxytyramine 3,4-Dihydroxyphenylacetate

MAO COMT
Homovanillic acid

Figure 8-21. Biosynthesis and metabolism of dopamine. COMT, catechol O-methyltransferase; MAO, monoamine oxidase;
DOPA, L-3,4-dihydroxyphenylalanine.

producing reduced levels of this enzyme is also associated

with decreased prefrontal cortical function, a finding in TABLE 8-10. Classification of Bipolar Disorders
schizophrenia, bipolar disorder, attention-deficit/hyperactiv-
ity disorder (ADHD), panic disorder, phobias, obsessive- TYPE DESCRIPTION
compulsive disorder, and anorexia nervosa. Other similarities
or related dissimilarities are seen in studies of families with Bipolar I Mania and major depression
independently occurring schizophrenia and bipolar disorder. Bipolar II Hypomania and major depression
Chromosomal studies implicate similar abnormalities in both Bipolar III Cyclothymia
Bipolar IV Antidepressant-induced hypomania
disorders. Dopamine expression is affected similarly in both Bipolar V Major depression with a family history of
disorders, and several genes in the dopamine pathway are bipolar disorder
being investigated intensely. Likewise, the dopaminergic Bipolar VI Unipolar mania
pathway is of considerable interest to investigators for other
disorders. Along with Parkinson disease, which has already
been discussed, dopamine is hypothesized to play a role in
ADHD and Tourette syndrome as well. Both schizophrenia
and bipolar disorder demonstrate elevated levels of vesicular
monoamine transporter (VMAT2 protein; SLC18A2 gene) in
Bipolar Disorder
the brainstem. This protein regulates neurotransmitter trans-
port but is distributed differently in brains affected by the two Bipolar disorder, or manic-depressive illness, is a major illness
disorders. Its proper function is essential for correct activity characterized by mood swings between periods of mania, or
of the monoaminergic systems, and it is the site of action of hypomania, and depression. The two major forms of this
several drugs, including reserpine and tetrabenazine. Finally, disorder are bipolar disorder I and bipolar disorder II (Table
the left side of the hippocampus is larger in brains affected 8-10). The similarities have suggested to some that these dis-
with bipolar disorder, but the hippocampus is smaller in orders may be variations of the same underlying etiology;
schizophrenia-affected brains. however, sufficient differences exist to warrant separation
(Table 8-11).
Bipolar disorder affects 1 to 2 million people in the United
States per year and more than 121 million worldwide. It is
PHARMACOLOGY the most common psychotic disorder, affecting approximately
1% to 1.5% of individuals in all age groups. Several biologi-
Monoamine Oxidase (MAO) Inhibitors
cal factors are observed in bipolar disorder, but the role they
MAO inhibitors were the first drugs used as antidepressants. play is unclear. They include oversecretion of cortisol, an
They inhibit MAO and prevent catabolism of catecholamines.
efflux of calcium into brain cells, abnormal hyperactivity of
They work more rapidly than tricyclic antidepressants, which
the prefrontal cortical glutaminergic system of the brain, and
prevent uptake of norepinephrine and serotonin. MAO
inhibitors also block tyramine catabolism, which results in a change in circadian rhythm.
increased blood pressure. The effort to identify susceptibility genes for major
psychiatric disorders, including schizophrenia and bipolar
 156 Neurologic Diseases

because symptoms of these may be similar. For example,

TABLE 8-11. Distinguishing Features between bipolar disorder symptoms may be confused with ADHD in
Bipolar I and Bipolar II children. To complicate the diagnosis further, some of these
disorders may coexist, such as ADHD and bipolar disorder or
BIPOLAR I BIPOLAR II schizophrenia and bipolar disorder.
The first-degree relatives of individuals with bipolar I dis-
Manic At least one with or At least one episode order are seven times more likely to have the disease than
episodes without of hypomania and the general population (1% to 1.5%). Offspring of an affected
depression at least one parent have a 50% risk of having a major psychiatric disor-
In 60–70% of episode of major
cases, manic depression der. Studies have not implicated a specific gene association in
episodes Most function this disease, although a few have been suggested in a general
precede or follow normally between mechanism shared between several disorders such as schizo-
depressive episodes phrenia and bipolar disorder.
episodes More chronic course Association studies demonstrate that an increased inci-
Average of four than bipolar
episodes per I—more depressive dence of bipolar disorder occurs in individuals born during
year episodes, shorter the winter and with complications at birth. Bipolar disorder
Significant negative periods of wellness also occurs more frequently among individuals with higher
effects on social than bipolar I socioeconomic status. Additionally, there is an increased inci-
and work life Highly associated dence among individuals who lost a parent in early child-
Duration of 1 week with risk for suicide
to months, if hood. Diabetes is diagnosed three times more often in
untreated individuals with bipolar disease. Table 8-12 shows that not
Depressive Duration of 6–12 Difficult to distinguish
only is diabetes more prevalent in individuals with bipolar
episodes months, if between unipolar disorder, but migraines, suicides, hypothyroidism, and sub-
untreated (major) depression stance abuse are also commonly seen in these affected
and bipolar II individuals.
depression Disturbances in neurotransmitter function have been
Typically lasts 2–3
months
studied using several of the methods already described. A
Depressive episodes gene of particular interest has been that for the dopamine
tend to develop D4 receptor (DRD4) located on chromosome 11p15.5. This
gradually gene contains an unusual 48-bp repeat that is present 2
to 11 times, yielding different functional polymorphisms;
the most common allele contains four repeats (DRD4.4).
The gene is expressed in many areas of the body but is
highly expressed in the frontal area of the brain and nucleus
accumbens, areas that are associated with a lack of moti-
affective disorder, has been difficult and certainly lags behind vation and affective and emotional behaviors. Early associa-
other complex diseases such as diabetes mellitus and tion studies were often contradictory and highlighted the
Alzheimer disease, for which vulnerability genes have been issues apparent with small sample sizes. More recent meta-
identified. As noted above, advances in gene identification analysis studies, or the combination of data from other
are occurring with schizophrenia, but they have not pro- studies to yield a larger sample with stronger statistical
vided a clear picture of how the disease develops and power, demonstrate that the DRD4.2 polymorphism (the
progresses. The greatest obstacles to progress with identi- DRD4 gene with two 48-bp repeats) is significantly associ-
fication of genes involved in psychiatric disorders include ated with unipolar depression and combined unipolar and
the following: bipolar depression. Stated differently, individuals with the
● Incomplete penetrance and nonmendelian inheritance DRD4.2 allele have a higher risk of depression. The DRD4.7
● Inadequate definition of phenotypes allele is associated with ADHD and schizophrenia. How
● Etiologic heterogeneity and diagnostic comorbidity the dopamine receptor is regulated is not fully understood.
● Insufficient standards in valid diagnostic definitions However, one function of the receptor is the inhibition of
As a result of these types of challenges, many studies have adenylyl cyclase, causing a reduction in ATP-to–cyclic AMP
been unable to corroborate loci associated with the disease conversion. Some reports suggest the DRD4.2 receptor, as
that are easily supported in other studies. well as other allelic forms, functions less effectively than
Notwithstanding these difficulties, MZ studies strongly the normal receptor, resulting in a blunted intracellular
support a genetic component for about 60% of bipolar response to dopamine, and thus accounts for the clinical
disorder–affected individuals. The interpretation of data is features of depression.
complicated, however, by the co-occurrence of other psychi- Early-onset disease occurs in individuals with first symp-
atric diseases at a higher rate in families with bipolar disorder. toms occurring during childhood or adolescence. Disease
These include anxiety disorders, schizophrenia, ADHD, and in children differs slightly from adult disease. The initial
major depression. In fact, proper diagnosis may be masked events of early-onset disease are general depression that
 Complex Diseases of the Brain 157

TABLE 8-12. Commonly Seen Associations with Bipolar Disorders Compared with Prevalence
in the General Population

ASSOCIATIONS WITH PREVALENCE IN PATIENTS WITH

BIPOLAR DISORDER BIPOLAR DISORDER PREVALENCE IN GENERAL POPULATION

Diabetes 10–11% 3.4%

Suicide 15–20% overall with no treatment 0.01% (USA)
Bipolar I: >50%
Children: 25%
Migraines Bipolar I: 14% 11% overall (USA, western Europe)
Bipolar II: 77% 6% males
15–18% females
Substance abuse >60% 10% (U.S. adults)
Most commonly alcohol followed by 20% (estimated; physicians’ patients)
marijuana or cocaine
Hypothyroidism 10.4% (lithium-associated) 4.6% (USA)
4.5% males 2–5% (iodine deficiency worldwide)
14% females

develops later into bipolar I or bipolar II disorder. Manic

TABLE 8-13. Major Types of Mania
events usually begin at an average age of 18 years (Table
8-13). Early-onset disease is associated with a family
TYPE CHARACTERISTICS
history of bipolar disorder, a higher incidence of comor-
bidity, and a more severe disease in children than in
adults. Adult-onset disease may appear at age 40 or later. Hypomania Euphoria
Severe mania Euphoria, grandiosity, high levels of
It is also possible for adult-onset disease to occur after
sexual drive, irritability, volatility,
years of repeated unipolar major depression or to accom- psychosis, paranoia, aggression
pany other medical or neurologic problems. An example Extreme mania Most of the displeasures; few pleasures
is the development of bipolar disorder following a stroke.
Adult-onset disease is less likely to be associated with a
positive family history of bipolar disorder than is early- PHARMACOLOGY
onset disease.
Two effective treatments for bipolar disorder are the use of Lithium
lithium and valproate. These drugs up-regulate BCL2 expres- Lithium is effective in 60% to 80% of all hypomanic and
sion, which has an anti-apoptotic function, in the frontal manic episodes. It acts on signal transduction mechanisms
cortex and hippocampus. Since neuroimaging studies suggest such as G protein, glycogen synthase kinase-3β, protein
kinase C, adenylyl cyclase, and phosphoinositide hydrolysis,
cell loss in these regions of the brain, one hypothesis is that
to change neuronal signaling patterns.
bipolar disorder occurs from abnormal apoptosis in these
Lithium affects the function of NMDA receptors by
regions of the brain. Thus, lithium and valproate may stabilize regulating glutamate-induced calcium entry by an unknown
mood by stimulating alternative cell survival pathways and mechanism. Suppression of receptor function may occur by
increasing neurotrophic factors. inhibition of glycogen synthase kinase. Lithium is
concentrated in the thyroid by active transport against a
concentration gradient and inhibits secretion of T3 and T4
that can lead to hypothyroidism and goiter.

PHARMACOLOGY
Valproate
BIOCHEMISTRY Valproate is an antiseizure agent that is also effective for
many individuals with bipolar II disorder and mania. Some
B-Cell CLL/Lymphoma 2 (BCL2) studies show better compliance with valproate than with
There are 25 members of the BCL2 gene family, classified lithium. It increases GABA available to the CNS and
into two subgroups: prolongs recovery of inactivated Na+ channels. In neurons,
■ Anti-apoptotic: BCL2, BCLxL, and others it inhibits repetitive firing through interaction with voltage-
■ Proapoptotic: BAX, BAK, BOK, BID, BAD, and others sensitive Na+ channels. Valproate will also alter fatty acid
BCL2 regulates cytochrome c release and mitochondrial metabolism, impair β-oxidation in mitochondria, and disrupt
membrane permeability. the urea cycle, leading to hyperammonemia.
 158 Neurologic Diseases

later in onset and does not present with all of these symp-
KEY CONCEPTS toms. Sanfilippo syndrome is also an MPS disease in which
■ Neurologic diseases may have distinctive phenotypic findings infants are normal and presentation begins at around age 2.
suggestive of the disease, and these may or may not be pathog-
2. At age 2.5 months, an infant is diagnosed with San-
nomonic: café-au-lait spots, Lisch nodules, cherry-red spots of
the macula, angiokeratomas, macro-orchidism, and others.
filippo syndrome. A sibling died at age 4 with the
same disease 8 months after diagnosis. Which of
■ Lysosomal storage diseases are progressive as substrate accu- the following best describes the etiology of these
mulates in the brain, skeleton, heart, liver, and spleen. symptoms?
■ Two major classes of lysosomal storage diseases are sphingo-
A. FMRP is absent in the brain
lipidoses and mucopolysaccharidoses.
B. Gangliosides accumulate in nerve cells
■ Sphingolipidoses are caused by deficiencies of enzymes neces- C. Glycosaminoglycans accumulate in connective tissue
sary for degradation of sphingolipids in lysosomes. D. Hypoxanthine and guanine are not salvaged
■ Mucopolysaccharidoses are caused from the accumulation of E. Merlin accumulates in vestibular schwannomas
glycosaminoglycans—heparan sulfate and dermatan sulfate. Answer. C
The deficient enzymes are involved in degradation of these
molecules. Explanation: Sanfilippo disease is one of several MPS dis-
eases with a similar etiology. They are lysosomal storage
■ Triplet repeats may occur in the promoter region, exon, intron,
diseases. For Sanfilippo, one of four enzymes may be defi-
or 3′ noncoding region of a gene in a disease-specific manner.
All have neurologic manifestations.
cient leading to an inability to degrade heparan sulfate. For
Hurler, Scheie, and Hunter syndromes, both heparan sulfate
■ Triplet repeats amplify during germ cell development in a parent- and dermatan sulfate are affected. FMRP is the protein
of-origin–specific manner. affected in fragile X syndrome. Gangliosides accumulate in
■ Complex neurologic diseases have associations with more than nerve cells in gangliosidoses. Hypoxanthine and guanine
one gene and encompass environmental factors. are not salvaged in Lesch-Nyhan syndrome, an HPRT
■ Epigenetic factors, primarily through studies of DNA methylation deficiency. Merlin accumulates in neurofibromatosis 2.
and histone modifications, are being identified that alter disease
presentation several generations after the insult. 3. An apolipoprotein profile was determined for 100
■ Neurologic disease once considered “adult onset” can be identi-
patients with stage II peripheral vascular disease. A
fied much earlier (early teen years) for some diseases with subgroup of these patients was at risk for develop-
advanced technologies. ing Alzheimer disease but demonstrated no symp-
toms at the time of evaluation. Which of the following
apolipoproteins has the greatest predictive value for
this subgroup?
A. ApoAI
●●● QUESTIONS B. ApoB48
1. At age 6.5 months, an infant presents with physical C. ApoB100
and mental deterioration, difficulty in swallowing, D. ApoCIII
progressive loss of vision and hearing, and increas- E. ApoE4
ing hypotonia leading to paralysis. Which of the Answer. E
following should be considered in a differential
diagnosis? Explanation: Apolipoprotein E (ApoE) is associated with
late-onset Alzheimer disease. Among the APOE alleles,
A. Fragile X syndrome APOE ε2, ε3, and ε4 occur most often and APOE ε4/ε4 is
B. Hunter syndrome associated with the earliest onset. This same subset of indi-
C. Juvenile-onset Parkinson disease viduals (ε4/ε4) is at a higher risk for atherosclerosis. Indi-
D. Sanfilippo syndrome viduals with APOE ε2/ε2 are at a higher risk for cardiovascular
E. Tay-Sachs disease disease. ApoB48 and ApoB100 are isoforms. ApoB48 is
Answer. E limited to chylomicrons and ApoB100 is associated with
liver lipoproteins (very-low-density, low-density, and
Explanation: This child presents with symptoms of Tay-
intermediate-density lipoproteins). ApoC is related to plasma
Sachs disease. The important consideration for a differential
triglycerides. ApoAI is the major component of high-density
is the age of the child. Fragile X symptoms generally appear
lipoprotein.
at or near puberty and do not include these symptoms.
Infants with Hunter syndrome are normal at birth and symp-
toms are a progressive deterioration due to a lysosomal
storage disease in which mucopolysaccharides are not Additional Self-assessment Questions can be Accessed
properly degraded. Juvenile-onset Parkinson disease is at www.StudentConsult.com
Cardiopulmonary 9
Disorders
CONTENTS diseases, such as familial hypercholesterolemia and cystic
fibrosis (CF), and rare disorders with complex and heteroge-
neous genetic etiologies, represented by long QT syndrome
CARDIAC AND VASCULAR-RELATED DISORDERS
and α1-antitrypsin deficiency, are featured. When possible, a
Familial Hypercholesterolemia discussion on therapeutic intervention is included, although
Long QT Syndrome no cures currently exist for these diseases.
PULMONARY-RELATED DISORDERS

Cystic Fibrosis ●●● CARDIAC AND VASCULAR-

α1-Antitrypsin Deficiency RELATED DISORDERS
Familial Hypercholesterolemia
This chapter presents an overview of several heritable cardio- Familial hypercholesterolemia (FH) is caused by a mutation
pulmonary disorders in humans. Inherited disease among the altering the structure and function of a cell surface recep-
cardiopulmonary disorders with well-characterized genetics tor that is specific for low-density lipoprotein (LDL). When
is uncommon relative to that in other organ systems, perhaps this receptor is reduced, absent, or malfunctioning, it results
reflecting a high degree of mortality associated with severe in persistently elevated levels of cholesterol in the blood
heart or lung disease. Here, both classic, common monogenic that ultimately lead to early coronary heart disease.

PATHOLOGY
Classifying Lipoproteins
There are several ways to classify lipoproteins. The most widely used systems recognize the differences in densities during
ultracentrifugation and electrophoresis.

ELEVATED SERUM SERUM

TYPE LIPOPROTEIN LABORATORY FINDINGS TC* TG* EXAMPLE

I Chylomicrons Very high triglycerides Normal ↓↓ Lipoprotein lipase deficiency

IIa LDL High cholesterol ↑↑ Normal Familial hypercholesterolemia, familial
apolipoprotein B
IIb LDL and VLDL High cholesterol and ↑↑ ↑ Familial hypercholesterolemia, familial
triglycerides combined hyperlipidemia
III IDL High cholesterol and ↑ or ↑ Dysbetalipoproteinemia
triglycerides normal
IV VLDL High triglycerides ↑ ↑↑ Familial hypertriglyceridemia
V Chylomicrons Very high triglycerides and ↑ ↑↑ Severe hypertriglyceridemia
and VLDL high cholesterol

IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; TC, total cholesterol; TG, triglycerides; VLDL, very-low-density lipoprotein.

*↓↓ = greatly decreased; ↑ = increased; ↑↑ = greatly increased.

 160 Cardiopulmonary Disorders

Exons 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
NH2
I II III IV V VI VII A B C COOH
Protein Ligand binding EGF precursor homology
domains C9 complement Clotting factor homology
homology
Signal O-linked Cytoplasmic
sequence sugars
Transmembrane

Figure 9-1. LDL receptor gene and protein structure. The domains of the protein are shown and are labeled in the lower
portion. The seven cysteine-rich, 40-amino-acid repeats in the LDL binding domain are assigned numerals I to VII. Repeats IV
and V are separated by eight amino acids. The three cysteine-rich repeats in the domain that is homologous with the EGF
precursor are lettered A to C. Arrowheads indicate the positions where introns interrupt the coding region. Exon numbers are
shown between the arrowheads.

LDL Receptor synthesis. One consequence of restricting the cell’s uptake of

The LDL receptor gene (LDLR) is found on chromosome 19 LDL molecules is that serum cholesterol levels remain high
p13.1-13.3 and spans 45 kb. The gene contains 18 exons with a risk of accumulating in arterial walls. Serum LDL
constituting a 5.3-kb mRNA and encodes a single-chain gly- appears to be the major source of cholesterol in atheroscle-
coprotein that consists of 839 amino acids (Fig. 9-1). Exons rotic plaques, and the two most devastating sequelae of ath-
2 through 6 make up the ligand-binding site, which is com- erosclerotic plaques in arterial walls are myocardial infarction
posed of seven tandem repeats of roughly 40 amino acids. and stroke.
Each repeat harbors six cysteine residues. Also, the C-terminal
ends of each repeat are characterized by a group of negatively
charged amino acids that play a direct role in binding basic
amino acid–rich segments of apolipoprotein B-100. PHYSIOLOGY
An interesting feature of the LDLR gene is that it shares
exons with other genes. Exons 7 to 14 of the LDLR gene Acute Myocardial Infarction
encode a region homologous with the epithelial growth factor Most acute myocardial infarction (AMI) occurs from
(EGF) precursor. In fact, the EGF precursor contains the same thrombotic occlusion and coronary atherosclerosis.
eight exons. Other LDLR exons are found in at least three Atherosclerosis may preexist for years, but precipitation of
different gene families. The sharing of exons between the LDL acute thrombotic occlusion occurs because of the instability
receptor gene and the other genes is referred to as exon shuf- of plaques. AMI can be seen with hemorrhage, fissuring,
fling. Exon shuffling hypothesizes that introns permit func- and plaque rupture. In more than 90% of patients, Q-wave
infarcts, characterized by an elevated ST segment, result
tional domains encoded by discrete exons to shuffle between
when occlusive thrombi persist and a large area of the
different proteins, thereby allowing proteins to evolve as
myocardium is blocked. Non–Q-wave infarcts, characterized
mosaic combinations of preexisting functional units. The LDL by ST segment depression, result from incomplete thrombi
receptor is a vivid example of such a mosaic protein. where less of the myocardium is affected and necrosis is not
elicited.
LDL Metabolism
LDL receptors are localized in clathrin-coated pits found pri-
marily in liver cells and bind the apolipoprotein B-100
ligand—the only protein found in LDL. As cholesterol-rich
LDL molecules are endocytosed into the cell, they dissociate PHYSIOLOGY
from the receptor, and the detached receptor protein is recy-
cled to the cell surface (Fig. 9-2). The LDL molecule is incor- Stroke
porated into a lysosome and degraded. The resulting free A cerebral infarction, or stroke, occurs when there is an
cholesterol can then be incorporated into cell membranes or interruption of blood flow to the brain.
metabolized into bile salts or steroids. Ischemic stroke occurs when a blood vessel is blocked,
An important aspect of the normal processing of LDL is reducing the flow of O2 and nutrients to areas beyond the
the mechanism by which surface receptors mediate feedback blockage, and waste material beyond the blockage cannot
be removed. This is the most common cause of stroke.
control of cholesterol synthesis. Excessive levels of free cho-
Hemorrhagic stroke occurs when a blood vessel ruptures,
lesterol inhibit cholesterol and LDL receptor syntheses, thus
causing hemorrhaging that reduces blood flow and leading
reducing LDL uptake and promoting cholesterol storage. Free to decreased mean arterial pressure and thus reduced
cholesterol represses 3-hydroxy-3-methylglutaryl coenzyme blood flow to tissues.
A (HMG-CoA) reductase, the rate-limiting step in cholesterol
 Cardiac and Vascular-Related Disorders 161

Insulin and triiodothyronine (T3) increase LDL binding to

LDL Apoprotein
B-100 LDL receptor receptors, whereas glucocorticoids decrease this binding.
within coated pit Therefore, individuals with diabetes or hypothyroidism expe-
rience an increase in circulating LDL and hypercholesterol-
Recycled emia along with a corresponding increased atherosclerosis
Lysosome receptor
Endosome risk. Individuals receiving glucocorticoids, such as dexameth-
asone, may also have increased hypercholesterolemia because
Inhibits of decreased receptor binding.
LDL receptor
synthesis
Inhibits Genetics of Familial Hypercholesterolemia
cholesterol FH is a single-gene disorder due to mutations in the LDLR
synthesis gene that result in either absent or defective receptors. It is a
common autosomal dominant disease that spares no racial or
Excess ethnic group. More than 600 mutations have been identified
cholesterol Stimulates
Cholesterol cholesterol that disrupt LDLR function. Phenotypic expression is variable
Bile acids, storage owing in part to the many different mutations that occur at
steroid hormones the LDLR locus. Point mutations or deletions in the gene may
render a nonfunctional or an absent protein depending on
the location of the point mutation or extent of the deletion.
Figure 9-2. Circulating LDL binds to specific receptors LDLR protein may be normal in certain respects (e.g., its
synthesized in the cell. The receptors localize in binding capacity for LDL is normal in vitro) but unable to
depressions (“coated pits”) in the cell surface. LDL
escape from the endoplasmic reticulum and therefore unable
particles are engulfed by endocytosis, and the coated pit
pinches off to form a vesicle (endosome). LDL dissociates to reach the cell membrane (Fig. 9-3). Alternatively, the
from the receptor, and the latter is recycled to the cell protein may insert into the cell membrane, but its capacity to
surface. The LDL is delivered to a lysosome, where bind LDL will be severely impaired or nonexistent.
enzymatic action results in free cholesterol that is used to Phenotypic effects are manifested in both heterozygotes
meet cellular needs (such as steroid hormone production). and homozygotes with FH. Heterozygotes have half the
The cellular level of cholesterol is controlled by at least number of normal LDL receptors while FH homozygous indi-
three feedback loops. Excessive levels of cholesterol serve
viduals have no or exceedingly few receptors. Notably in this
to (1) inhibit cholesterol synthesis, (2) inhibit receptor
synthesis (hence, inhibit LDL uptake), and (3) stimulate disease, there is a direct correlation between the number and
the storage of cholesterol in the form of cholesteryl activity of functioning LDL receptors, levels of serum LDL
esters. cholesterol, and the age of onset and severity of the resulting
atherosclerosis. The clinical manifestations of each scenario
are described below.

Heterozygous Familial Hypercholesterolemia

Heterozygous FH is the second most common single-gene-
determined disorder in humans, affecting 1 in 500 persons in
the United States (Table 9-1). As a rule, heterozygotes remain
asymptomatic until the third to fourth decade, although
PATHOLOGY hypercholesterolemia is present from birth. With no reduced
Atherosclerotic Lesions reproductive fitness, the persistence of the high disease allele
frequency is explained. Manifestations of the condition
Atherosclerosis is responsible for the majority of cases of
include xanthomas of the tendons, particularly the Achilles
myocardial and cerebral infarction, and it exists in two forms.
Fatty streak, or early, lesions are the more common form. and tendons around the knees and elbows (Fig. 9-4) and in
They usually are confined to the intima, creating flat, yellow the hand. Tendon xanthomas are deposits of cholesterol and
areas on the artery surface. Initially, lesions are composed of are virtually pathognomonic. Their incidence increases with
foam cells, macrophages, and T cells but later also can age and they eventually manifest in about 80% of heterozy-
include smooth muscle cells. gotes. Less specific signs, found also in persons with normal
Fibrous plaque, or advanced, lesions also exist in the lipid levels, are xanthelasma (palpebral xanthomas) and arcus
intima, creating raised, pearly gray areas on the surface and cornealis (Fig. 9-5).
leading to narrowing and thickening of artery. The plaque is Far more serious are symptoms of coronary disease, which
covered by dense connective tissue at the luminal surface develop in most patients during the third to fourth decades
(fibrous cap) and composed of lipid-laden smooth muscle
of life. Frank infarctions begin to occur in the fourth decade
cells, macrophages, and T cells. The plaque covers an area
and peak in the fifth and sixth decades. By age 60, as many
of foam cells, necrotic debris, and cholesterol. Fibrous
plaques become a complicated lesion once there is as 85% of affected individuals have sustained an infarction.
extensive degeneration and calcification. FH heterozygotes are expected to produce defective
or absent LDLR protein from one allele but normal LDLR
 162 Cardiopulmonary Disorders

LDL
LDL 3 4
receptor

LDL
receptor
synthesis Figure 9-3. Familial
hypercholesterolemia is caused by
DNA Nucleus
several types of genetic defects, all
Transcription involving the gene for the LDL receptor
RNA molecule. Sometimes the gene is
either missing or so damaged that
the receptor is not transcribed and
1 processed into a functional mRNA
Ribosome
(1). Alternatively, the receptor may
be translated and folded but is still
sufficiently abnormal that it is not
transported to the cell surface (2) or, if
Golgi it reaches the surface, cannot migrate
apparatus along the cell membrane to the coated
pits, which are the only sites where
2 LDL can be internalized (3). In still
other defects, the receptor reaches the
Folded Newly formed coated pits, but its capacity to bind LDL
receptor receptor is reduced (4). In all cases, the ultimate
result is the same: reduced cellular
intake of LDL, leading to higher serum
LDL levels and atherogenesis.

TABLE 9-1. Incidence of Common

Single-Gene Disorders

SINGLE GENE DISORDER INCIDENCE

Hemochromatosis 1 in 200 to 1 in 500

Hypercholesterolemia 1 in 500
Sickle cell anemia 1 in 600 (African ancestry)
Cystic fibrosis 1 in 1600 (European
ancestry)
Tay-Sachs disease 1 in 3500 (Ashkenazi
Jewish ancestry)
Huntington disease 1 in 5000
Phenylketonuria 1 in 10,000

Figure 9-4. Xanthomas on elbow. (From Feit J, et al.

Hypertext Atlas of Dermatology. Available at: http://
proteins from the other allele. It is possible, however, espe- www.muni.cz/atlases.)
cially with the high incidence of heterozygosity, for two indi-
viduals carrying different mutant alleles to produce a child
carrying both mutations. The mutant alleles may be unable present in childhood. Severe, persistent atherosclerosis leads
to complement each other in the compound heterozygote, to early-onset coronary heart disease; death from myocardial
leading to a severe form of FH. infarction before age 30 is common.
Although homozygosity is rare, homozygous FH has been
Homozygous Familial Hypercholesterolemia an important paradigm of human lipoprotein metabolism
Homozygous FH is a rare disease, occurring in approximately from several different perspectives. First, this condition estab-
1 in 1 million persons born with the complete absence of lished the role that LDL plays in human atherosclerosis.
functional receptors. These patients have severe hypercholes- Second, FH established the first genetic basis for athero-
terolemia (600–1000 mg/dL; normal, 150–230 mg/dL) at sclerosis. Third, this disease has emerged as a model for
birth and develop raised, yellowish, cutaneous xanthomas by understanding the variable phenotypic expression of a
age 4. Tendon xanthomas and arcus cornealis are typically complex disorder. Finally, FH has played a key role in
 Cardiac and Vascular-Related Disorders 163

reduce and maintain cholesterol levels. Within 5 years after

successful liver transplantation, xanthomas in many patients
have regressed and coronary artery lesions have stabilized.
Some lesions may even have regressed. Transplantation is
most successful when done prior to the onset of cardio-
vascular complications resulting from the course of the
disease.

BIOCHEMISTRY
Niacin
Niacin is a vitamin. It is pyridine-3-carboxylic acid, which is a
component of the coenzymes NAD+ and NADP+. More than
A 200 enzymes require these coenzymes for electron transfer
in redox reactions. NAD+ frequently functions in catabolic
reactions to produce energy; NADP+ functions more often in
anabolic reactions such as cholesterol and fatty acid
synthesis. Most niacin comes from the diet, since synthesis
from tryptophan is inefficient. A mild deficiency causes
glossitis; a marked deficiency causes pellagra.

PHYSIOLOGY
Bile Acids Are End Products of
Cholesterol Metabolism
Cholesterol is excreted in bile as free cholesterol and bile
acids. Bile acids and phospholipids solubilize cholesterol in
B bile. When bile acids cannot be recycled, they can be
synthesized in the liver and stored in the gallbladder. Those
Figure 9-5. Xanthelasma (A) and corneal arcus (B). (A from bile acids that are transferred to the small intestine but not
Feit J, et al. Hypertext Atlas of Dermatology. Available at: reabsorbed at the portal vein are then converted to
http://www.muni.cz/atlases.) secondary acids. Some secondary acids are reabsorbed in
the colon.
Some drugs, such as colestipol, which cause decreased
bile acid reabsorption in the intestine, will cause an increase
in serum cholesterol conversion to bile acids. This, in turn,
developing new therapies for forestalling, if not preventing, increases cholesterol synthesis.
human atherosclerosis.

Experimental Therapies for

Familial Hypercholesterolemia PHARMACOLOGY
FH heterozygotes are treated with more conventional thera-
pies than FH homozygotes: diet plus a combination of Statins
niacin, an inhibitor of HMG-CoA reductase, and a bile The statin family of drugs inhibits HMG-CoA reductase in the
acid sequestrant can reduce the total cholesterol and LDL liver. They are the most commonly used cholesterol-lowering
cholesterol concentrations in FH heterozygotes by as much drugs. Lovastatin and simvastatin are prodrugs, whereas
as 50%. Because 50% to 70% of LDL receptors are located others are in the active form. Statins may be used in
in the liver, the most efficacious modern therapeutic option combination with other drugs for greater effect.
for FH homozygotes has been liver transplantation, which Statins are metabolized by the cytochrome P-450 3A4
provides cells with normal, active LDL receptors. The pro- system. Drugs inhibiting P-450 3A4 increase plasma statin
found effect of liver transplantation has been to normalize concentration and can increase the toxicity risk. Grapefruit
juice contains a substance that decreases statin metabolism
the plasma lipoprotein concentrations as well as to induce
by the liver; statin accumulation can cause liver damage.
regression of the tissue deposition of cholesterol. Cholesterol
Drugs that induce P-450, such as barbiturates, will increase
levels decrease postoperatively to normal or near-normal statin metabolism.
levels, and many patients require no additional pharmaco- Statins are teratogenic.
logic intervention, such as therapies with statins, to further
 164 Cardiopulmonary Disorders

Development of novel gene therapy protocols will benefit First, the gene is controlled by its own promoter, which
from efficient routine means of delivering genomic DNA to will drive expression at a physiologically relevant level.
cells. Several gene therapy strategies have been investigated Second, the gene is expressed from a genomic DNA sequence,
in animal models with varying levels of success. A limitation including all intron sequences. This allows alternative splic-
of some earlier strategies was that vector systems carrying ing to occur that can produce alternative forms of the
DNA into cells could incorporate only small fragments of protein in a tissue-specific or developmentally regulated
DNA while entire genes often exceed this limitation. This was fashion. Third, critical noncoding elements that control gene
coupled with low efficiencies of delivering large (>100-kb) expression at a transcriptional level are present and cor-
DNA inserts to cells. Original vector systems relied on viruses, rectly oriented toward the promoter to control expression.
such as adenovirus, lentivirus, or retrovirus, with limited These three features can be easily combined in a gene
capacity for additional DNA incorporation or unacceptable delivery vector with the capacity to deliver other complete
immunogenicity (see Chapter 13, Gene Therapy). Newer genomic DNA loci.
systems employ bacterial artificial chromosomes (BACs) that
will accept large DNA inserts needed to replace entire genes.
The efficiency of the BAC DNA delivery is still low, but effi-
ciency and stable incorporation and expression of genes in
Long QT Syndrome
human cells are improving. The long QT (LQT) syndromes are primarily a collection
One approach to improve efficiency has been to modify of related cardiac ion channel defects that alter myocardial
a BAC to become infectious. A BAC was identified contain- repolarization, culminating in prolonged electrocardiographic
ing the complete 45-kb LDLR genomic DNA locus within QT intervals and a form of ventricular tachycardia (Fig.
a 135-kb insert. The insert contains all 18 LDLR exons, 9-6) termed torsades de pointes (TdP). Prolonged QT may
the introns, and the promoter. Immediately adjacent to the occur from either a decrease in repolarization of cardiac
promoter are three steroid response elements (SREs) critical currents or an increase in depolarizing cardiac currents,
for promoter regulation by steroids. This BAC was converted although the former occurs more commonly owing to reduc-
by molecular manipulation to an infectious vector incor- tions in potassium currents (IKr or IKs). The ion channel
porating elements from herpes simplex virus type 1 (HSV-1) abnormalities predispose a person to arrhythmia that may
and Epstein-Barr virus (EBV). The modified BAC was then result in sudden syncope, or to a sudden drop in blood
used to infect human fibroblasts in culture from receptor- pressure resulting in temporary loss of consciousness. This
negative FH patients. Transfection of the infectious BAC is perhaps the most common clinical sign seen in the LQT
containing the LDLR gene, promoter, and controlling ele- syndromes. The syncopal events can be solitary and rare
ments was highly efficient compared with the noninfectious or recur frequently. If not corrected, the TdP may degener-
BAC construct. In addition, and most importantly, the ate to ventricular fibrillation and sudden death. In the
LDLR gene insert restored LDLR expression to appropriate United States, the LQT syndromes have a prevalence of
levels. 1 in 7000 individuals. Indeed, it is estimated that congenital
LQT syndrome may be responsible for nearly 4000 cases
of sudden childhood death annually. Accordingly, LQT syn-
dromes may also account for a percentage of sudden infant
death syndrome (SIDS) cases.
BIOCHEMISTRY
At least 12 genes are associated with LQT syndrome
Steroid Response Elements (Table 9-2). Five of the genes are associated with two
Two different response elements have been identified for syndromes: Romano-Ward (RW) syndrome and Jervell and
steroids. The SRE with a consensus sequence of Lange-Nielsen (JLN) syndrome. Prior to molecular charac-
5′-GGTACAnnnTGTTGT-3′ is bound by the androgen terization of the gene mutations within individuals and
receptor (AR), glucocorticoid receptor (GR), progesterone families with LQT syndrome, the two syndromes were
receptor (PR), and mineralocorticoid receptor (MR). recognized as unique, thus underscoring the phenotypic
The estrogen response element (ERE) with a consensus heterogeneity that became clearer with molecular studies.
sequence of 5′-AGGTCAnnnTGACCT-3′ is recognized only Both syndromes present with LQT syndrome but differ in
by the estrogen receptor. the presence or absence of hearing and in the mechanism
The mechanism of transmission of cues from receptors to of inheritance. Normal hearing and autosomal dominant
response elements is unknown.
transmission characterize RW syndrome, whereas JLN syn-
drome features sensorineural hearing loss and autosomal
recessive inheritance. Two additional LQT syndromes have
been proposed as candidates for RM but currently have
Familial hypercholesterolemia is a classic monogenic loss- not been designated as such. In RW and JLN syndromes,
of-function disease, and homozygous FH has long been five genes produce potassium channel proteins. A Na+
considered a candidate for a gene replacement strategy. The channel protein gene is involved with LQT3, also a member
application of molecular genetics to BACs and the LDLR of the RW syndrome family. More recently, a calcium channel
gene provides several advantages for gene therapy strategies. defect has been linked to LQT8.
 Cardiac and Vascular-Related Disorders 165

P T

Q S

QT intervals

Figure 9-6. Torsades de pointes (French, “twisting of points”) is a cardiac arrhythmia seen with prolonged QT intervals in long
QT syndrome (LQTS).

TABLE 9-2. Comparison between Long QT Syndromes

SYNDROME SYNDROME GENE

TYPE AD AR NAME PROTEIN CHROMOSOME INITIATING FACTORS

LQT1 RW JLN1 KCNQ1 KVLQT1 11p15.6 Exercise, such as swimming, and

emotional stress; responds to
β-blockers
LQT2 RW KCNH2 HERG 7q35-36 Startled by noise, such as alarm
clocks and ringing telephone
LQT3 RW SCN5A Nav 1.5 3p21-24 Occurs during sleep; more likely
to be fatal than LQT1 or LQT2;
responds better to mexiletine
than other forms; also called
Brugada syndrome
LQT4 Sick sinus ANKB Ankyrin-B 4q25-27 Exercise and emotional stress
syndrome
with
bradycardia
LQT5 RW JLN2 KCNE1 Mink 21q22.1-22.2 Sympathetic stimulation
LQT6 RW KCNE2 MiRP1 21q22.1-22.2 Certain medications and exercise
LQT7 Andersen KCNJ2 Kir2.1 17q23 Hypokalemia; also known as
Andersen syndrome; patients
can develop periodic paralysis
(usually associated with
hypokalemia) and skeletal
developmental abnormalities

Recent Additional LQTS Genes Identified

LQT8 Timothy CACNA1C CACNA1C 12p13.3

LQT9 Limb-girdle CAV3 Caveolin 3 3p25
muscular protein
dystrophy,
type 1C
LQT10 SCN4B SCN4B 11q23
LQT11 AKAP9 A-Kinase 7q21-22
anchor
protein-9
LQT12 SNTA1 α1-Syntrophin 20q11.2

Note: For gene names, KCN designates potassium channel and SCN designates sodium channel.
166 Cardiopulmonary Disorders

PHYSIOLOGY Romano-Ward Syndrome

The most common forms of LQT syndrome are LQT1, LQT2,
Cardiac Action Potentials and LQT3. Among these, LQT1 is the most common and
Differences in cardiac action potential occur in different represents more than 60% of cases. LQT2 represents about
areas of the heart. These differences are caused by the 35% of cases, and LQT3 less than 5% of cases. The other
different types and distribution of channels, and they reflect forms are quite rare.
differences seen in initiation time, shape, and duration of the The autosomal dominant forms of LQT syndrome are
potential. Four major time-dependent and voltage-gated
responsible for the majority of cases. Three distinct ST-T wave
membrane currents are seen in cardiac action potentials:
■ The Na+ current (INa) is responsible for rapid depolarization
phenotypes associated with five different genes are recog-
of the action potential in the atria, ventricles, and Purkinje
nized as RW syndrome (see Table 9-2). As with most forms
fibers. of LQT syndrome, characteristic “triggering” events are
■ The Ca++ current (ICa) is responsible for rapid associated with precipitating cardiac events. LQT1, for
depolarization of the action potential in the sinoatrial (SA) example, seems to be triggered by exercise, particularly swim-
and atrioventricular (AV) nodes. This current also initiates ming, and by emotional stress. Incredibly, 99% of cardiac
contractions in cardiomyocytes. events occurring while swimming involve individuals with
■ The K+ current (IK) is responsible for repolarization of the LQT1.
action potential in cardiomyocytes. LQT2, representing approximately 35% of cases, is due to
■ The “pacemaker current” (If) is partially responsible for the
mutations in the KCNH2 gene on chromosome 7. Auditory
pacemaker activity of the SA and AV nodal cells and
stimuli such as a ringing telephone or an alarm clock may
Purkinje fibers.
provoke LQT2 onset. Over 80% of cardiac events associated
with auditory stimuli occur in individuals with LQT2.
LQT3, associated with 5% of cases, is due to a mutation in
PHYSIOLOGY the SCN5A gene on chromosome 3. This gene codes for the
cardiac Na+ channel. Sleep or undisturbed rest apparently
QT Interval triggers LQT3, suggesting that slowed heart rate is a risk factor.
The time in seconds between the start of the Q wave and RW syndrome accounts for most LQT syndrome cases;
the end of the T wave on an electrocardiogram (ECG) is the however, nearly 30% of clinically diagnosed RW individuals
QT interval. It represents the time it takes ventricles to do not have mutations in one of the five genes described
contract and recover. Heart rate is dependent on the QT above. Hence, additional genetic loci, representing genetic
interval. Bazett’s formula is the most popular clinical method heterogeneity of the syndrome, appear to be involved in
to describe the QT interval/heart rate relationship:
the etiology. Penetrance is also incomplete in RW syndrome,
and 30% to 50% of individuals harboring a disease-causing
QTc = QT RR
mutation in one of the five RW genes show no symptoms.
where QTc is the QT interval corrected for heart rate, and RR Overall, LQT syndrome is a genetically heterogeneous and
is the interval (measured in seconds) from the onset of one mechanistically complex disorder.
QRS complex to the onset of the next complex. The formula
overcorrects at high heart rates and undercorrects at slow Jervell and Lange-Nielsen Syndrome
heart rates. Normal values range from 0.30 to 0.44 seconds JLN syndrome is a rare, autosomal recessive form of LQT
(0.45 seconds in women). syndrome that features congenital bilateral sensorineural
hearing loss. The estimated prevalence for JLN syndrome is
3 in 1 million individuals. Whereas heterozygous mutations
PHARMACOLOGY in the KCNQ1 gene result in the LQT1 phenotype of RW
syndrome, the homozygous (or compound heterozygous)
Acquired Long QT Syndrome mutations precipitate JLN syndrome. In keeping with the
Acquired LQTS generally results from drug use or metabolic relative frequencies of the gene defects found for RW syn-
abnormalities. Common causes include the following: drome, roughly 90% of all JLN syndrome cases are due to
■ Drugs: all block the IKr current mediated by the KCNH2- mutations in the KCNQ1 gene. Because of the incomplete
encoded potassium channel (HERG protein). This is not penetrance found in the heterozygous state, parents of a child
apparent before exposure to the drug. Major classes of with JLN syndrome are asymptomatic.
drugs that can cause LQTS are antiarrhythmic drugs, Another gene, KCNE2, is involved in 10% of JLN syn-
certain nonsedating antihistamines (terfenadine and drome. Both forms of JLN feature autosomal recessive inheri-
astemizole were removed from the market), macrolide tance. KCNE1 encodes a “minimal potassium ion channel”
antibiotics, antipsychotics, and antidepressants.
protein, MinK. These proteins are β subunits that coassemble
■ Electrolyte abnormalities: hypokalemia and
with α subunits (KvLQT1) produced by KCNQ1. The β sub-
hypomagnesemia.
■ Eating disorders.
units are ancillary proteins that modulate the gating kinetics
■ Stroke. and enhance stability of multimeric channel complexes.
Different mutations within the KCNE1 gene result in LQT5
 Pulmonary-Related Disorders 167

that presents either as RW or JLN. In JLN syndrome, the other clinical signs is a related, milder manifestation of the
mutations affect both inner ear and cardiac channels. same pathophysiologic mechanism.

LQT4 and LQT7 Clinical Features of Cystic Fibrosis

The last two genes associated with LQT syndrome but not RW Clinically, CF is identified by a triad of abnormal conditions
or JLN include a potassium channel protein and the ankyrin-B primarily manifested as pulmonary problems, gastrointestinal
protein. LQT7, also known as Andersen syndrome, is charac- problems, and elevated Cl− in sweat (>60 mEq/L) (Table 9-3).
terized by periodic paralysis, prolongation of the QT interval, In addition, reproductive problems are significant in males—
cardiac arrhythmias, and mildly dysmorphic features such as more than 95% of whom may be sterile.
low-set ears, micrognathia, hypertelorism, syndactyly, short The most commonly affected organs in CF are the lungs
stature, and clinodactyly. It is unique among the channelopa- and pancreas. However, lung disease is the primary cause
thies in that it may affect both cardiac and skeletal muscle, thus of morbidity and mortality (Fig. 9-7). In general, classic CF
explaining the co-occurrence of periodic paralysis and LQT in patients manifest early symptoms of dry, hacking, nonpro-
the same individual. For LQT7, the KCNJ2 gene produces the ductive cough with wheezing. Later symptoms include a
K+ channel J2 protein, KIR2.1, found in both skeletal and productive cough, rales, wheezing, repeated infections,
cardiac muscle. Mutations reducing KIR2.1 prolong the termi- decreased appetite, failure to grow, and clubbing. These symp-
nal phase of the cardiac action potential; by reducing the toms increase progressively without proper management of
extracellular setting of K+, there are delayed afterdepolariza- infections and nutritional status.
tions (DADs) and spontaneous arrhythmias. Ventricular
arrhythmias are common but only rarely degenerate into a TABLE 9-3. Problems Associated with
hemodynamically compromising rhythm, such as torsades de Cystic Fibrosis
pointes or ventricular fibrillation. Unlike with other forms of
LQT syndrome, sudden death is not associated with LQT7. SITE ASSOCIATED PROBLEM
It is suggested that mutated ankyrin-B in cardiomyocytes
causes an abnormal cytoskeleton arrangement resulting in Pulmonary Mucus-obstructed airways
fewer functional Na+ channels with altered kinetics and pro- system Bacterial infections
longed cardiac repolarization. In both LQT3 and LQT4, Na+ Early symptoms
channels reopen late, but the plateau potentials differ at −20 Dry, hacking, nonproductive cough
mV and −40 to −50 mV, respectively. Increased respiratory rate (wheezing)
Intermediate symptoms
Productive cough with rales,
Management for Long QT Syndrome wheezing
For many individuals—those with no syncope or complex Repeated infections
arrhythmias—no treatment is required. However, for indi- Decreased appetite, weight loss
viduals with syncope, complex arrhythmias, or a family Failure to grow
Clubbing
history of sudden cardiac death, β-adrenergic blockers, such Advanced symptoms
as propranolol and atenolol, are recommended. These drugs Chronic, productive cough
slow the heart rate and can prevent syncope in about 90% Exertional dyspnea
of individuals with LQT syndrome. LQT3, the Na+ channel Cyanosis
defect, responds best to the Na+ channel blocker mexiletine. Lung abscess
Bone pain
Individuals who have not been responsive to medications Osteoarthropathy
and are at risk of suffering serious or sustained abnormal
Gastrointestinal Mucus-obstructed pancreatic ducts
arrhythmias may receive an implantable cardioverter- system Decreased pancreatic enzymes
defibrillator (ICD). The ICD is usually implanted in the left Intestinal blockage
pectoral region and monitors the cardiac rate. When the heart Poor weight gain
rate exceeds a programmable rate, a biphasic shock wave is Easy bruising secondary to vitamin K
delivered to restore normal rhythm. deficiency
Chronic diarrhea in infancy
Rectal prolapse
Hypoproteinemia
●●● PULMONARY-RELATED Pancreatitis
DISORDERS Diabetes
Sweat glands Hyponatremia
Cystic Fibrosis Hypochloremia
Cystic fibrosis (CF) is a disease that affects the epithelia of Heat exhaustion and dehydration
during exercise, ↑ in hot weather,
several organ systems, including the respiratory tract, exo-
and fever
crine pancreas, male gonads, intestinal tract, hepatobiliary
Reproductive Blocked or absent vas deferens and
tract, and sweat glands. It is caused by defects in the CF
system aspermia
transmembrane regulator conductance protein (CFTR). Con- Cervical polyps
genital bilateral absence of the vas deferens (CBAVD) without
 168 Cardiopulmonary Disorders

Median Percent Predicted FEV1 vs Age, 1990 and 2004 Box 9-1. DIFFERENTIAL DIAGNOSES FOR
CYSTIC FIBROSIS
100
1990
2004
Cystic fibrosis should be considered in:
90
Infants with
Meconium ileus
80
Predicted (%)

Hypoproteinemia and anemia

Hyponatremia of unknown etiology
70 Older children with
Rectal prolapse
60 Failure to thrive, poor growth and weight gain, nutritional
problems, chronic diarrhea, malabsorption
50 Recurrent and refractory respiratory disorders, including
nasal polyps
40
Adolescents and adults with
Recurrent pancreatitis
6 8 10 12 14 16 18 20 22 24 26 28 30
Recurrent sinusitis/bronchitis
Age (y)
Recurrent bronchiectasis
Figure 9-7. Lung function in individuals with CF, measured Nasal polyps
as forced expiratory volume per 1 second (FEV1), decreases Male infertility
by about 2% per year. The lower a person’s FEV1, the more
severe the lung disease. (From the Cystic Fibrosis
Foundation. Patient Registry 2008 Annual Report. Bethesda,
MD, Cystic Fibrosis Foundation, 2009.)
Male infertility, seen as azoospermia, is nearly universal in
male patients with CF and most often takes the form of
CBAVD. Sperm ducts degenerate or atrophy as a consequence
of prolonged blockage by thick mucus secretions. In females,
fertility is reduced because of thick desiccated cervical mucus.
MICROBIOLOGY Meconium ileus is found at birth in nearly 20% of all patients.
Other problems commonly encountered with CF include
Cystic Fibrosis and Respiratory Infections nasal polyps, rectal prolapse, cirrhosis, and diabetes mellitus
Respiratory infections are a major concern for individuals (Box 9-1). Whereas CF used to be a fatal childhood disease,
with CF. Infection and swelling damage the lungs and cause aggressive symptom monitoring and treatment has raised the
lung function (FEV1) to decrease. Infections occur more overall median age of survival to 37.4 years (Fig. 9-8).
often in damaged lungs.
Molecular Pathophysiology of Cystic Fibrosis
The basic defect expressed in CF is a failure of Cl− transport
across cell membranes. Figure 9-9 depicts transport of ions
Respiratory Infections vs Age across cell membranes forming the epithelial lining of the
airways. In normal airway cells, the ion traffic utilizes two
100 channels of Cl− conductance at the luminal surface. One
90 channel is dependent on cyclic AMP (cAMP), and the other
80
is activated by Ca++. CFTR governs the cAMP channel.
70
CFTR is a large chloride ion channel composed of 1480
Percentage

60
amino acids. It comprises five functional domains, including
50
two membrane-anchoring segments each characterized by six
40
membrane-spanning portions that form the core of the ion
30
20
channel, two nucleotide-binding domains capable of binding
10 ATP to modulate channel function, and a regulatory (R)
0 domain, whose multiple capacities for phosphorylation
0–1 2–5 6–10 11–17 18–24 25–34 35–44 45; suggest that it serves as a switch that governs CFTR function.
Age (y) As might be expected from the clinical manifestations, CFTR
Overall percentage in 2004: is expressed on the apical surface of epithelia in the lungs,
P. aeruginosa 57.3 S. aureus 51.7 MRSA 14.6 pancreatic ducts, intestines, and sweat gland ducts.
H. influenzae 16.2 S. maltophilia 11.6 B. cepacia 2.9
Defective CFTR proteins result in an impaired ability to
secrete chloride ions (see Fig. 9-9). The disturbance of Cl−
Figure from the Cystic Fibrosis Foundation. Patient Registry 2008 Annual transport across the luminal surface is associated with a com-
Report. Bethesda, MD, Cystic Fibrosis Foundation, 2009. pensatory influx of Na+ ions to retain electrical neutrality. The
 Pulmonary-Related Disorders 169

Survival From Age 1 Year, by Year of Birth

100
98
96
94

Surviving (%)
92
90
88
86 1995–2004
1990–1994
84 1985–1989
Figure 9-8. Mean survival age of 82 1980–1984
individuals with CF. (From the Cystic 80
Fibrosis Foundation. Patient Registry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
2008 Annual Report. Bethesda, MD, Age (y)
Cystic Fibrosis Foundation, 2009.)

Figure 9-9. Ion transport across

epithelium. A, In normal airway cells,
Na; Na; Na;
there are two Cl− channels for
conductance. One channel is CFTR Cl: Cl:
cAMP
dependent on cyclic AMP, and the other Cl: Cl: Cl:
Cl: Cl: Cl:
is activated by Ca++. CFTR governs the Cl;:
K; K K;
cAMP channel. B, In individuals with Na; Na; ATP
Na; Ca;; Ca;;
CF, the defective CFTR protein results Ca;;
in an ability to secrete Cl− ions and a Cl: Cl: Cl:
compensatory influx of Na+ ions to
retain electroneutrality. The Na; Na; Na;
accompanying water influx causes ATP ATP ATP
dehydration at the cellular surface and Na; Na; Na;
ADP ADP ADP
leads to the sticky mucus characteristic
K; K; K; Amiloride
of the disease. C, Two strategies are
shown to normalize the electrolyte Blood Lumen
imbalances in CF: amiloride to lessen
the Na+ influx and ATP (or UTP) to
stimulate the Ca++-dependent Cl− input. A B C

accompanying water influx causes dehydration at the cellular heterozygous carriers. A reproductive advantage for the het-
surface, leading to the sticky mucus characteristic of the erozygote (Aa) over the normal homozygote (AA) would
disease. The sticky mucus in the lung causes mechanical account for the high frequency of the lethal recessive gene
obstruction and chronic inflammation of the airways. for CF in white populations. The hypothesis currently favored
is that heterozygous carriers are more resistant to infantile
Inheritance of Cystic Fibrosis gastroenteritis than noncarriers, and thus a single mutant
CF is one of the most common autosomal recessive disorders allele might protect the carrier from profuse diarrhea and
in whites. It is the most common lethal monogenic disease. fluid loss. This describes a heterozygote advantage that is
One infant in 2000 to 2500 is born with the recessive disor- seen in several disorders in which the heterozygous genotype
der, and there are approximately 30,000 affected individuals has a higher relative fitness than either the homozygous
in the United States. This means that approximately 1600 dominant or homozygous recessive genotype. One of the best
affected newborns may be expected each year in the United known examples of heterozygote advantage is sickle cell trait
States. Almost all patients with CF are whites; blacks (1 in (see Chapter 6).
15,000) and Asian Americans (1 in 31,000) are rarely afflicted.
As seen with other autosomal recessive disorders, the fre- CF Gene and CFTR
quency of heterozygous carriers is greater than the frequency The CFTR gene, responsible for CF, is located on the long
of affected homozygous individuals. One in 22 to 28 Cauca- arm of chromosome 7. The gene is large, containing more than
sians is a carrier of CF, depending on the population screened. 230,000 base pairs, and it has 27 exons, producing a 6.5-kb
The majority of affected children—more than 99%—come mRNA. More than 1500 different mutations have been
from marriages of two normal parents, both of whom are described at the CF locus. The most common is an in-frame
 170 Cardiopulmonary Disorders

N-linked carbohydrate

NH2
NBD
Cytosol ATP
binding
domain
PO4 CO2H NBD
D F508
mutation

PO4 R domain

Figure 9-10. CFTR is composed of 5

PO4 PO4
domains. The most common mutation
in CF is the Δ508 deletion, found in the
nucleotide-binding domain (NBD).

3-bp deletion (termed ΔF508) that causes omission of a single

phenylalanine at amino acid 508 within one of the ATP TABLE 9-4. Classification of CFTR Mutations
binding domains. CFTR protein harboring the ΔF508 muta-
tion does not fold properly and hence fails to move through EFFECT OF
the endoplasmic reticulum to the Golgi apparatus, where it MUTATION MUTATION OF
CLASS CFTR PROTEIN MECHANISM
normally undergoes posttranslational maturation (Fig. 9-10).
Accordingly, the defective protein is absent from its final
destination on the surface of luminal epithelial cells in CF I Reduced or absent Nonsense,
synthesis frameshift, or
patients. The inability of the altered CFTR to move beyond splice-junction
its intracellular location to the cell membrane surface mutations
explains why heterozygous carriers with this mutation have II Block in protein Missense
only normal cell surface channels and no interference in processing mutations,
Cl− transport. amino acid
deletions
Most of the CF mutations are single-base changes or small III Block in regulation of Missense
deletions in the CFTR gene. Remarkably, the ΔF508 deletion CFTR chloride mutations
accounts for 70% of classic CF cases in Caucasians. Overall, channel
it can be found in approximately 30% to 80% of the patients, IV Altered CFTR chloride Missense
depending on the ethnic group. The remaining mutations channel mutations
conductance
occur at other sites in the CF gene and are classified by the
type of mutation (Table 9-4). This profusion of “private muta-
tions” occurring at low frequencies has increased the diffi-
culty of devising a comprehensive strategy to screen for CF.
Such allelic heterogeneity helps explain the variations seen for genetic testing (Table 9-5). This panel detects the causal
in the qualitative and quantitative phenotypic expression mutations in 97% of Ashkenazi Jewish, 88% of other white,
of CF. 69% of black, and 57% of Hispanic patients. Hence, for
In an effort to both standardize and maximize the effi- the great majority of CF patients, genetic testing is highly
ciency of DNA-based diagnoses and carrier testing for CF, informative.
the American Academy of Medical Genetics recommends The ΔF508 deletion is a severe mutation, whereas other
that a panel of the 25 most common CF mutations be used mutational changes appear to be less severe, since the clinical
 Pulmonary-Related Disorders 171

TABLE 9-5. 25 Alleles Recommended for CF Carrier Screening

1078delT 3120+1G>A A455E G85E XR334W

1717-1G>A 3659delC ΔF508 I148T R347P
1898+1G>A 3849+10kbC>T ΔI507 N1303K R553X
2184delA 621+1G>T G542X R1162X R560T
2789+5G>A 711+1G>T G551D R117H W1282X

alleles. Such testing is invaluable in population screening

Box 9-2. CBAVD AND CFTR MUTATIONS programs and to at-risk relatives and their reproductive part-
ners. Ideally, carrier screening should be offered to couples
Mutations of the CFTR gene are suggested by the following: before they become pregnant to allow time to consider
Absence of vas deferens on palpation
options if they are carriers. Studies show, however, that indi-
Azoospermia
viduals are more likely to seek testing if there is a family
Low ejaculated semen volume
Evidence of abnormalities of seminal vesicles or vas
history of CF or after a pregnancy occurs.
deferens on rectal ultrasound examination As suggested, many but not all CF mutations can be
detected in screening panels, and particular panels can be
designed to recognize the most common mutations in a par-
course can range from mild to severe. This is best seen with ticular population. A negative report for one or both members
regard to pancreatic dysfunction, where certain severe muta- of a couple does not exclude the possibility of an affected
tions consistently predict pancreatic insufficiency and other offspring. The risk, known as the residual risk, is dependent
base changes prescribe some pancreatic function. Such a on other factors such as racial or ethnic background of each
genotype-phenotype relationship is less readily apparent with parent, the specific mutations being tested, and whether both
the pulmonary symptoms. In fact, even individuals sharing parents are tested.
the same deleterious CFTR genotype may exhibit differences
in lung disease progression and symptomology, indicating that Gene Therapy for Cystic Fibrosis
other factors—either genetic or environmental—may have a Since the pulmonary complications of CF are life threatening,
role in CF lung disease expression. Finally, CBAVD is nearly the lung is the principal target of gene therapy. Although lung
universal in males with CF but may also occur in males transplantation has become more common, it is not a viable
without other CF symptoms (Box 9-2). These individuals option for all CF patients and it carries significant risks that
harbor one mild and one severe CFTR mutation. In the restrict the number of individuals considered for this surgery.
absence of two severe mutations, CF is not clinically observed. Currently, the 1-year survival rate for lung transplantation is
This suggests that derivatives of wolffian duct structures are 80% to 90% and 5-year survival is 60% to 70%. Successful
very sensitive to CFTR dysfunction. outcomes of transplantation depend on timing of surgery,
Mutations in the CFTR gene may overlap phenotypes pre- medical status of other organs, and psychosocial support
sented by other disorders, not only CBAVD. Chronic rhino- systems. Infections present a special problem for CF lung
sinusitis (CRS) is a consistent feature of individuals with CF. transplantation patients. It is desirable to target pulmonary
In some patients, the severe ΔF508 mutation on one allele epithelial cells—and not other cells—for corrective gene
combined with a less deleterious mutation presents a clinical therapy because the long-term consequences of overexpres-
picture of chronic rhinosinusitis without other CF features. sion of a cAMP-regulated Cl− channel in other cell types are
Similarly, CRS is also seen in individuals who have a single not yet known. Targeting is facilitated by the fact that the
CFTR allele mutation but who do not meet other criteria airway epithelium is contiguous with the external environ-
required for diagnosis of CF. For these reasons, practitioners ment. For this reason, gene delivery by viruses with tropism
should consider CFTR mutations when evaluating other for the airways, such as adenoviruses, currently has the most
CF-like and CF-associated presentations such as recurrent appeal.
sinusitis, bronchitis, bronchiectasis, nasal polyps, recurrent
pancreatitis, refractory asthma in children, recurrent pneumo-
nia, hypoproteinemia and anemia in infants, and children MICROBIOLOGY
with rectal prolapse.
Viral Gene Therapy Vectors

Molecular Testing for Cystic Fibrosis Viruses are efficient at transferring DNA into a host cell. Viral
vectors must be modified to avoid eliciting an immune
In genetic counseling programs, an important consideration
response—the greatest disadvantage of viral vectors—by
is the development of simple, inexpensive means of unam-
the host. Commonly used vectors are retrovirus, adenovirus,
biguously detecting heterozygous carriers of inherited reces- adeno-associated virus, herpes simplex virus, and lentivirus.
sive disorders. CF testing has largely been achieved by The size of the insert is a limiting factor for choice of vector.
DNA-based molecular genetic testing for common disease
 172 Cardiopulmonary Disorders

Adenovirus-based vectors have been used to introduce α1-antiprotease, but by convention, most biomedical scien-
normal human cDNAs of CFTR into the lung epithelium of tists, physicians, and patients refer to the protein as AAT.
animal models, and normal expression of membrane-lined Unequivocally, however, the key physiologic role played by
CFTR has been demonstrated. Since the adenovirus does not AAT in the lungs is the inhibition of neutrophil elastase,
integrate into the genome of the lung epithelial stem cells, it an enzyme that normally destroys bacteria. In the absence
is lost with cellular turnover. Reinfection therefore is neces- of AAT, neutrophil elastase will destroy elastic fibers in lung
sary but can cause immunologic reactions. There also may be connective tissue.
the risk of a replication-competent infectious virus being Emphysema was originally believed to occur from an
generated as a result of recombination with ubiquitous wild- imbalance between protease and antiprotease activity. When
type virus. In general, the possible risk factors have excluded a marked genetic deficiency of AAT became associated with
application of the strategy of gene therapy in humans by severe pulmonary disease, it became clear that AAT defi-
adenovirus vectors. ciency is a risk factor for lung disease. However, because the
A nonviral gene transfer vector has been used to transfer onset, rate of progression, and severity of pulmonary disease
a normal CFTR gene to epithelial cells. Compacted DNA vary among individuals with severe AAT deficiency, it is also
nanoparticles containing only the CFTR gene sequences are clear that additional genetic or environmental factors are
used to more efficiently cross the cell membrane and enter etiologically relevant.
the nuclei, where recombination can occur. These nanoparti-
cles have shown few immunogenic or toxic effects in animals AAT Gene
and humans and extend the strategies being used to develop The AAT gene is found at chromosome 14q32.1, is 12.2 kb
new gene therapies designed to correct defective genes in length, and contains seven exons. The gene has at least
in situ. three different names in the literature including AAT, PI, and
SERPINA1; all can be used interchangeably.
The AAT gene is highly polymorphic. More than 125 dif-
ferent mutations have been identified in the gene, and a
BIOCHEMISTRY subset of these impacts serum AAT levels. Three codomi-
nantly expressed AAT alleles—M, S, and Z—are primarily
Nonviral Gene Vectors important to lung, as well as liver, disease. By convention,
Nonviral vectors avoid some problems seen with viral these variants are assigned the symbol Pi, designating prote-
vectors. ase inhibitor, followed by a superscript capital letter (PiM, PiS,
■ DNA vaccination: recombinant gene is injected into and PiZ) to define the allele. Historically, the capital letter
tissues (i.e., muscle) indicates the relative electrophoretic mobility of the mutant
■ Liposomes: gene fused with liposomes; can cross
protein, where M represented moderate, S slow, and Z the
blood-brain barrier slowest gel mobility.
■ Poly-cation conjugates: DNA condensed into
The PiM, or M, allele is the normal allele. There are actually
nanostructure; can pass through membranes; cells can be
a number of M alleles that do not reduce the serum AAT
targeted by use of conjugated receptor ligands
■ Bacteria: Salmonella with altered genes targets cells (i.e.,
concentration, which normally is 20 to 53 μmol/L. Hence,
macrophages) to deliver DNA they are simply neutral polymorphisms of the AAT gene. The
M allele is the most common form in whites, with an allele
frequency of 95% and a homozygote (MM) frequency of
approximately 90% (Table 9-6). The S and Z alleles are more
rare and are associated with reduced levels of AAT activity
α1-Antitrypsin Deficiency
and hence account for AAT-deficient disease. The S variant
One of the etiologies for chronic obstructive pulmonary results from a valine-for-glutamate substitution at amino acid
disease (COPD) is unrestrained proteolytic activity in the
connective tissue of the lungs, especially the elastin compo-
nent. The gradual destruction of pulmonary elastic tissue
results from a diminished presence of α1-antitrypsin (AAT). TABLE 9-6. AAT Genotype Frequencies in Whites
Emphysema is a type of COPD with decreased AAT activity. of Northern European Ancestry and
AAT is a major serum serine protease inhibitor produced Associated AAT Serum Activities
in the liver. The 394-amino-acid protein is the most abundant
antiprotease in the lung, constituting roughly 95% of all AAT/PI ENZYME ACTIVITY
alveoli antiprotease activity. In blood, AAT is associated GENOTYPE FREQUENCY (% CONTROL)
with the α1-globulin fraction and accounts for 90% of the
antiprotease activity based on its reactivity with trypsin. MM 0.90 100
Human antitrypsin actually inhibits the activity of a broad MZ 0.038 60
SS 0.001 50–60
spectrum of proteins, including trypsin, chymotrypsin, elas-
SZ 0.0012 (1/800) 30–35
tase, skin collagenase, plasmin, thrombin, and bacterial pro- ZZ 0.0004 (1/2500) 10–15
teases. Because of this, the protein is more accurately labeled
 Pulmonary-Related Disorders 173

264 (V264E) of the AAT gene. The Z form is defined by a evidence that, in addition to the abnormalities in protein
glutamate-to-lysine substitution at position 342 (E342K) and processing and secretion, Z protein has a reduced ability to
represents the most severely impaired AAT variant. Homozy- inhibit elastase. Overall, the ZZ genotype typically results in
gosity of the PiZ (ZZ) has the greatest deficiency of protease an AAT activity of only 10% to 15% of normal values (see
inhibitor (serum levels of 3.4–7 μmol/L, indicating 10% to Table 9-6). Such individuals with familial emphysema are
15% of the activity found in the normal, or MM, state). This highly susceptible to the development of lung disease as early
form is associated with nearly all AAT-deficiency disease. as age 35 years.
However, emphysema also occurs with moderate frequency
in individuals heterozygous for the PiS and PiZ alleles, desig- Pathogenesis of Lung Disease
nated SZ. Because the AAT genes are codominant, any com- in ZZ Individuals
bination of a deficiency allele and an M allele (MS or MZ) Not all ZZ individuals with severe AAT deficiency develop
results in at least 50% AAT activity and, accordingly, is not emphysema. Interestingly, serum levels of AAT in ZZ asymp-
associated with pulmonary disease. tomatic and emphysema-symptomatic individuals may be the
Several families have been reported in which a “silent,” or same. The manifestations of clinical illness evidently require
null, allele appears to be present. The rare silent allele (des- exposure to certain precipitating environmental factors.
ignated PiNull) results in the complete absence of synthesis of Tobacco smoke is a major and unambiguous trigger for overt
AAT; individuals homozygous for the null allele develop pulmonary disease in ZZ individuals (Fig. 9-11). Characteris-
emphysema by their mid-20s. Affected individuals are gener- tically, ZZ emphysema-symptomatic individuals have been
ally homozygous for a deletion that results in a frame shift heavy smokers for many years. The smoking habit seems to
and premature termination of translation. subtract an average of at least 10 years from their life span.
In ZZ patients, the onset of dyspnea occurs at a median age
ZZ Genotype of 40 in ZZ smokers compared with 53 years in ZZ nonsmok-
The frequencies of molecular variants show considerable ers. The insidious onset of shortness of breath in smokers
variation among ethnic groups and geographic areas. The ZZ progresses relentlessly to a typical presentation of pulmonary
genotype has the highest incidence in Scandinavia, where emphysema.
approximately 1 in 1700 persons (0.06%) is homozygous for In health, the neutrophil elastase is continually inhibited
this defective allele. DNA haplotype analysis suggests that the by AAT and, to a lesser extent, by α2-macroglobulin. AAT
original Z mutation arose in Scandinavia. This detrimental Z enters lung tissue by simple diffusion from the serum. In the
allele is essentially absent in African blacks, Asians, and Native
Americans, indicating that the deleterious homozygous state,
and thus AAT deficiency, is largely a disorder of whites.
In the United States, 0.01% to 0.05% of the white popula- Smoke
tion is ZZ homozygotes. Although one person in 2500
has α1-antitrypsin deficiency, about one person in 25 is a
heterozygous carrier. Thus, there are 100 times as many
heterozygous carriers as there are affected individuals, a Activates leukocytes Alveolar macrophages
and neutrophils are
scenario similar to that seen in CF. increased in the lung
The Z variant of AAT differs from the normal M protein in
interesting ways. This single-amino-acid substitution (E342K)
affects the folding and ultimately the conformation of the Inflammatory
response initiated
molecule. Most misfolded Z antitrypsin aggregates near the Proteolytic
end of the endoplasmic reticulum, where newly synthesized activity increases
proteins ordinarily bud off and pass into the Golgi apparatus
Oxygen free
to be packaged for transport. The basic defect, accordingly, is radicals increase
a blockage in Z antiprotease processing in the endoplasmic
reticulum, resulting in the intracellular accumulation and
polymerization of mutant AAT with subsequently lowered Proteases Protease inhibitors
are produced are inactivated
levels of circulating antiprotease. Presumably because of the
accumulation of AAT polymers, liver disease, characterized
by abnormal liver enzymes, fibrosis, or cirrhosis, occurs in a
minority of patients. Destruction of
Additionally, the Z protein isolated from hepatocytes con- alveolar tissue
tains carbohydrate side chains that differ markedly from
normal. The Z protein has an increase in mannose and a Figure 9-11. Tobacco smoke activates leukocytes to
increase proteolytic activity. It also causes alveolar
decrease in both sialic acid and galactose residues. Appar- macrophages and neutrophils to release chemotactic
ently, the excess of mannose residues on the carbohydrate substances that initiate an inflammatory response. This
side chains prevents the final formation of carbohydrate side response creates oxygen free radicals that inactivate protease
chains with terminal sialic acid residues. Finally, there is inhibitors. Thus alveolar tissue damage proceeds unchecked.
 174 Cardiopulmonary Disorders

absence of AAT, the unrestrained action of proteolytic

enzymes damages the lung parenchyma and vascular bed MICROBIOLOGY
(Fig. 9-12).
The heavy cigarette smoker with an AAT deficiency is in Macrophages and Inflammation
double jeopardy because macrophages are found in greater Macrophages have an important role in inflammatory
numbers in the lungs of smokers than of nonsmokers. Mac- responses initiated by foreign substances. They are
rophages release a chemotactic factor that attracts neutrophils recruited from surrounding tissues or derived from
to the lungs, resulting in an increased concentration of pro- monocytes that migrate to the site. At the site, macrophages
are exposed to stimuli, such as interferon-γ, cytokines, and
teases. Further, macrophages release a variety of substances
lipopolysaccharides from bacteria, and morphologic and
that provoke an inflammatory response with a concomitant
functional changes occur. Macrophages secrete proteases
increase in oxidative metabolism. Consequently, the released (collagenase, elastase) and plasminogen activator. They are
freely diffusible O2 free radicals from macrophages and neu- known as activated macrophages when phagocytosis and
trophils react with the active site of AAT—a methionine- microbicidal activity increase. Activated cells produce
serine peptide bond at AAT methionine residue 358. The interleukin (IL)-8, a neutrophil chemotaxin. Leukotrienes and
inhibition of elastase by AAT depends on the integrity of the superoxide may also be released.
methionine-serine bond in the active site. Cigarette smoke
leads to the oxidation of the methionine residue in the active
site, which effectively destroys the bond between methionine
and serine. The cleaved, ineffective AAT molecule is eventu- Overall, cigarette smoke can be implicated in many ways
ally catabolized in the liver and spleen. in the pathogenesis of emphysema and AAT deficiency,
including activation of macrophages, recruitment of neutro-
phils, enhanced release of proteolytic enzymes by macro-
phages and neutrophils, and oxidative inactivation of the
protease inhibitors.

Therapy for α1-Antitrypsin Deficiency

The presence of methionine at the reactive site of AAT
renders the molecule more susceptible to inactivation by
oxidants present in cigarette smoke or released by phago-
cytes. Using recombinant DNA technology, the methionine
residue at position 358 could be replaced by valine in the
AAT gene (Table 9-7). It has been demonstrated that the
genetically modified AAT is more resistant to oxidant damage
than the native molecule. In principle, ZZ-susceptible indi-
viduals can be given transfusions of the modified AAT with
its enhanced capacity to inhibit neutrophil elastase. Several
A constructs have shown promising results in preclinical and
early clinical trials.
The most successful treatment strategies begin with life-
style alterations, such as discontinuing smoking, bronchodila-
tion, oxygen therapy, and pulmonary rehabilitation. As the
disease progresses, AAT replacement therapy is available for
those with signs of emphysema to minimize lung damage and
to slow the progression of the disease. Human AAT is obtained

TABLE 9-7. Modification of AAT Reactive Site by

Change of Methionine to Valine to
Decrease Inactivation by Oxidants

PROTEASE
B INHIBITOR TARGET

Figure 9-12. AAT deficiency leads to an increased risk of α1-Antitrypsin Pro-Met*-Ser-Ile-Pro-Pro Elastase
emphysema. Alveolar walls break down, resulting in Engineered Pro-Val*-Ser-Lie-Pro-Pro Elastase
overinflation and permanent enlargement. A, Normal lung. oxidation-
B, Emphysema lung. (Reproduced by permission of Talecris resistant mutant
Biotherapeutics, Inc., http://www.prolastin.com.) *Reactive site.
 Questions 175

from human serum and administered intravenously. As the

disease approaches the severe state, surgery options such as KEY CONCEPTS
lung volume reduction surgery and lung replacement become ■ Familial hypercholesterolemia is caused by a defect in the LDL
the best options. receptor. Defects can occur in any site of gene expression,
product processing, and presentation on the membrane. Com-
pound heterozygotes can occur.
■ LDL receptor gene demonstrates “exon shuffling” in evolutionary
development to the current gene.
MICROBIOLOGY ■ Excess cholesterol in circulation can be identified in xanthomas,
xanthelasmas, corneal arcus, and heart disease.
Risks of Human-Derived Products
■ Most mutations for long QT syndromes are channelopathies, and
There is a risk of transmitting infectious agents with human-
potassium channels are most often affected. LQT syndromes
derived products. Viruses include hepatitis B, hepatitis C,
generally have initiating factors and may be the cause of some
human immunodeficiency virus (HIV-1, HIV-2), parvovirus,
sudden infant deaths.
herpes simplex virus type 1, cytomegalovirus, and Epstein-
Barr virus. Nonviral pathogens may include Creutzfeldt- ■ Cystic fibrosis is the most common autosomal recessive disorder
Jakob disease. Some strategies to inactivate pathogens are among Caucasians and is caused by mutations in a transmem-
nanofiltration, absorption, precipitation, chromatography, brane conductance regulator protein. Effects are present in exo-
pasteurization, and heat inactivation. crine glands.
■ CFTR mutations are associated with congenital bilateral absence
of the vas deferens and chronic rhinosinusitis syndrome.
■ CF screening uses a panel of the 25 most prevalent mutations.
Negative results could be “false” negatives.
ANATOMY & PHYSIOLOGY ■ α1-Antitrypsin (AAT) deficiency is one etiology of chronic obstruc-
tive pulmonary disease. AAT normally inhibits neutrophil elas-
Estimating Static Lung Volume following Lung tase. AAT deficiency allows neutrophil elastase to destroy lung
Volume Reduction Surgery connective tissue. Smoking exacerbates AAT deficiency.
Lung volume reduction surgery (LVRS) removes diseased
lung tissue—usually 20% to 30%—because disease alters
the mechanics of breathing. This allows the remaining lung
to function with more normal mechanics. In emphysema, it
is important to remove the diseased lung because the ●●● QUESTIONS
disease destroys elastin in the extracellular matrix.
1. A 10-year-old child with severe heterozygous famil-
∆v L ial hypercholesterolemia has a total cholesterol of
C=
∆PTP 416 mg/dL and an LDL cholesterol of 360 mg/dL.
Three treatments were implemented: dietary control,
Therefore, for the above equation, static compliance (C) is sitosterol, and bezafibrate. Each treatment lasted 3
greatly increased with the same transpulmonary pressure months, resulting in a 28% to 50% decrease in LDL
(PTP); the diseased lung enlarges (VL) greater than a normal cholesterol. Which of the following is most likely
lung but has less elastic recoil. A graph of emphysema and deficient in this patient?
normal static pressure volume will show a greater slope for
A. Actin
emphysema.
B. Dihydropyridine receptor
C. LDL receptor
D. Myosin binding protein C
E. Ryanodine receptor
There is compelling evidence that the ZZ individual is Answer. C
more susceptible to cirrhosis of the liver and liver cancer
Explanation: This child has familial hypercholesterolemia.
than are normal individuals in the general population.
A defect in the LDL receptor prevents LDL from binding
Liver disease apparently relates to the sustained aggrega- and transport into the cell. For this reason it remains in
tion of the Z-type AAT in liver cells, where AAT is pri- the blood. Actin is associated with myosin for contractility,
marily synthesized. Even with a modicum of success of allows cell motility, and is part of the cytoskeleton. The
the current genetically engineered AAT, it will be neces- dihydropyridine receptor functions as a voltage-gated cal-
sary to prevent accumulation of the Z protein in the liver. cium channel and is also critical for excitation-contraction
Current research efforts are directed at deactivating, or coupling in a voltage-sensitive and calcium-independent
“turning off,” the Z form of the AAT gene, as well as manner; mutations in this receptor cause hyperkalemic
fostering secretion of the abnormal protein. Replacement periodic paralysis. The ryanodine receptor is a skeletal
of the diseased liver with a liver transplant remains a muscle receptor that is fundamental to the process of
feasible option. excitation-contraction coupling and skeletal muscle calcium
 176 Cardiopulmonary Disorders

homeostasis. Myosin binding protein is a myosin- Explanation: Four hundred probands have a total of 800
associated protein found in the cross-bridge–bearing zone possible alleles at the KCNQ1 locus. Each individual has
(C region) of A bands in striated and cardiac muscle; LQT1 syndrome. The inheritance in these individuals sug-
mutations in this protein are associated with hypertrophic gests autosomal recessive inheritance because parents are
cardiomyopathy. unaffected. The presence of 79 bi-alleles demonstrates that
some individuals had two different alleles. Two different
2. A 28-year-old female with a four-generation family
alleles in individuals with autosomal recessive inheritance
history of early-onset coronary artery disease and
who demonstrate the disease is a compound heterozygous
myocardial infarctions presents for evaluation. Lab-
condition. The two alleles may affect the protein differently
oratory results are as shown:
such that neither can produce a normal product. Another
example of this occurs in familial hypercholesterolemia.
PATIENT NORMAL 4. A 24-year-old male presented with a history of
reduced ejaculatory volume and infertility. Physical
Total cholesterol 383 mg/dL 120–140 mg/dL examination revealed normal testes and epididymis
Triglycerides 65 mg/dL 40–150 mg/dL but the vas deferens were bilaterally absent. FSH,
HDL cholesterol 49 mg/dL >40 mg/dL LH, prolactin, and testosterone levels were normal.
LDL cholesterol 321 mg/dL 60–160 mg/dL
Semen analysis demonstrated reduced volume and
Glucose 88 mg/dL <100 mg/dL
ALT/AST Normal — azoospermia. Which of the following is the most
TSH Normal — likely diagnosis in this male?
Urinalysis No protein No protein A. Cystic fibrosis
B. Hemochromatosis
Which of the following transport mechanisms is pri- C. Kallmann syndrome
marily affected in this patient? D. Klinefelter syndrome
E. Testicular adrenal rest tumor
A. Active transport
B. Lysosomal degradation Answer. A
C. Osmosis Explanation: Males with cystic fibrosis (CF) generally present
D. Passive diffusion with infertility, but this may also occur in other disorders. If
E. Receptor-mediated endocytosis there is one mild and one more severe mutation, congenital
Answer. E bilateral absence of the vas deferens may occur but the indi-
vidual may have only mild CF symptoms and not have been
Explanation: This patient has elevated LDL cholesterol,
diagnosed. Iron deposition in the hypothalamus and pituitary
suggesting a defective LDL receptor, which is the mutation
can lead to sexual dysfunction. Kallmann syndrome, which
for familial hypercholesterolemia. In this case LDL should
is most commonly associated with anosmia, has decreased
be removed by binding to LDL receptors, located predomi-
gonadotropin-releasing hormone leading to hypogonadism
nantly in the liver. ApoB100 is exclusive to LDL and is
and reduced infertility. In Klinefelter syndrome the seminifer-
required for binding to the receptor and endocytosed at
ous tubules are hyalinized; testosterone levels will be low and
clathrin-coated pits. The endosomes fuse with lysosomes
follicle-stimulating hormone (FSH) and luteinizing hormone
for degradation that releases cholesterol. The receptor is
(LH) levels will be high. Testicular adrenal rest tumors (TARTs)
recycled to the surface and cholesterol is used for steroid
are not tumors but benign hyperplasia of adrenal germ cells
hormones, bile acids, lipoproteins, and regulator actions.
that have descended with the testes during development.
Lysosomal degradation is affected because there is a
They are associated with congenital adrenal hyperplasia
decrease in endocytosis, but this is a secondary rather than
and are demonstrated in inadequate glucocorticoid therapy
a primary effect.
such that adrenocorticotropic hormone is high and feedback
3. A study of KCNQ1 genes in 400 unrelated probands to the hypothalamus/pituitary is inadequate.
with long QT syndrome (LQT1), who had no affected
parent, identified 79 bi-allelic mutations in the gene. Additional Self-assessment Questions can be Accessed
Which of the following is expected in this population at www.StudentConsult.com
of unrelated affected individuals?
A. A large number of compound heterozygotes
B. Decreased reproductive fitness with allelic mutations
C. Earlier age of onset of symptoms with multiple
mutations
D. More affected heterozygotes than homozygotes
E. Variations in the triplet repeat expansion at the locus
Answer. A
Renal, Gastrointestinal, 10
and Hepatic Disorders
CONTENTS Finally, several disorders of the liver are discussed. The
intestine must absorb metal ions correctly or an overload or
deficiency may occur, as discussed for iron and copper. Both
RENAL DISORDERS
are toxic if not transported correctly and excreted properly.
Renal Agenesis Some of the same genes are involved in the transport of both
Multicystic Renal Dysplasia metals.
Cystinuria

GASTROINTESTINAL DISORDERS ●●● RENAL DISORDERS

Hirschsprung Disease Urinary tract abnormalities are present with approximately
HEPATIC DISORDERS
40% of all congenital abnormalities and are frequently asso-
ciated with syndromes. Likewise, there is an increased risk
Hemochromatosis for urinary tract abnormalities with structural anomalies of
Menkes Syndrome most organ systems. Chronic renal failure in infants and chil-
Wilson Disease dren results from congenital errors of development in a
majority of cases (Box 10-1). With complex adult phenotypes
associated with renal failure, such as hypertension, more
subtle defects of development are suggested. Among the
many disorders described in the intestine are more than 250
syndromes and single-gene disorders that confer an increased
risk for urinary tract abnormalities.

The hepatic, renal, and gastrointestinal systems are linked by

function, and considering them together provides an oppor-
tunity to view several disorders that affect more than one
organ either primarily or secondarily. This chapter begins by ANATOMY & EMBRYOLOGY
addressing renal development and a few disorders that may Kidney Abnormalities
cause kidney development to fail or occur inappropriately. A
Kidney anomalies in shape and size occur in 3% to 4% of
metabolic disorder affecting reabsorption within the kidney
newborns and include:
and intestine draws attention to the fact that transport across ■ Renal agenesis
membranes may occur by similar mechanisms in different ■ Malrotated kidneys: the kidney maintains its embryonic
organs, causing similar or different problems. position; often associated with an ectopic kidney
A well-known disorder of intestinal development, Hir- ■ Ectopic kidneys: most are located in the pelvis and result
schsprung disease, emphasizes the role of progenitor cell from a failure to “ascend”
function; in this case, the progenitor cells are neural crest ■ Horseshoe kidney: the poles of the kidney fuse; usually

cells. Although the hallmark of Hirschsprung disease is mega- asymptomatic

colon, this disease may occur in association with syndromic ■ Duplications of the urinary tract: duplications of the

disorders and as such is a concern of physicians evaluating abdominal part of the ureter and renal pelvis are common;
children with chromosomal abnormalities. Equally interesting a supernumerary kidney is rare
■ Ectopic ureter: the ureter opens anywhere except into the
is that the gene that causes Hirschsprung disease is the same
bladder
gene involved in normal development of the kidney and ■ Ureteric ectopia: the ureter is not incorporated into the
other organs; however, when mutated, it may also result in posterior part of the bladder
multiple endocrine neoplasia and familial medullary thyroid ■ Cystic disease
carcinoma.
 178 Renal, Gastrointestinal, and Hepatic Disorders

Box 10-1. CLINICAL FEATURES ASSOCIATED WITH TABLE 10-1. Examples of Genes Important in
A RENAL ANOMALY Kidney Development

Edema Oligohydramnios GENES PROTEIN FUNCTION

Exstrophy of the bladder Imperforate anus
Anuria-oliguria Ambiguous genitalia
Aniridia Hemihypertrophy GDNF Glial cell line–derived Binds to RET-GFRα1
Supernumerary nipples Preauricular pits and ear tags neurotrophic factor and regulates
Hypertension Polydipsia ureteric development
Recurrent urinary infections Prune-belly abdomen WNT WNT4—expressed in
Abdominal mass Polyuria family mesenchymal
pretubular
aggregates; required
for nephrogenesis
WNT6, WNT7,
WNT11—expressed
Box 10-2. CLINICAL CONDITIONS ASSOCIATED in ureteric bud
WITH DIFFERENTLY NAMED CHROMOSOME WNT11—involved in
22p11 DELETIONS ureteric bud
branching
BMP Bone morphogenic BMP4—expressed in
Velocardiofacial syndrome
family protein mesenchyme; may
DiGeorge sequence antagonize ureteric
CATCH-22 (cardiac defect, typical facial dysmorphism, bud development;
mental deficiency, and chromosome 22q11.2 deletion) controls GDNF
Conotruncal anomaly face syndrome expression
Opitz G/BBB syndrome FOXD1 Expressed in renal
CHARGE association with Cayler cardiofacial syndrome stroma and required
for ureteric branching
and nephrogenesis
WT1 Wilms tumor protein Crucial for
nephrogenesis;
Renal Agenesis regulates SPRY1
SPRY Sprouty Expressed in ureteric
Organogenesis of the kidney is initiated when the ureteric family bud
bud forms as an outgrowth of the wolffian duct. The failure
of the kidney to develop—agenesis—differs from atrophy of
renal tissue, although the latter may mimic agenesis. True
agenesis results from failure of the ureteric bud to develop or
maldevelopment of the metanephric blastema between the to 4.9 in 10,000 births for unilateral renal agenesis. Obviously,
25th and 29th days of development. Along with failure of the bilateral agenesis is fatal, and the infant is either stillborn or
kidney to develop, both ureters and renal arteries are absent. dies within a few hours of birth. Unilateral agenesis may be
Renal agenesis may be associated with chromosomal abnor- asymptomatic and an incidental finding (Fig. 10-1). In these
malities such as trisomy 21, trisomy 22, trisomy 7, trisomy cases, the adrenal gland is absent in less than 10% of cases.
10, and Turner and Klinefelter syndromes. It also has been However, the ipsilateral fallopian tube in females and the
associated with microdeletions of chromosome 22q11 (Box seminal vesicle in males may be absent. Most cases of renal
10-2) and chromosome 11p13; the latter deletion contains agenesis are spontaneous, although a few reports offer
the Wilms tumor gene (WT1) that produces an essential tran- evidence of autosomal dominant or recessive inheritance.
scription factor for proper urogenital development. In addition to failure of the kidney and ureters to develop,
The molecular mechanisms determining kidney formation oligohydramnios occurs and leads to other deformations such
are complex, and although they are not well understood, it is as a constricted chest cavity, underdeveloped lungs, and pul-
increasingly clear that inductive signaling pathways direct the monary hypoplasia. This occurs because amniotic fluid is
process. Several pathways important in kidney development mainly derived from fetal urine. This deformation sequence
are important in other organs. Some will be recognized as presents as Potter facies, characterized by hypertelorism,
proto-oncogenes that were discussed in Chapter 5 (Table low-set ears, receding chin, tapering fingers, and infraorbital
10-1). Of the known genes involved in development, muta- folds (Box 10-3; Fig. 10-2). Potter facies is not pathognomonic
tions in the human SPRY2 (sprouty) gene result in complete for renal agenesis, since any abnormality causing oligohy-
agenesis, reduced size, or lobularization or cystogenesis of the dramnios may cause this presentation.
ureteric bud. In one case, inappropriate SPRY2-mediated sig-
naling was identified in an ectopic organ with a complete
ureteric bud.
Multicystic Renal Dysplasia
The incidence of renal agenesis varies, ranging from 0.4 to An abdominal mass in a newborn most commonly results
3.9 in 10,000 births for bilateral renal agenesis and from 0.4 from unilateral or bilateral multicystic kidneys. Normally, the
 Renal Disorders 179

Unilateral Bilateral
Normal renal agenesis renal agenesis

Figure 10-1. Unilateral and bilateral

renal agenesis. (Courtesy of Dr.
Philippe Jeanty, Women’s Health
Alliance, Nashville, TN.)

Box 10-3. TYPES OF STRUCTURAL DEFECTS

IN DEVELOPMENT

Malformation: Poor tissue formation

Deformation: Unusual forces on normal tissue
Disruption: Breakdown of normal tissue
Dysplasia: Abnormal organization of normal cells

Figure 10-3. Multicystic renal dysplasia. The kidneys are

asymmetric with variably sized cysts. Small ureters lead to a
hypoplastic bladder. (Courtesy of Edward Klatt, MD, Mercer
Medical School, Savannah, GA. Used by permission of the
Spencer S. Eccles Health Sciences Library, University of Utah
Health Sciences Center; http://www-medlib.med.utah.edu.)

metanephric blastema differentiates into renal parenchyma

under the influence of the ureteric bud. In the absence of
normal induction of the metanephric blastema, multicystic
dysplasia occurs (Fig. 10-3). For unknown reasons, the left
kidney is involved more often than the right.
Most cases of unilateral multicystic renal dysplasia (MRD)
Figure 10-2. Potter facies. (Courtesy of Edward Klatt, MD,
Mercer Medical School, Savannah, GA. Used by permission undergo spontaneous involution for unknown reasons,
of the Spencer S. Eccles Health Sciences Library, University although it is suspected that cystic fluid is reabsorbed. In
of Utah Health Sciences Center; http://www-medlib.med.utah. other cases, unilateral MRD may be asymptomatic until
edu.) adulthood. Bilateral MRD is usually associated with
 180 Renal, Gastrointestinal, and Hepatic Disorders

oligohydramnios and Potter facies and usually results in

stillbirth or death within the first few days. Numerous non- TABLE 10-2. Malformations and Anomalies
renal malformations may be associated with MRD involving Reported with Multicystic Renal
Dysplasia
the gastrointestinal, neurologic, cardiovascular, and muscu-
loskeletal systems. Dysmorphologies may also be identified
SYSTEM FEATURES
(Table 10-2).
The incidence of unilateral MRD is 1 in 4300 live births.
Together, unilateral and bilateral MRD have an incidence of Urogenital Bladder wall diverticulum
Contralateral renal agenesis
1 in 3600 live births. Familial multicystic renal dysplasia has
Cystic dysplasia of the testis
been described several times. An example of such a family is Ectopic kidney
the occurrence of two children affected with unilateral MRD Fibromuscular dysplasia
and a father who was also discovered to have unilateral MRD. Fused renal ectopia
The mother had terminated one pregnancy because of bilat- Horseshoe kidney
Hypoplasia
eral MRD with a Potter anomaly. Neither the two children
Patent urachus
nor the father nor the aborted fetus had any additional Seminal vesicle abnormalities
malformation. This and other cases support an autosomal Ureterocele
dominant inheritance, but most cases are associated with Gastrointestinal Duodenal atresia
chromosomal abnormalities (Box 10-4). Esophageal atresia
Hirschsprung disease
Imperforate anus
Cystinuria Inguinal hernia
Omphalocele
Historically, cystinuria was one of the original inborn errors Tracheoesophageal fistula
of metabolism identified by Sir Archibald Garrod in 1908.
Neurologic Anencephaly
Cystine is produced endogenously from the metabolism of Caudal agenesis
dietary methionine, as seen in Chapter 4 (see Fig. 4-5). It is Caudal regression syndrome
transported across the epithelial cells of the gastrointestinal Congenital deafness
tract and reabsorbed at the brush border of the proximal renal Hydrocephalus
tubules. Amino acids are readily filtered by the glomerulus Mental retardation
Microcephaly
and undergo nearly complete reabsorption by proximal Microphthalmia
tubular cells. Only 0.4% of the filtered cystine normally Myelomeningocele
appears in the urine. A common pathway exists for cystine, Spina bifida
lysine, arginine, and ornithine, and therefore a defect results Cardiovascular Aortic stenosis
in failure of reabsorption for all four amino acids. However, Coarctation of the aorta
cystine is the least soluble of these amino acids, and as a Patent ductus arteriosus
result it is the only one that is pathogenic. Pulmonary stenosis
Dietary cystine is absorbed from the small intestine in a Truncus arteriosus
Ventricular septal defect
manner similar to that of the kidneys. Therefore, a defect is
expected to affect intestinal absorption as well as reabsorp- Musculoskeletal Clinodactyly of the fifth finger
Congenital dislocation of the hip
tion from the kidneys. However, in the absence of cystine Flexion deformities of the fingers
absorption, cysteine may still be synthesized from methionine Syndactyly
and homocysteine metabolism. The consequence of increased Talipes equinovarus
and insoluble cystine in the urinary tract is the formation of
renal stones—nephrolithiasis. Onset can occur at any time Dysmorphic Features
beginning after about age 1 year, but most stones present with
the painful symptoms of urinary obstruction in the second to Bipartite uterus
third decade. Twenty-five percent of affected individuals first Cleft palate
have stones in the first decade of life; 30% to 40% of affected Epicanthal folds
individuals experience stones during the second decade. Hymenal atresia
Hypertelorism
Although renal calculi, or kidney stones, are often the only Low-set ears
presentation, several uncommon manifestations may also Macroglossia
occur, such as frequent urinary tract infections, chronic Macrosomia
backache, and hematuria. Micrognathia
As noted for other metabolic disorders, cystinuria is an Preauricular pits
Pigment defects of iris and hair
autosomal recessive disorder. Originally, three types of cys- Short neck
tinuria were distinguished. In cystinuria type I, all four amino
acids—cystine, lysine, arginine, and ornithine—are excreted
in the urine in high concentration. Types II and III demon-
strate an incomplete recessiveness in which heterozygotes
 Gastrointestinal Disorders 181

have elevated amino acid excretion and variable intestinal dysplasia can be associated with different disorders, including
absorption. All three forms were thought to be allelic and several that involve the gastrointestinal system. In this section,
expressed from the SLC3A gene (solute carrier family member a disorder of intestinal function is discussed.
3A) on chromosome 2 until a second gene was identified in
a different cohort of individuals. The second gene encodes a
subunit that associates with the active transporter produced
Hirschsprung Disease
by SLC3A; this gene, SLC7A9, is now recognized as being Hirschsprung disease, or aganglionic megacolon, is a congeni-
responsible for all non–type I cystinuria. A new terminology tal disorder characterized by the absence of neural crest–
has been proposed to classify cystinuria as type A, formerly derived neurons along part of the distal large intestine. This
type I, and type B, formerly types II and III (Table 10-3). is also referred to as a neurocristopathy—a term used to
Cystinuria is one of the most common errors in amino acid describe lesions related to aberrations in neural growth,
transport, occurring at a frequency of 1 in 7000 individuals. migration, or differentiation that occur during embryologic
Most of these individuals have type A cystinuria. Type B development. The abnormalities or dysfunctions of neural
disease is relatively rare, although an increased incidence of crest cells include carcinoid tumors, neuroblastoma, neurofi-
cystinuria exists among individuals of Libyan Jewish ancestry bromatosis, pheochromocytoma, and Hirschsprung disease.
resulting from a founder effect. Founder effects occur in small Normally, neural crest cells originating primarily at the vagal
populations originating from very few founders that recruit level of the hindbrain and sacral regions of the spinal cord
few newcomers to the community, preferring instead to marry migrate to regions of the intestine and rectum. Failure of these
within the community. Approximately 1 in 2500 persons of cells to migrate or differentiate properly results in the disease.
Libyan Jewish descent has type B disease (Fig. 10-4). The
carrier frequency in this population is around 1 in 25, the
same as the frequency for cystic fibrosis, which is the most PATHOLOGY & PHYSIOLOGY
common autosomal recessive disorder among whites. Hirschsprung Disease
Owing to improper neural crest cell development in the
intestine, the Meissner submucosal and Auerbach myenteric
plexuses are missing. Functional obstruction and dilatation
PHARMACOLOGY proximal to the affected segment occurs, with dilatation
occurring proximal to the aganglionic segment. Mucosal
Treatment of Cystinuria
inflammation or stercoral ulcers may also occur.
Several drugs are used to treat cystinuria, all of which have Constipation, leading to an enlarged colon, is secondary to
free sulfhydryl groups that can form mixed disulfides with failure of the aganglionic segment to relax in response to
cysteine. The mixed disulfides are much more soluble than proximal distention of the internal anal sphincters following
cystine. Tiopronin and d-penicillamine bind to excess cystine rectal distention.
and increase solubility. However, the patient must be The rectum is always affected, and the intestinal
monitored for efficacy and tolerance. submucosa fails to stain with acetylcholinesterase.
Mercaptopropionylglycine and captopril are not as
effective as tiopronin or d-penicillamine, but they have fewer
side effects. Box 10-4. SYNDROMES ASSOCIATED WITH
MULTICYSTIC RENAL DYSPLASIA

Beckwith-Wiedemann syndrome
●●● GASTROINTESTINAL DISORDERS Trisomy 18
Waardenburg syndrome
As demonstrated for cystinuria, mutations in a single gene
Joubert syndrome
may have effects in organs within different systems, such as VACTERL (vertebral, anal, cardiac, tracheal, esophageal,
intestine and kidney, because the proteins function in differ- renal, and limb) association
ent places. Similarly, disorders such as multicystic renal

TABLE 10-3. Classification of Cystinuria

CLINICAL FEATURE OF HETEROZYGOTE

TYPE GENE LOCATION Urinary Excretion Intestinal Absorption

I or A SLC3A1 Chr 2p16.3 Normal Abnormal

II, III or B SLC7A9 Chr 19q13.1 Moderate increase in cystine and dibasic amino acids Nearly normal to severely
impaired
 182 Renal, Gastrointestinal, and Hepatic Disorders

Urinary
excretion 2259 2156 2199 15247 268 Creatinine
mmol/g

bp ND

196
177

Figure 10-4. Mendelian inheritance of mutation G259R of

SLC7A9 in a non–type I cystinuria consanguineous family.
Haplotypes (vertical open and filled bars) were obtained with
markers from cystinuria non–type I locus on chromosome 19.
Open chromosomes are normal but not necessarily identical,
and filled chromosomes are identical cystinuria-transmitting
chromosomes. The sum of the urinary excretion of cystine, Figure 10-5. Long-segment Hirschsprung disease. The
lysine, arginine, and ornithine is reported for each individual arrow indicates the area of the transition zone between the
as μmol/g of creatinine. Shown is detection of mutation enlarged area that has ganglion cells (normal) and the small
G259R by the DdeI site generated using BR1 and the forward area that does not (Hirschsprung disease). Following barium
mutagenesis primer BF2 (5′-CTGCCTTTGGCCATTATCCTC-3′; enema and radiography, a biopsy confirms the presence or
the underscored character indicates the mutated nucleotide). absence of ganglion cells. (From Moore KL, Persaud TVN.
The undigested (ND) band is 196 bp. The mutation results in The Developing Human, 7th ed. Philadelphia, WB Saunders,
two bands of 177 bp and 19 bp (not shown) after the 2003, p 282.)
mutated allele is digested by DdeI.

Typically, newborns and infants with Hirschsprung disease groups, the incidence for whites, blacks, and Asians is 0.75,
have intestinal obstruction and subsequent colon distention 1.05, and 1.4 per 5000 live births, respectively.
resulting from a lack of peristalsis. The disease is variable, and Hirschsprung disease is a multigenic disease and is associ-
the region of the colon affected varies. Aganglionosis is ated with mutations in at least 9 to 11 genes (Table 10-4). In
restricted to the rectosigmoid colon, also referred to as short- addition to those shown in the table, the gene for the glial
segment disease, in 80% of individuals; in 15% to 20% of cell line–derived neurotrophic factor receptor (GRFA1) and
these cases, aganglionosis extends proximally to the sigmoid PAX3, a DNA-binding protein important in early neurogen-
colon. Total intestinal aganglionosis with the absence of gan- esis, have been identified in animal models. Many of these
glion cells from the duodenum to the rectum is rare, repre- genes control morphogenesis and differentiation of the enteric
senting only 5% of cases; this is referred to as long-segment nervous system. Of particular interest among these genes is
disease (Fig. 10-5). the RET gene.
Hirschsprung disease is the most common form of func- RET is expressed in tissues of neural crest origin and is a
tional intestinal obstruction in infants, with an incidence of 1 susceptibility gene for several inherited diseases, including
in 5000 in live births. The disease is more common in males, the multiple endocrine neoplasia (MEN) syndromes and
with a ratio of 4 to 1, and most of this is short-segment Hirschsprung disease. This gene was originally identified as a
disease. In contrast, for long-segment disease, the male-to- proto-oncogene, encoding a receptor tyrosine kinase (Fig.
female ratio is only 1.5 to 2. In 70% of cases, Hirschsprung 10-6). The transmembrane protein is composed of a signal
disease occurs as an isolated disease, but it may also occur peptide, a cysteine-rich region, a transmembrane region, a
with a chromosomal abnormality, and the incidence can vary conserved intracellular tyrosine kinase domain, and an extra-
with the different associated genetic syndromes. For example, cellular cadherin-related binding domain. This transmem-
classic congenital central hypoventilation syndrome is associ- brane receptor is the signaling component of receptor
ated with Hirschsprung disease in 16% to 20% of affected complexes with four ligands: glial cell line–derived neuro-
individuals. Twelve percent of Down syndrome patients have trophic factor (GDNF), neurturin (NRTN), artemin (ARTN),
Hirschsprung disease. In Bardet-Biedl syndrome, Hirschsprung and persephin (PSP). Members of the GFRα family bind to
disease is found in 2% of affected individuals. In cartilage- RET, permitting the binding of a specific ligand. RET/GFRα1
hair hypoplasia, it is present in 9%. Among different racial binds GDNF, RET/GFRα2 binds NRTN, RET/GFRα3 binds
 Hepatic Disorders 183

TABLE 10-4. Genes Associated with c-RET

Hirschsprung Disease
CAD
GFRα1
PROTEIN
GENE LOCATION FUNCTION FREQUENCY
CAD

RET Chr 10q11.2 Tyrosine 70–80% long

kinase segment GDNF
CAD
receptor 50% familial
15–20%
sporadic
Cell
GDNF Chr Glial cell <10% membrane
CAD
5p12-13.3 line–derived
neurotrophic
factor
NRTN Chr 19q13.3 Neurturin, RET <1% PKA-ACAP 1 P
ligand complex 2 P SRC
EDN3 Chr 20q13 Endothelin B <10%
EDNRB Chr 13q22 Endothelin B <10%
TK
receptor
ECE-1 Chr 1p36.1 Endothelin- <1%
converting
enzyme 3 P
SOX10 Chr 22q13.1 SRY/HMG box <1% TK
transcription
factor 4 P
PHOX2B Chr 4p12 Paired-like <1% Promotes cell survival,
homeobox 5 P proliferation, differentiation,
2b motility, or oncogenesis
6 P
SIP1 Chr 2q22 SMN- 6 Cases
interacting
protein
Figure 10-6. RET signaling receptor. GDNF binds
Data from Puri P, Shinkai T. Pathogenesis of Hirschsprung’s disease and its preferentially to the sphingolipid-rich region of GFRα1 and
variants: recent progress. Semin Pediatr Surg 2004;13:18–24. activates the SRC family of tyrosine kinases. Binding of GDNF
to GFRα1 also causes recruitment of cRET and dimerization,
resulting in activation of tyrosine kinases and phosphorylation
of six tyrosines in the cytoplasmic domain. Four of the
tyrosines are binding sites for signaling proteins important in
a variety of cellular functions. CAD, cadherin-like domains that
ARTN, and RET/GFRα4 binds PSP. The enteric nervous bind Ca++; GDNF, glial cell line–derived neurotrophic factor;
system fails to develop in the absence of RET-GFRα1/GDNF GFRα1, GDNF family receptor α1; TK, tyrosine kinase.
signaling. Less dramatic effects are seen in the absence of
RET-GFRα2/NRTN signaling.
Mutations in the RET gene are also responsible for MEN Wilson disease will be discussed along with Menkes syn-
type II cancer syndromes: MENIIA, MENIIB, and familial drome, which demonstrates several similarities to Wilson
medullary thyroid carcinoma (FMTC). Each of these has an disease. As is the case for AAT deficiency, disorders of the
autosomal dominant mechanism of inheritance. RET muta- liver are not necessarily restricted to the liver and in fact may
tions in Hirschsprung disease tend to be inactivating, or loss- not be a chief complaint at presentation. Hemochromatosis
of-function, mutations, whereas those for MENII are activating. and Wilson disease can have devastating effects on the liver
As seen in Table 10-4, other mutations compromising the owing to iron and copper overload; however, they also have
developmental expression or activity of the RET pathway serious consequences in other systems. Menkes syndrome is
also cause Hirschsprung disease. However, the co-occurrence due to a mutation in a gene quite homologous to the gene
of MENII and Hirschsprung disease in some families with the causing Wilson disease. However, the effects are more pro-
same mutations suggests a common mechanism for both nounced in neurologic manifestations and catabolism because
disorders. of copper deficiency. These examples emphasize the power of
genetics in complex metabolic disorders involving the liver.
●●● HEPATIC DISORDERS
Hemochromatosis
There are three primary genetically determined diseases that
target the liver with acute, subacute, or chronic manifesta- The function of iron is controlled by the need for hemoglobin
tions. One of these, α1-antitrypsin (AAT) deficiency, is dis- synthesis. Most of the iron in the body is recycled repeatedly
cussed in Chapter 9. Here, hereditary hemochromatosis and as transferrin-bound iron is transported to marrow precursors
 184 Renal, Gastrointestinal, and Hepatic Disorders

TABLE 10-5. Classification of Types of Hemochromatosis (HFE)

TYPE GENE LOCATION PROTEIN INHERITANCE

Classic HFE or HFE-1 HFE Chr 6p21.3 Recessive

HFE-2
HFE-2A HJV Chr 1q21 Hemojuvelin Recessive
HFE-2B HAMP Chr 19q13 Hepcidin Recessive
HFE-3 TFR2 Chr 7q22 Transferrin receptor-2 Recessive
HFE-4 SLC40A1 Chr 2q32 Ferroportin Dominant

with the most common form of iron overload disorder, known

TABLE 10-6. Factors Influencing Iron Absorption
as hemochromatosis. Mutations in the transferrin receptor 2
gene (TFR2) are much less common than HFE mutations but
FACTORS INCREASING FACTORS DECREASING present a clinical picture very similar to HFE-associated
INTESTINAL IRON INTESTINAL IRON
ABSORPTION ABSORPTION
hemochromatosis. A third gene is hemojuvelin (HJV), which
is mutated in most cases of juvenile hemochromatosis. Juve-
Inadequate diet Regular blood transfusions nile hemochromatosis is rare but is associated with more
Impaired absorption High-iron diet severe iron overload than with mutations in either the HFE
Achlorhydria Iron-loading vitamins or TFR2 genes. Each of these genes is expressed in the liver,
Gastric surgery
Celiac disease
and it is now clear that the liver plays a critical role in the
Pica regulation of iron absorption through an iron regulatory
Increased iron loss hormone known as hepcidin. Specifically, hepcidin is central
GI bleeding in determining the amounts of iron that must be mobilized
Duodenal and gastric ulcers from macrophages, enterocytes, and hepatocytes.
Hiatal hernia
Gastritis from drugs or toxins
Iron absorption is influenced by a variety of factors that
Diverticulosis affect the expression of enterocyte iron transport molecules
Hookworm (Table 10-6). A brush border ferric reductase, DCYTB, reduces
Meckel diverticulum dietary iron to the ferrous state (Fe++). The brush border iron
Anemias, decreased transporter divalent metal transporter 1 (DMT1) mediates the
erythropoiesis
Hypoxia actual uptake of iron from the intestinal lumen across the
Inflammation apical membrane and into the enterocyte. Iron that is not
Pregnancy needed by the body is stored in the enterocyte as ferritin and
eventually lost by ultimate cell turnover. Iron is transferred
across the basolateral membrane and into the circulation by
that become erythrocytes, which are then ingested by macro- the iron transport protein ferroportin 1, SLCA401 (also known
phages in the reticuloendothelial system after a life span of as IREG1) and hephaestin, a ferroxidase with homology to
about 120 days. Iron is removed from hemoglobin by heme ceruloplasmin. Hepcidin expression regulates these activities.
oxygenase, and most is returned to the plasma, where it is
bound to transferrin again. Only a small quantity of iron
leaves this cycle and enters the liver and other tissues, where
it participates in the synthesis of other hemoproteins such as BIOCHEMISTRY
cytochromes and myoglobin.
Iron overload occurs by two basic mechanisms: too much Ferritin
is absorbed or too many erythrocytes are destroyed. In the The ferritin concentration in blood is related to the amount of
first case, iron in excess of the iron-binding capacity of trans- iron stored in tissues and therefore is used as a marker of
ferrin is deposited in parenchymal cells of the liver, heart, and iron load. Ferritin is an iron-containing protein complex that
some endocrine tissues. In the second case, iron accumulates is found principally in the intestinal mucosa, spleen, bone
in the reticuloendothelial macrophages. If this capacity is marrow, and liver.
exceeded, iron is then stored in the parenchyma. It should be Translation is regulated by the iron regulator protein (IRP).
apparent that the first situation is far more serious and can In low concentrations of iron, IRP binds to the iron response
element (IRE) located in the 5′ untranslated region of ferritin
lead to tissue damage and fibrosis if not corrected. Both types
mRNA and thus inhibits translation. Conversely, at high
of overload can be dangerous and lead to damage, but mac-
concentrations of iron, iron binds to IRP and changes its
rophages function to protect organs as long as possible. conformation, releasing it from IRE; ferritin mRNA is then
Several genes play an important role in the regulation of translated.
iron absorption (Table 10-5). Mutations in HFE are associated
 Hepatic Disorders 185

Hepcidin expression in the liver is up-regulated when iron

stores are increased and down-regulated when iron stores PHYSIOLOGY & PATHOLOGY
are decreased. Once expressed, hepcidin interacts directly
with ferroportin 1 at the intestine basement membrane, Transferrin
resulting in internalization and degradation of this trans- Transferrin is produced predominantly in the liver but also
membrane protein; hence, the iron associated with ferro- in the testes and central nervous system; it carries iron
portin is then released back into the cell. Thus, as hepcidin from the intestine, reticuloendothelial system, and liver
increases to decrease ferroportin at the basement membrane, parenchymal cells to all proliferating cells in the body.
Owing to the mildly alkaline pH of the extracellular fluid,
iron increases in the cell and iron transfer to the body
the iron-transferrin complex binds to membrane-bound
decreases. Since it is known that the expression of DMT1
transferrin receptors with high affinity. During endocytosis,
and DCYTB is affected by cellular iron concentrations, this iron is released from transferrin because of the decreased
suggests that signals of excess iron from the body affect pH in the endosome. The apotransferrin (transferrin without
the ferroportin 1 protein before expression of DMT1 and iron) and transferrin receptor are then recycled to the cell
DCYTB is affected. Stated differently, if iron is not trans- membrane surface, where the change in pH (more alkaline)
ported across the basolateral membrane, the iron concentra- causes dissociation of the apotransferrin from the receptor.
tion within the enterocyte increases, which leads to decreased
ferric reduction at the apical surface and decreased iron
transport into the enterocyte (Fig. 10-7). This interaction
replaces the previously held hypothesis that the crypt cells
of the duodenum regulate iron absorption. During iron overload, another receptor, TFR2, is expressed
Iron is transported in the body by transferrin, which binds and its expression is greater than TFR1 receptors. TFR2
two iron molecules. Transferrin and HFE compete for binding receptors bind more transferrin than TFR1, and this increased
sites at the transferrin receptor TFR1, found on hepatocytes TFR2-nonbinding also increases hepcidin expression. Thus,
and other cells. It is proposed that transferrin has a higher increased HFE availability and TFR2 expression can lead
binding affinity for TFR1 than does HFE and that higher to decreased absorption at the basolateral surface of the
transferrin levels lead to increased free HFE on the cell enterocyte by increased expression of hepcidin.
surface. It is further proposed that increased free HFE stimu- Mutations in either HFE or TFR2 produce iron overload
lates increased hepcidin. To summarize, once iron is absorbed because these proteins regulate hepcidin expression. Hepci-
through ferroportin, it binds to transferrin, which transports din levels are lower but detectable with these mutations, and
it to the liver. On the hepatocytes are transferrin receptors therefore iron absorption can still be regulated minimally;
that may bind HFE and transferrin proteins, but transferrin is however, regulation is insufficient to decrease the high rate
the preferred protein. As HFE is displaced or unable to find of absorption or reduce the iron stored. Mutations in hemo-
TFR1 binding sites, hepcidin expression is up-regulated by a juvelin, on the other hand, are more severe, and no hepcidin
mechanism that is not clearly understood. Hepcidin, then, is present. As might be expected, a double mutation in HFE
causes ferroportin internalization and a reduction in iron and TFR2 produces a severe phenotype.
absorption. Hereditary hemochromatosis (HH) is an autosomal reces-
sive, late-onset disease featuring altered iron metabolism.
Specifically, increased iron absorption in the gastrointestinal
tract leads to excessive iron deposits in the primary storage
targets, namely, the liver, pancreas, heart, and endocrine
organs (Fig. 10-8), resulting in a toxic situation for the organ.
There is no major mechanism for iron excretion. The heredi-
tary form of the disease differs from secondary hemochroma-
PHYSIOLOGY & BIOCHEMISTRY tosis due to acquired iron excess, as might be seen with
repeated transfusions.
Iron Absorption Clinically, symptoms for the classic hereditary form of
Dietary iron is absorbed in the duodenum from heme and hemochromatosis generally do not manifest until the fifth or
nonheme sources but by different mechanisms. Nonheme sixth decade of life. Early symptoms are generally nonspecific
iron is usually in the ferric (Fe+++) form, which is easily and can include fatigue, arthralgia, erectile dysfunction, and
complexed with anions, thereby reducing its solubility and increased skin pigmentation. With progression, hepatospleno-
absorption. Absorption is increased by glucose, fructose, megaly and tenderness occur and lead to liver fibrosis and
some amino acids, and vitamin C. Vitamin C complexes with cirrhosis. The incidence of hepatocellular carcinoma increases
iron, reducing it to the ferrous (Fe++) form, thereby
after liver damage. Iron deposition in the heart causes cardio-
enhancing its absorption.
myopathy. The accumulation of iron also initiates endo-
Heme iron, derived from myoglobin and hemoglobin, is
more readily absorbed than nonheme iron. Iron absorption
crinopathies, including diabetes mellitus, hypopituitarism,
is decreased in the presence of PO4, HCO3−, and bile hypogonadism, and hypoparathyroidism. In addition, affected
acids. individuals experience increased infections owing to decreased
hepcidin, which has antimicrobial properties.
 186 Renal, Gastrointestinal, and Hepatic Disorders

Hepatocyte High body Low body

Enterocyte
membrane iron demand iron demand

Low diferric transferrin

Low
hepcidin

Diferric
High diferric transferrin transferrin
A

High High hepcidin

hepcidin
B C

Low hepatocyte iron

Gut
Liver
Hepcidin
Low
hepcidin

Low hepcidin
High hepatocyte iron

High
hepcidin

Ferroportin 1 Iron TFR1 Apotransferrin Diferric transferrin

DMT1 and DCYTB HFE TFR2 Monoferric transferrin

Figure 10-7. Model for the regulation of intestinal iron absorption. Changes in body iron usage alter the amount of diferric
transferrin in the circulation (A). This molecule is preferentially taken up by cells requiring iron. The change in diferric transferrin
concentration is detected by transferrin receptor (TFR) 2 and the hemochromatosis protein (HFE)/TFR1 on the plasma
membrane of hepatocytes and leads to changes in the expression of hepcidin (B). Hepatocyte iron stores also affect hepcidin
production by altering the amount of TFR1 on the plasma membrane. Circulating hepcidin interacts with ferroportin 1 at the
basolateral membrane of the villus enterocytes of the small intestine, causing the iron transporter to be internalized and
degraded and reducing iron export (C). The accumulation of iron within the cell decreases the expression of divalent metal
transporter 1 (DMT1) and DCYTB at the brush border. Any changes in iron absorption affect diferric transferrin levels, thereby
completing a negative feedback loop that regulates body iron homeostasis. (Redrawn with permission from Frazer DM,
Anderson GJ. Iron imports. I. Intestinal iron absorption and its regulation. Am J Physiol Gastrointest Liver Physiol
2005;289:G631–G635.)
 Hepatic Disorders 187

been identified in Australian aborigines, Melanesians, and

Polynesians, but these are linked to HLA haplotypes com-
monly observed in Europeans, thus suggesting admixture.
Hemochromatosis is occasionally recognized in blacks with
C282Y alleles with a frequency of 1 in 6000, significantly
less than in individuals of European ancestry. Non-HFE-
associated hemochromatosis among blacks apparently results
from an unidentified gene.

BIOCHEMISTRY
Metal Ions
More than 25% of all enzymes contain metal ions or require
them for activity. The most commonly required in enzymatic
reactions are iron and magnesium. Cobalt (in coenzyme B12)
and manganese are also required.
Most are divalent ions. Iron and manganese change
Figure 10-8. Hereditary hemochromatosis. Hepatocellular oxidation states during reactions.
iron deposition (blue) is stained with Prussian blue stain in an
early stage of the disease. Parenchymal architecture is
normal. (From Kumar V, Abbas A, Fausto N. Robbins &
Cotran Pathologic Basis of Disease, 7th ed. Philadelphia, WB
Saunders, 2005.) PATHOLOGY
Iron Overload among Africans
Iron overload in Africans results from a predisposition to iron
Expression of hemochromatosis in women occurs later than loading. This condition was formerly called Bantu siderosis.
in men, presumably owing to loss of iron during menses and It occurs among Africans who drink beer from
pregnancy. As a result, onset of a full phenotypic clinical nongalvanized steel drums and differs from HFE-associated
expression for hemochromatosis generally occurs after meno- hemochromatosis. Iron loading occurs in Kupffer cells as
pause. Unlike men who initially have cirrhosis or diabetes, well as hepatocytes. However, cardiomyopathy and diabetes
women at the outset have vague symptoms of fatigue, arthral- are less frequent. Serum ferritin levels are elevated, but
gia, and pigmentation. The differences in these presentations transferrin levels may not reflect the extent of overload.
may contribute to more men being diagnosed than women There is a concern that affected individuals may be more
susceptible to infections and tuberculosis than individuals
until progressive hepatic involvement becomes significant in
with HFE-associated hemochromatosis.
women.
Nearly 90% of all HH patients are homozygous for an HFE
missense mutation that changes cysteine at amino acid residue
282 to tyrosine (C282Y). A second HFE variant that substi-
Menkes Syndrome
tutes aspartate for histidine at amino acid 63 (H63D) also is
associated with HH and typically occurs in the homozygous Copper is required in trace amounts for several reactions
state or as a compound heterozygote with C282Y. HFE het- (Box 10-5). It is absorbed in the stomach and duodenum
erozygotes may also accumulate iron but rarely, if ever, dem- and bound to albumin for transport to the liver. Two highly
onstrate clinical signs. homologous genes are involved in copper homeostasis:
The frequency of C282Y homozygotes in individuals of ATP7A, expressed in most cells, and ATP7B, expressed in
northern European ancestry is as high as 1 in 250, indicative
of a high allele frequency and characteristic of a very common
disease. However, HFE-associated hemochromatosis serves as
a classic example of incomplete penetrance in that roughly Box 10-5. ENZYMES REQUIRING COPPER AS
half of C282Y homozygotes demonstrate some degree of A COFACTOR
iron overload and only 10% develop pathologic indicators
of iron overload. For the majority of clinically identified HH Lysyl oxidase—connective tissue formation
patients, it is clear that HFE mutations serve as predisposing Superoxide dismutase—free radical scavenging
Cytochrome c oxidase—electron transfer
entities and may be necessary but not sufficient for disease
Tyrosinase—electron transfer
expression.
Monoamine oxidase—neurotransmitter formation
Among the many populations that have been studied, Dopamine β-hydroxylase—conversion of dopamine to
mutations in C282Y have not been found in individuals from noradrenaline
Africa, Asia, Southeast Asia, or Micronesia. A few alleles have
 188 Renal, Gastrointestinal, and Hepatic Disorders

hepatocytes, brain, kidney, and placenta. The liver has a addition of copper to certain enzymes. However, when cells
high affinity for copper-bound albumin, and in hepatocytes are exposed to excessive copper, the copper-transporting
the ATP7B protein facilitates copper incorporation in protein is rapidly relocated to the plasma membrane, where it
apoceruloplasmin, followed by release of copper-rich ceru- functions in copper efflux to decrease the concentration of
loplasmin in the circulation; thus, copper is a rate-limiting copper in the cytoplasm. The normal function of ATP7A is to
element for ceruloplasmin formation. move copper from the intestinal mucosa into the blood-
Ceruloplasmin, a ferroxidase, is important also for iron stream, where it is bound to albumin proteins and transported
transport. By oxidizing ferrous iron to the ferric form, ceru- to tissues. If the ATP7A protein is nonfunctional, the uptake of
loplasmin promotes iron loading onto transferrin, which copper from the intestines is impaired and copper deficiency
binds only the ferric form of the metal. Oxidizing ferrous results. Mutations in this gene result in Menkes syndrome,
to ferric iron also serves an antioxidant function and thus which is sometimes referred to as kinky hair disease. It is an
helps prevent or reduce oxidative damage to proteins, lipids, X-linked disorder characterized by early retardation in growth,
and DNA. Aceruloplasminemia shares features with other peculiar hair, and focal cerebral and cerebellar degeneration.
neurodegenerative diseases. Severe neurologic impairment begins within a month or two
of birth and progresses rapidly to decerebration.
These manifestations underscore the critical function of
copper for the proper function of several enzymes. Lysyl
BIOCHEMISTRY & PATHOLOGY oxidase is important for the cross-linking of collagen and
Ceruloplasmin elastin, and deficiencies lead to problems in connective tissue,
such as bone. The enzyme dopamine β-hydroxylase (or dopa-
Ceruloplasmin is an α-globulin. Ninety percent of copper is
mine β-monooxygenase) requires copper to catalyze the con-
bound to ceruloplasmin; each molecule binds six copper
atoms, and albumin carries the remaining 10% with less
version of dopamine to noradrenalin. Individuals with Menkes
avidity (i.e., albumin donates copper more readily). syndrome have a reduced activity of this enzyme with high
Ceruloplasmin concentration is decreased with liver levels of dopamine and low levels of noradrenalin and its
disease. Its absence, however, does not produce marked neuronal metabolite dihydroxyphenylglycol. Cytochrome
changes in copper metabolism, although it will cause a oxidase is part of complex IV of the electron transport chain
gradual accumulation of iron in the liver and other tissues. in mitochondria; it contains bound copper atoms. Since copper
This occurs because the ferroxidase activity of ceruloplasmin is insufficient, the activity of the enzyme is reduced and less
is important for oxidizing ferrous iron to ferric iron. Without ATP is produced, leading to lipid catabolism in the body to
oxidation, iron is not bound to transferrin. Copper deficiency provide adequate energy for essential biochemical reactions.
is accompanied by a hypochromic, microcytic anemia
In addition, lower cytochrome oxidase activity leads to neu-
similar to that produced by iron deficiency.
romuscular disorders and progressive brain degeneration.

The main difference in copper transport that is facilitated

Wilson Disease
by ATP7A and ATP7B lies in the direction of transport. There are only two routes for copper excretion: trap excess
ATP7A facilitates transport from the enterocyte into the cir- in the enterocyte or excrete excess in bile. Excess copper
culation and the basolateral surface of the cell. ATP7B func- intake induces metallothionein production in enterocytes that
tions to move copper out of the hepatocyte and into bile at capture it for storage followed by normal shedding. The physi-
the apical surface of the cell. Both proteins provide copper ologic route for balancing copper is through incorporation
to copper-dependent enzymes as they migrate through the into ceruloplasmin and compartmentalization into a vesicular
trans-Golgi network, the final portion of the Golgi apparatus. membrane compartment in preparation for excretion into
However, under conditions of elevated extracellular copper, bile (Fig. 10-9). Mutations in the ATP7B gene, located on
ATP7A proteins relocate to the plasma membrane and ATP7B chromosome 13q14.3-q21.1, prevent release of copper from
relocates to an intracellular vesicle to assist in copper efflux. hepatocytes and cause an inability to use or excrete the
Two genes are implicated in copper transport in the brush metal. As an additional consequence, apoceruloplasmin is
border of intestinal cells: CTR1 and DMT1. The latter has degraded and ceruloplasmin levels decrease (Table 10-7).
been discussed with iron transport and may transport as many This can be a significant event because ceruloplasmin is
as eight metals, has four or more isoforms, and carries out required for iron homeostasis and neuronal maintenance in
transport for multiple purposes. CTR1 (copper transporter 1, the central nervous system. Ceruloplasmin also serves an
also known as SLC31A1) is located on the basolateral surface acute-phase reactant with increased levels during inflamma-
as well as the apical surface, since copper can enter entero- tion, infection, and trauma. Without copper incorporation,
cytes from the blood. The mechanism of copper absorption is only apoceruloplasmin is produced.
not as well understood as that for iron. However, it is known Wilson disease was first described in 1912 as progressive
that, once in the enterocyte, most copper is bound to metallo- lenticular degeneration characterized by bilateral softening of
thionein or other proteins having a high affinity for copper. the lenticular nucleus and by liver cirrhosis. The original char-
Both ATP7A and ATP7B proteins are localized predomi- acterizations of this disease focused more on its neurologic
nantly in the trans-Golgi network, where they facilitate the aspects than the liver aspects. However, it has since become
 Hepatic Disorders 189

Figure 10-9. Model of copper uptake

and metabolism in hepatocytes.
Crossing the plasma membrane
through either CTRL1 or DMT1, most Plasma
copper is shuttled to the trans-Golgi
network (TGN) by the chaperone WND
HAH1/ATOX1, which delivers copper
to the P-type ATPase located in the CP
trans-Golgi network. In the case of CP CP
hepatocytes, this ATPase is ATP7B—the
defective protein in Wilson disease. In
enterocytes, the ATPase is ATP7A—the
defective protein in Menkes syndrome. TGN
GSH Bilis
The chaperone protein, CCS, delivers
copper to cytosolic copper/zinc
superoxide dismutase (SOD), which
MT
dismutates superoxide into hydrogen
peroxide. COX 17 delivers copper to Cu/Zn
SOD
the mitochondria, where it is required
for cytochrome c oxidase. Glutathione HAH1
(GSH) may also be a chaperone by CCS
binding copper and delivering it to COX 17
metallothionein (MT) and to some
copper-dependent apoenzymes, such HCTRL/
as SOD. (From Arrendondo M, Núñez DMT1
MT. Iron and copper metabolism. Mol Cu;
Aspects Med 2005;26:313–327.)

TABLE 10-7. Comparison Between Menkes Syndrome and Wilson Disease

GENE LOCATION DEFECT LABORATORY FINDINGS TREATMENT

Menkes syndrome ATP7A Chr Xq12-q13 Intestinal absorption Decreased serum copper Daily copper
of copper levels histidine
Decreased liver copper levels injections
Increased intestinal copper
levels
Decreased ceruloplasmin
Wilson disease ATP7B Chr 13q14.3-q21.1 Biliary excretion of Decreased serum copper Copper chelation
copper levels
Increased liver copper levels
Increased urinary copper
levels
Decreased ceruloplasmin

clear that the disease is the result of copper accumulation in in different populations. For example, the prevalence is 1 in
the liver, and only after liver disease has initiated does neu- 10,000 individuals in China, Japan, and Sardinia. As this sug-
rologic involvement begin. gests, mutations are panethnic, but the frequency of certain
Wilson disease is an autosomal recessive disease resulting mutations is ancestry dependent. The H1069Q mutation
from mutations in the adenosine triphosphatase gene, ATP7B, accounts for 35% to 45% of mutated alleles in individuals
located on chromosome 13q14.3-q21.1. Many mutations with European ancestry. Mutations in exons 8, 14, and 18
have been described that affect the transport of copper across account for 60% of the mutations detected in British popula-
the trans-Golgi network and into vesicles. These mutations tions. The R778L mutation accounts for about 57% of the
specifically cause diminished copper excretion, leading to its Wilson disease alleles in persons of Asian ancestry. Mutations
accumulation in the liver, brain, and eye. Onset of the disease in exons 8 and 12 account for 57% of Wilson disease alleles
may be as early as age 3 years in families with affected in the Chinese population. Wilson disease alleles H714Q and
members and ranges to age 50 years before diagnosis. delC2337 are identified in up to 45% of individuals with
The heterozygote frequency of Wilson disease mutations is Russian ancestry. These data underscore the importance of
1 in 90 in the general population, with the disease occurring family history in determining the correct mutation through
in 1 in 30,000 individuals. Expression of the disease varies specific laboratory instructions for screening.
 190 Renal, Gastrointestinal, and Hepatic Disorders

Clinically, Wilson disease is variable but characterized by affected individuals, they are not pathognomonic, since they
recurrent jaundice, fatigue, malaise, arthropathy, and rashes. may be associated with other causes of liver disease such as
Fulminant hepatic failure may occur with severe coagulation autoimmune hepatitis and cholestasis.
problems, encephalopathy, acute Coombs-negative hemoly- The first line of treatment is copper chelation. Dysarthria
sis, and renal failure. Chronic liver disease poses additional and tremors are more amenable to chelation therapy than
issues with separate consequences, including portal hyperten- dystonia. Renal tubule disease is usually improved with chela-
sion, hepatosplenomegaly, ascites, and low serum albumin. tion therapy. In severe disease, however, individuals may not
As the disease progresses, neurologic manifestations of respond to chelation and may require liver transplantation.
Wilson disease increase as hepatic manifestations begin to
decline. After the age of 18 years, 70% of clinical features are
neurologic and about 20% are liver associated, classified as
acute and chronic hepatitis, cirrhosis, and fulminant hepatic NEUROSCIENCE & ANATOMY
failure. Lenticular degeneration is associated with dystonia,
its most common feature. Additional features include dysar- Basal Nuclei (Ganglia) and Lenticular Nucleus
thria, tremors, dysphagia, and psychiatric disturbances. Eighty The basal nuclei (ganglia) consist of five interconnected
percent of all individuals with Wilson disease and 98.1% of nuclei: the caudate nucleus, putamen, subthalamic nucleus,
individuals with neurologic symptoms have Kayser-Fleischer globus pallidus, and substantia nigra. The lenticular nucleus
rings, seen as discoloration of Descemet’s membrane of is composed of the globus pallidus and putamen.
the corneal endothelium (Fig. 10-10). Although common in The organization of the basal nuclei (ganglia) leads to an
order of connections. Specific cells in the putamen or
caudate project to the globus pallidus or substantia nigra
and allow cortex-to–motor neuron transmission. The nucleus
is located between the caudate nucleus and the island of
Reil, or insula, with its anterior portion attached to the head
of the caudate nucleus.

MICROBIOLOGY
Coombs Test
Two Coombs tests are used to detect and characterize
antibodies: the direct and indirect. The indirect is almost
never needed routinely.
The direct Coombs test is conducted to investigate the
cause of hemolytic anemia, whether it is to diagnose
hemolytic disease of the newborn or hemolytic anemia in
A adults or to investigate hemolytic transfusion reactions. A
positive Coombs test supports autoimmune hemolysis (IgG,
complement, or both are on the surface of erythrocytes).
Many diseases can initiate autoantibody formation leading
to hemolysis. Drugs can also initiate autoantibody formation
(e.g., quinidine, methyldopa, and procainamide).

PHARMACOLOGY
Chelating Properties
Chelating agents are heavy-metal antagonists with the ability
to complex with metals to prevent or reverse their binding to
ligands. An ideal chelator has the following characteristics:
B ■ Water soluble
■ Resists biotransformation

Figure 10-10. Kayser-Fleischer rings. Yellow-brown ■ Reaches sites of metal storage

discoloration of the Descemet membrane surrounding the ■ Forms nontoxic complexes with toxic metals
cornea resulting from copper deposition. (Reproduced with ■ Active at physiologic pH
permission of the Verlag Online Journals of Ophthalmology. ■ Can be excreted
Available at: http://www.atlasophthalmology.com.)
 Questions 191

before she learned she was pregnant. Which of the

KEY CONCEPTS following best describes this infant’s renal and anal
■ Renal abnormalities are associated with many systems. anomalies (see Box 10-3)?
■ Many syndromes and single-gene diseases are associated with A. Deformation
renal abnormalities. B. Disruption
■ Renal and gonadal development are related. C. Malformation
D. Sequence
■ Abnormal renal development affects the amount of amniotic fluid
E. Syndrome
and fetal health.
Answer. C
■ The same transporter is required for cystine, lysine, arginine, and
ornithine intestinal absorption and kidney reabsorption. Muta- Explanation: Malformations occur during the first trimester
tions in the transporter affect cystine and cause cystinuria of development. Statins are teratogens that have the great-
because it is insoluble, whereas the other amino acids are more est effect during morphogenesis. (See Pharmacology Box,
soluble. Chapter 9, p 186.)
■ Hirschsprung disease is an aganglionic disease caused by
2. Two hundred unrelated patients with cystinuria pre-
failure of neural crest cells to migrate properly. It is classified
viously diagnosed on the basis of urine excretion of
as short segment, the most common presentation, or long
cystine were invited to participate in a study of the
segment.
disease. Genes involved with absorption of cysteine
■ The RET oncogene is involved in Hirschsprung disease and also and other dibasic amino acids were analyzed for
in multiple endocrine neoplasia (MENIIA and MENIIB) and famil- mutations. Data revealed few individuals with type A
ial medullary thyroid carcinoma. Other genes are mutated in cystinuria. Most individuals had type B cystinuria
some cases, but RET is mutated in most cases. and among those, one allele was present in 70%
■ Hemochromatosis is caused by mutations in several genes affect- of the individuals. Which of the following is best
ing iron absorption, transport, and binding at the hepatocytes. reflected by these data?
Most common is the HFE mutation (classic hemochromatosis).
A. Deformation
■ Hemochromatosis may develop secondary to another condition B. Disruption
in which there is RBC hemolysis, which releases hemoglobin, or C. Founder effect
after multiple transfusions, which may occur in individuals with D. Sequence
hemoglobinopathies. E. Syndrome
■ Menkes syndrome and Wilson disease are caused by defects in
Answer. C
copper homeostasis.
Explanation: The most common form of cystinuria is type I
■ Excess copper is lost in enterocytes or it is incorporated into
ceruloplasmin and prepared for excretion into bile. It may also
or type A; the designation depends on the literature source.
be deposited in the Descemet membrane surrounding the Non–type I cystinuria is more rare. In the case presented,
cornea, where it is called Kayser-Fleischer rings. most of the individuals in this population present with the
rarer form, supporting a founder effect mutation that is spe-
cific to this group. Often these types of mutations remain
high in a geographical area rather than dispersing to other
areas because of religious practices requiring marriages in
the same religion or geographical barriers that hinder emi-
gration and immigration. Disruption, deformation, sequence,
●●● QUESTIONS and syndrome are terms applied to describe how a presen-
1. A newborn infant has multiple anomalies, including tation occurred developmentally. The proper term that could
aplastic left radius and thumb, thoracic scoliosis, be applied here, but is not given as an option, is malforma-
fusion of ribs on left thoracic and lumber region tion because the disorder results from an intrinsic error in
vertebrae, esophageal stricture, anal atresia, and development (i.e., a transporter).
renal dysplasia corresponding to the VACTERL
association. The mother had taken lovastatin (10 mg/ Additional Self-assessment Questions can be Accessed
day) for 11 weeks after her last menstrual cycle at www.StudentConsult.com
Disorders of Sexual 11
Differentiation and
Development
CONTENTS by the effects of mixed genotypes, or mosaicism, in an
individual. These examples demonstrate a primary effect on
sexual development.
NOMENCLATURE
Finally, attention is given to disorders having a primary
effect on the adrenocortical pathway and a secondary effect
GONADAL DIFFERENTIATION AND DISORDERS OF
on sexual development. These outcomes revisit the underly-
SEXUAL DEVELOPMENT
ing biochemical dogma of accumulating substrate with the
Turner Syndrome inability of enzymes to produce product. In these cases, accu-
Klinefelter Syndrome mulating substrates can also be used in the production of sex
Chromosomal Mosaicism steroids and thus secondarily affect sexual development and
Triple-X Female function.
DISORDERS OF SEX STEROIDOGENESIS

Ovotesticular Disorder of Sexual Development ●●● NOMENCLATURE

46,XY Disorder of Sexual Development During the past several years there has been a universal
46,XX Disorder of Sexual Development with Maternal
movement away from terms associated with conditions of
Virilization
intersex and abnormal gonadal development. While terms
DISORDERS OF COMBINED ADRENOCORTICAL AND such as “hermaphrodite” have prevailed since Greek mythol-
SEX STEROIDOGENESIS ogy, a more psychosocially sensitive terminology is replacing
many less appropriate terms. In this chapter, the newer
3β-Hydroxysteroid Dehydrogenase Deficiency
21-Hydroxylase Deficiency
terminology for disorders of sexual development is used
11β-Hydroxylase Deficiency (Table 11-1).

●●● GONADAL DIFFERENTIATION

AND DISORDERS OF
Historically, the area of genetics that perhaps engenders the
SEXUAL DEVELOPMENT
most enthusiasm among students of all ages involves disor- Gonadal development begins during the fifth week of gesta-
ders adversely affecting normal sexual development. The best tion as a thickened ridge on the medioventral border of the
known are those caused by nondisjunction of the sex chro- urogenital ridge. During the sixth week, the primordial germ
mosomes. Unlike the outcome for a fetus or newborn that is cells, which migrated into the yolk sac during gastrulation,
monosomic or trisomic for an autosome, the same mechanism increase mitotically and migrate back through the dorsal mes-
affecting sex chromosomes results in viable individuals with entery of the hindgut to the gonadal ridge, where they become
normal or near-normal life expectancy. incorporated into the primary sex cords. The primary sex
This chapter provides highlights of genes involved in sex cords begin developing to define the outer cortex and inner
differentiation in the developing embryo and fetus, along with medulla. Before the sixth week of gestation, sex is indistin-
examples of how certain mutations change the expected guishable; primordial germ cells are undifferentiated epiblast
outcome. In addition to specific genes involved, the best cells specified during gastrulation to become germ cells. Germ
known chromosomal anomalies—Turner syndrome and cells not arriving in the gonad prior to sex differentiation
Klinefelter syndrome in particular—are discussed, followed usually degenerate, but occasionally they may persist and
 Gonadal Differentiation and Disorders of Sexual Development 193

TABLE 11-1. Revised Terminology for Disorders Pseudoautosomal

of Sexual Development region (PAR):
pairs and recombines SRY
with X chromosome
PREVIOUS REVISED

SRY is near but not in

Intersex Disorder of sexual
PAR. Recombination
development (DSD) may translocate this
Female pseudohermaphrodite 46,XX DSD gene to the X
Male pseudohermaphrodite 46,XY DSD 11.3 chromosome.
True hermaphrodite Ovotesticular DSD
XX male or XX sex reversal 46,XX testicular DSD 11.2
XY sex reversal 46,XY complete gonadal Euchromatic
dysgenesis 11.1
11.1 region

11.2

12 Heterochromatic
later develop into extragonadal germ cell tumors. The differ-
region
ence in sex differentiation is a reflection of gene expression
from the X and Y chromosomes. Under the influence of Y
chromosome genes, testis organization begins in the sixth and Y
seventh week. Ovary differentiation begins at about the Figure 11-1. Anatomy of the Y chromosome. The
twelfth week. pseudoautosomal region of the Y chromosome contains
Early cytogenetic evidence indicated that the Y chromo- genes also found on the X chromosome.
some possessed a gene or genes controlling the destiny of a
bipotential gonad to become an ovary or testis. Several can-
didate genes were investigated. The hypothetical gene sought 841-bp transcript resulting in a 204-amino-acid protein.
was called the testis determining factor (TDF). The location Within the SRY protein is a centrally located high mobility
of the gene producing this factor was localized to the short group (HMG) octamer that binds to the minor groove of the
arm on the Y chromosome through a study of patients with DNA helix, inducing a large conformational change. This
translocations and deletions of the Y chromosome. The dis- explains the observation that almost all SRY mutations in
covery of 46,XX males with a translocation of the Yp termi- 46,XY females have been localized to the HMG region:
nus to the Xp led to the discovery that the terminal region Mutant SRY proteins may not effectively bind to the DNA
of Yp was essential for testicular differentiation. to cause the conformational change needed for other genes
At the termini of Y chromosomes are pseudoautosomal to be transcribed.
regions (PARs). The few genes within these regions are the
same on the X and Y chromosomes and are inherited in an
autosomal manner, as the name implies. Males have two
copies of these genes, one allele on the Y chromosome and BIOCHEMISTRY
one on the X chromosome, just as females have one allele on
Amino Acid Motifs
each of the X chromosomes. During meiosis I, Xp and Yp
align at the PAR and recombination may occur. In transloca- Several amino acid motifs are commonly found in
transcription factors. In a helix-turn-helix motif, which
tions involving genes adjacent to the pseudoautosomal region,
includes homeodomain proteins (proteins with a recurring
molecular analyses revealed a 1.1-kb transcript expressed
60-amino-acid motif, important in controlling development),
only in testes, and this gene was designated as the sex- a recognition helix binds in the major groove of DNA and is
determining region of the Y chromosome (SRY) (Fig. 11-1). stabilized by the turn and the other helix.
In the case of 46,XX males, recombination between Xp and The zinc finger motif includes general transcription factors
Yp translocated the SRY gene to the Xp chromosome along such as TFIIIA, and SP1. This motif binds in the major
with genes in the pseudoautosomal region; some of these groove of DNA and uses a repeating zinc motif to create the
PAR genes are also expressed specifically in testes. The SRY final structure.
gene is the gene encoding the TDF and has become the In the helix-loop-helix motif, including the transcription
general designation for the gene product, which may also be factors MYOD, MYC, and MAX, two helices are separated by
referred to as the SRY protein. a loop that acts as a sequence-specific DNA binding
domain.
The SRY gene contains only one exon and no introns. The
The leucine zipper motif, including FOS, JUN, and CREB,
5′ flanking promoter region of SRY differs from the vast
does not directly interact with DNA. It contains leucine
majority of genes. It contains no TATA or CCAAT boxes, is repeats on two protein α-helices that interdigitate in a
GC rich, and contains two zinc finger recognition sites for zipper-like fashion to stabilize the protein.
the SP1 transcription factor. The single exon produces an
 194 Disorders of Sexual Differentiation and Development

Intermediate WT-1 Genital SOX9 Bipotential WNT-4+Ovarian gene(s)N

Ovary
mesoderm SF-1 ridge DMRT1,2 gonad (SOX9 ↓)
WNT-4
WT-1
46,XY complete
2X DAX1 (AHC) SRY
gonadal dysgenesis
2X WNT-4
Congenital adrenal Mutations (SOX9 ↑)
hypoplasia, Deletions Gene(s)N
hypogonadotropic
hypogonadism

Sertoli DAX1 blocks AMH

cells up-regulation

Testes

SF-1 + SOX-9
WT-1 AMH
Testes Sertoli cells AMH Müllerian duct regression
AMH gene receptors

Leydig cells

SF-1 Genes encoding steroidogenic enzymes

Testosterone Stabilization of
the wolffian ducts
Androgen
5α-Reductase receptor
Male differentiation of
DHT the urogenital sinus
and external genitalia

Figure 11-2. Diagrammatic representation of genes involved in sex differentiation. AHC, adrenal hypoplasia congenita,
hypogonadotropic hypogonadism; AMH, antimüllerian hormone; DAX1 (AHC), critical region on the X chromosome gene 1;
SF-1, steroidogenic factor-1; WT-1, Wilms tumor suppressor. (Adapted from Larsen PR, Knonenberg HM, Melmed S, et al:
Williams Textbook of Endocrinology, 10th ed. Philadelphia, WB Saunders, 2003, p 864.)

The SRY gene was identified first because of the interest in heterozygote. Consequently, among the variable phenotypic
sex differentiation. However, within the developmental expressions, Sertoli cells will either not initiate differentiation
pathway, steroidogenic factor-1 (SF1) gene expression is nec- or not be maintained. An important target for SOX9 in sex
essary for SRY transcription (Fig. 11-2). If SRY is expressed, determination is antimüllerian hormone (AMH), also called
another gene, SOX9, is up-regulated and SRY expression müllerian inhibitory factor (MIF).
remains high throughout Sertoli cell development. SOX pro- Development of both male and female gonads and adrenal
teins are related to SRY and have HMG boxes that are 71% glands requires the expression of the SF1 gene; this gene
homologous to those of SRY. also regulates the steroid hydroxylase enzyme system. Failure
SOX9 expression is required not only for proper sex dif- of SF1 expression to occur in adrenals causes a cascade of
ferentiation but also for chrondrogenesis. Mutations in the events in which insufficient corticosterone produces signifi-
SOX9 gene result in a severe dwarfism syndrome, campto- cant consequences. SF1 is expressed in the Sertoli cells
melic dysplasia, characterized by bowing of long bones and after initiation of gonad development when mitosis is sup-
skeletal dysplasia including hypoplastic scapulae, unmineral- pressed and inhibin is produced. SF-1 acts synergistically
ized thoracic pedicles, and narrowed iliac bones (Fig. 11-3). with Wilms tumor suppressor-1 (WT-1) to up-regulate AMH.
Mutations in SOX9, which can be in both the regulatory and AMH diffuses from Sertoli cells to the paired müllerian
the coding regions, also lead to XY female sex reversal (46,XY duct primordia and effects apoptosis through serine/threonine
complete gonadal dysgenesis) owing to gonadal dysgenesis protein kinase surface receptors. Further internal and exter-
in about 75% of the cases. Inherited as an autosomal domi- nal male differentiation is regulated by testosterone synthesis
nant disorder, normal development does not occur in a in the Leydig cells, beginning at about 9 weeks, and by
 Gonadal Differentiation and Disorders of Sexual Development 195

dihydrotestosterone. Testosterone synthesis may be stimu- (AHC) is caused by loss-of-function mutations in DAX1,
lated by placenta-derived human chorionic gonadotropin resulting in hypogonadotropic hypogonadism in males. In
(hCG) prior to fetal pituitary luteinizing hormone (LH) the severest clinical presentation, infants have primary
expression, since hCG and LH share regions of high homo- adrenal insufficiency characterized by hyponatremia, hyper-
logy and can interact with the same receptors on Leydig kalemia, acidosis, and hypoglycemia—a life-threatening situ-
cells. ation if untreated. Less severe mutations may present with
hypogonadotropic hypogonadism at adolescence. Mutations
resulting in this presentation are due to decreased DAX1
ANATOMY & EMBRYOLOGY expression rather than to absence of expression in the
hypothalamus and pituitary.
Adrenal Development
The adrenal cortex and medulla have different origins. The
cortex is a mesoderm derivative. It first appears in week 6 as
a mesenchyme aggregation between the root of the dorsal BIOCHEMISTRY
mesentery and the gonad. Cells are derived from the
Receptor Serine/Threonine Kinases
mesothelium of the posterior abdominal wall.
The medulla is a neural crest cell derivative. Cells of the Receptor serine/threonine kinases are one of five classes of
medulla are innervated by presynaptic sympathetic fibers, catalytic receptors. They phosphorylate serine and/or
similar to sympathetic ganglia. threonine residues on proteins. An example of these is the
transforming growth factor-β (TGF-β) superfamily. This large
family includes members important during embryogenesis
and control of cellular differentiation in adults: antimüllerian
ANATOMY hormone (AMH), inhibins, activins, bone morphogenic
proteins, and other glycoproteins.
Bowlegs The receptors are single-membrane-spanning
Bowleg is the outward curvature of the lower limbs with glycoproteins and consist of two types, RI and RII. Ligands
medial concavity. It includes genu varum and tibia vara. bind to RII but not to RI. RI recognizes ligand is bound to RII
Normal physiologic bowing is observed in newborns and but cannot itself bind ligand; however, it transmits a signal
infants and, like idiopathic bowing, generally self-corrects. In when ligand is bound to RII. Formation of a stable RII-ligand/
camptomelic dysplasia, the femoral and tibial shafts are RI complex results in phosphorylation of RI at serines and
bowed. This may be secondary to generalized bone threonines. This activates the kinase activity of RI to
softening. Examples of disorders in which bowing occurs downstream effectors.
are rickets, osteogenesis imperfecta, secondary Substrates of activated RI include the SMAD family of
hyperparathyroidism, and hypo- or hyperphosphatasia. transcription factors. When phosphorylated by the TGF-
complex, the activated factor translocates to the nucleus,
binds to DNA, and effects transcription.
Sex reversal can also occur from a duplication of a region
on the X chromosome called DSS (dosage-sensitive sex rever-
sal). It is regulated by SF1 through feedback inhibition. DSS
contains a gene designated as DAX1 for DSS-AHC critical PHYSIOLOGY
region on the X chromosome. Prior to testis determination,
Human Chorionic Gonadotropin (hCG)
DAX1 is expressed in both gonads. As SRY expression
increases from the Y chromosome, DAX1 expression declines hCG shares structural homology with luteinizing hormone
in testes. In the ovary, where SRY expression is not normal (LH), follicle-stimulating hormone (FSH), and thyroid-
stimulating hormone (TSH). It functions after binding to a G
or expected in 46,XX females, DAX1 expression is present
protein receptor on target cells, and it may also interact with
and blocks the synergistic activity of SF-1 and WT-1 to
receptors for LH, FSH, and TSH because of shared
up-regulate AMH expression; ovary development occurs. To homologies.
reiterate, when SRY expression increases, DAX1 expression is The syncytiotrophoblast, which forms the placenta,
repressed. Thus accordingly, DAX1 expression does not occur produces hCG beginning in the second week after
in testes, where SRY expression is high, and ovary formation fertilization. Production begins to decline during the eighth
is prevented. An interesting situation occurs when the DAX1 week and thereafter occurs from the chorion, the outer
gene is duplicated on the X chromosome in a male. Here, the embryonic membrane. Its major function is to maintain the
amount of SRY is insufficient to antagonize the increased corpus luteum during the first trimester of pregnancy via LH/
DAX1 activity. DAX1 remains active and continues to prevent hCG membrane receptors. This function is important because
testis formation, resulting in a 46,XY female (46,XY complete the corpus luteum secretes progesterone and estrogen to
maintain pregnancy. Excess hCG is excreted in urine and is
gonadal dysgenesis).
the basis of most pregnancy tests. During the second
DAX1 is expressed in all tissues of the steroidogenic
trimester, the placenta begins secreting progesterone, which
axis: hypothalamus, pituitary, adrenal cortex, and gonads. plays a critical role in supporting the endometrium of
As might be expected, mutations in DAX1 can affect these pregnancy and hence the survival of the fetus.
tissues. The X-linked form of adrenal hypoplasia congenita
 196 Disorders of Sexual Differentiation and Development

secretion of sex hormones is largely responsible for the

immaturity of the external genitalia.
Turner syndrome is often diagnosed at birth by the pres-
ence of a webbed neck, edema of the hands and feet, and a
very low birth weight (below the third percentile) (Fig. 11-4).
Short stature, the lack of secondary sex characeristics, and
primary amenorrhea are significant features seen later in the
development of these females. Short stature, a hallmark of
the syndrome, is generally 3 standard deviations below the
age-adjusted mean. Other features may include micrognathia,
low posterior hairline, cubitus valgus, renal anomalies, and
coarctation of the aorta. There is also an associated increased
incidence of autoimmune diseases, including Hashimoto
thyroiditis, Crohn disease, and diabetes mellitus.

PHYSIOLOGY
Secondary Sexual Characteristics
Secondary sexual characteristics are generally those
changes that occur at puberty. For females, breast and
Figure 11-3. Camptomelic dysplasia is caused by mutations associated apocrine gland development follow stimulation
in the SOX9 gene. This gene plays an essential role in sex by estrogens secreted by ovaries. Estrogen also causes the
determination as well as in chrondrogenesis. In the latter,
pelvis to widen, and causes deposition of fat in the hips,
SOX9 protein binds to sequences in the collagen gene,
buttocks, and thighs. It also induces uterine growth,
COL2A1, to regulate its expression. (Used with permission of
proliferation of the endometrium, and menses. Pubic and
GE Healthcare: http://www.medcyclopaedia.com.)
axillary hair also grow, controlled by androgens secreted by
adrenals and ovaries.
Through testosterone and dihydrotestosterone, males
experience growth of the penis, testes, and pubic hair.
Understanding the generalities of sex development and
Testosterone directly increases muscle mass and size as
several disorders that can occur with mutations allows a
well as the mass of vocal cords, which leads to deepening
better appreciation of the survival of individuals missing an of the voice, and the mass of bones, which leads to
X chromosome or having additional sex chromosomes. increased strength and changes in skeletal shape.
5α-Reductase converts testosterone to DHT at peripheral
target tissues, such as the skin, and contributes to beard
Turner Syndrome
growth, acne, and temporal balding. DHT binds to the same
Turner syndrome is a sex chromosome disorder of sexual androgen receptor as does testosterone but with greater
development with a 45,X karyotype. It results from loss of affinity, and the DHT-receptor complex binds with greater
an X chromosome through nondisjunction, which character- avidity to DNA.
izes approximately 60% of the individuals with the classic
features of Turner syndrome (Box 11-1). The remaining 40%
encompasses a wide range of structural abnormalities of one
of the X chromosomes, including Xp and Xq deletions (Box
11-2). Many patients with Turner syndrome have a mosaic PHYSIOLOGY
pattern comprising two or more cell lines and have a milder
phenotype. Amenorrhea
Women with Turner syndrome have rudimentary ovaries, Primary amenorrhea is defined as the absence of pubertal
also called streak ovaries. Germ cells in the primitive gonad development by age 14 years, menarche by age 16 years,
begin to differentiate but then undergo degeneration, some- or menarche 4 years after onset of thelarche. The primary
times referred to as dysgenesis, and oocytes are lost during causes of primary amenorrhea are hypothalamic or pituitary
fetal development. By birth, the number of oocytes is dysfunction, ovarian dysfunction, anatomic disorders of the
severely diminished, and by adolescence ovarian tissue has uterus or vagina, and other causes such as hypothyroidism
or adrenal hyperplasia.
regressed to ridges of white streaks devoid of both germ
Amenorrhea is physiologic when it occurs inappropriately
cells and hormone-producing cells and consisting of fibrous
in a prepubertal female, during pregnancy and in early
connective tissue. The degeneration of ovaries has secondary lactation, and after menopause. At other times, it reflects a
consequences, notably, an inhibition of breast development pathologic condition that should be evaluated.
and the presence of infantile external genitalia. The decreased
 Gonadal Differentiation and Disorders of Sexual Development 197

Box 11-1. FEATURES OF TURNER SYNDROME

45,X karyotype
Webbed neck, coarctation of aorta, high-arched palate,
shield-like chest with widely spaced nipples, short
metatarsals, renal abnormalities; gonadal dysgenesis;
may also have cubitus valgus, renal abnormalities, edema
of hands and feet, micrognathia
Lack of ovarian development leading to deficient secretion
of sex steroids
Increased incidence of diabetes mellitus, inflammatory bowel
disease, and autoimmune disease

Box 11-2. TURNER SYNDROME GENOTYPES*

45,X
45,X/46,XX
45,X/47,XXX
45,X/46,XY
46,X,i(Xp) A B
46,X,i(Xq)
Figure 11-4. Turner syndrome. A, Lymphedema and
46,X,del(Xq)
webbed neck are frequent features in infants with Turner
46,X,del(Xp)
syndrome. Lymphedema in newborn females is an indication
45,X,46,X,i(Xq) for karyotyping. B, A 13-year-old girl with classic Turner
47,X,i(Xq),i(Xq) features, including short stature, webbed neck, delayed
sexual maturation and lack of secondary sexual
*Features of Turner syndrome may occur with different genotypes.
characteristics, cubitus valgus, and broad, shield-like chest
with widely spaced nipples. (From Nussbaum RL, McInnes
RR, Willard HF. Thompson & Thompson Genetics in Medicine,
6th ed. Philadelphia, WB Saunders, 2001, p 175.)
The frequency of Turner syndrome is 1 in 2000 to 1 in 2500
live-born females. There is high intrauterine mortality, and
98% to 99% of all 45,X fetuses are spontaneously aborted—
accounting for 20% of all chromosomally abnormal aborted
embryos. Given the high incidence of in utero loss, one might Turner females are short because they are missing one
expect 45,X fetuses at term to be severely affected, particu- SHOX allele. SHOX expression is most evident in the midpor-
larly mentally. Nevertheless, Turner syndrome is associated tion of limbs and the first and second pharyngeal arches.
with normal intelligence. Expression in these sites supports the involvement of this
Turner syndrome results from haploinsufficiency of certain gene in the development of other Turner stigmata beyond
genes on the X chromosome. Gene dosage considerations led short stature, such as cubitus valgus, genu varum, high-arched
to the prediction that the genes implicated in the Turner phe- palate, micrognathia, and sensorineural deafness. This also
notype escape lyonization. This further suggests that a func- explains the greater height attained by 46,X,i(Xp) females
tional Y chromosome homolog may exist, since two copies who possess many Turner syndrome features. The Xp isochro-
are suggested in XX females. As discussed earlier, genes pos- mosome contains SHOX alleles, and these females express
sessing these characteristics are those residing in the PAR of three SHOX alleles.
X and Y chromosomes. Genes in the PAR that are dosage Treatment for Turner syndrome usually includes growth
sensitive probably contribute to the short stature observed in hormone therapy to improve growth followed by estrogen
Turner syndrome. One such gene has been identified in the replacement to improve development of secondary sex
Xp22 and Yp11.3 regions containing a homeobox that is characteristics. Estrogen therapy is also important for reduc-
mutated in individuals with idiopathic short stature. This gene ing the risk of osteoporosis, which is common in Turner
is designated SHOX, for short stature homeobox-containing syndrome.
gene. There are two forms of this gene resulting from alterna- Turner syndrome is phenotypically mimicked in Noonan
tive splicing. SHOXa protein is expressed in skeletal muscle, syndrome, which features webbed neck, short stature, pectus
placenta, pancreas, heart, and bone marrow fibroblast. excavatum/carinatum, characteristic facies, cryptorchidism,
SHOXb protein expression is restricted to fetal kidney and and cardiac anomalies. However, whereas Turner syndrome
skeletal muscle and has its greatest expression in bone marrow results from an absent X chromosome, Noonan syndrome
fibroblasts. is inherited in an autosomal dominant manner and thus
 198 Disorders of Sexual Differentiation and Development

occurs in both males and females. At least 50% of Noonan

syndrome is associated with mutations in the PTPN11 Box 11-3. FEATURES OF
gene, a non–receptor protein tyrosine phosphatase. Muta- KLINEFELTER SYNDROME
tions affect the ability of the protein to convert from an
inactive to an active form. Adjacent to the catalytic domain 47,XXY and variants for karyotype
Prepubertal features: small testes, disproportionately long
of PTPN11 are two tandem SRC homology 2 (SH2) domains
legs, personality and behavioral disorders
that permit a PTPN11 to bind to other proteins with
Postpubertal features: small testes, gynecomastia, and other
SH2 and SRC homology 3 (SH3) domains and to remove signs of androgen deficiency (e.g., diminished facial and
phosphate groups from specific phosphotyrosine residues. body hair, small phallus, poor muscular development,
Among the substrates of PTPN11 is GRB2, which par- eunuchoid habitus, taller than average height)
ticipates in signal transduction for cell cycle regulation Increased risk of osteoporosis if untreated
pathways. Activating mutations in the SH2 or protein tyro- Increased risk of breast cancer and extragonadal germ cell
sine phosphatase domains of PTPN11 increase signal trans- tumors
duction through the MAPK pathway. The MAPK pathway
is one of several signaling systems used by growth hormone,
prolactin, and epidermal growth factor. These actions lead
to the clinical manifestations of Noonan syndrome. Although
fertility may be reduced in Noonan syndrome, both males In affected males, testes are about one-third the normal
and females are fertile. Individuals with Noonan syndrome size and gynecomastia may occur in 50% of individuals.
often have pulmonary stenosis, particularly those with Most patients are sterile owing to decreased testosterone
PTPN11 mutations, whereas females with Turner syndrome production. Microscopic studies of testes show degenera-
have coarctation of the aorta. tive changes in the seminiferous tubules (Fig. 11-5).
Studies suggest that the number of germ cells is normal
or near-normal at birth, but this is followed by a dramatic
loss leading to a defective testis with increasing hyaliniza-
tion of the seminiferous tubules and clumping of Leydig
ANATOMY cells.
The mechanism resulting in Klinefelter syndrome is directly
Comparison between Aortic Coarctation and related to dosage compensation. Many X chromosome genes
Pulmonary Stenosis are expressed in higher doses before and after inactivation
Both coarctation of the aorta and pulmonary stenosis are in the Klinefelter male. Although lyonization of additional
congenital defects. Coarctation of the aorta (narrowing of X chromosomes occurs just as in females, several charac-
the aorta) may be proximal or distal to the ductus arteriosus. teristics of the additional X chromosomes are atypical.
It is present in about 5% of individuals with congenital heart Recall that lyonization does not occur until the blastomere
disease and in about 20% of females with Turner syndrome stage. Prior to this, all X chromosome alleles are active
and may occur in individuals with NF1, Williams syndrome, and capable of expression, which implicates an elevated
and Sturge-Weber syndrome. level of many proteins as contributory to abnormal devel-
Pulmonary stenosis, in which the pulmonary valve restricts
opment. Similarly, between 15% and 20% of genes escape
blood flow from the right ventricle to the pulmonary artery,
inactivation and are expressed from the inactive and active
may occur alone or with other congenital heart defects. It is
present in about 10% of individuals with congenital heart
X chromosomes, again representing a difference in dosage
disease and places these individuals at risk for right ventricle between males and females. For example, as previously
hypertrophy. discussed, the SHOX gene in the pseudoautosomal region
of the X and Y chromosomes is not inactivated and the
increased number of SHOX alleles is associated with
increased height in individuals who are 47,XXY, 47,XYY,
and 47,XXX.
Another gene of interest is the steroid sulfatase (STS)
Klinefelter Syndrome
gene located on the X chromosome. This gene is expressed
Klinefelter syndrome, the most common cause of hypogonad- from the inactive X chromosome. Estrone sulfate is a major
ism in the male, occurs in males with the 47,XXY karyotype circulating plasma estrogen that is converted to the biologi-
at a frequency of 1 in 1000 live-born males. Another disorder cally active estrogen—estrone—by steroid sulfatase. This
of sexual development, Klinefelter syndrome is also known enzyme also hydrolyzes sulfate ester bonds in other steroids
as seminiferous tubule dysgenesis. It is usually detected at such as cholesterol and dehydroepiandrosterone (DHEA).
adolescence because of the lack of sexual maturation or later The level of STS expression is higher in fetal tissues than
because of infertility. Prepubertal suspicion of Klinefelter in adult tissues, and thus it is not surprising that the pla-
syndrome is generally associated with disproportionately cental syncytiotrophoblast produces large quantities of bio-
long legs, small external genitalia, and behavioral disorders logically active estrogens during pregnancy. Estrogens play
(Box 11-3). important roles in fetal development, but excessive estrogen
 Gonadal Differentiation and Disorders of Sexual Development 199

A B

Figure 11-5. Leydig cells in Klinefelter syndrome. A, Testicular biopsy shows small, hyalinized seminiferous tubules and
pseudoadenomatous clusters of Leydig cells. The patient had normal semen volume and severe oligospermia. B, Atrophy
of seminiferous tubules and reduced testicular volume give a false impression of Leydig cell hyperplasia in Klinefelter
syndrome. Studies have shown that the total number of Leydig cells is actually reduced. C, A few hyalinized
seminiferous tubules surrounded by Leydig cells contain abundant microvacuolated cytoplasm. Even though Leydig cells
may appear morphologically normal in Klinefelter syndrome, they are often functionally deficient and androgen levels are
often reduced, accompanied by elevated FSH and LH levels. (Used with permission of Dr. Dharam Ramnani at
Webpathology.com.)

exposure affects development—just as seen with excessive association between abnormal hormonal balance and
androgen exposure. breast cancer. Klinefelter individuals with unusually high
The limbs of males with Klinefelter syndrome are longer numbers of X chromosomes have been reported, such as
than average and facial hair is sparse (Fig. 11-6). Although 48,XXXY, 49,XXXXY, and 50,XXXXXY. With more than
some are passive with poor self-image, most have an IQ in two X chromosomes, the deleterious effect on IQ is
the normal range. Less than 1% of mentally retarded males increasingly severe. Despite numerous X chromosomes,
have a 47,XXY complement. A few 47,XXY males are free of however, the individual is male by virtue of the Y
any clinical signs of Klinefelter syndrome, with the exception chromosome.
of infertility. Testosterone replacement beginning in the early to the
Individuals with Klinefelter syndrome have a 20-fold mid-adolescent years is recommended for Klinefelter syn-
increased risk of developing breast cancer. The incidence drome. This therapy will not reverse gynecomastia but will
of breast cancer in patients with Klinefelter syndrome is support secondary sexual characteristics. Individuals with
comparable to the incidence in females, suggesting an Klinefelter syndrome are at an increased risk for osteoporosis
 200 Disorders of Sexual Differentiation and Development

Another condition genotypically similar to Klinefelter

syndrome is XYY syndrome, which affects 1 in 850 to 1
in 1000 males. Unlike Klinefelter males, these individuals
have normal sexual behavior and normal secondary sexual
characteristics. 47,XYY males are taller than average with
low weight compared with stature and may have minor
skeletal anomalies. They have larger facial features, large
hands and feet, severe acne as adolescents, and possibly
mild to moderate hyperactivity. There are reports that XYY
males are more aggressive and have behavioral and learning
problems.

Chromosomal Mosaicism
Clinical records include many mosaic forms of Klinefelter
and Turner syndromes, including 46XX/45X, 46,XY/45X,
46,XX/47XXY, 46,XY/47,XXY, and others. The actual phe-
notypic manifestation of mosaicism depends on the relative
proportion and distribution of the two types of cells in
the body. As a consequence of the many bizarre mosaic
combinations, a graded series of clinical Turner and Kline-
felter syndrome features has been found in individuals,
ranging from a severe presentation to a nearly normal
Figure 11-6. Klinefelter syndrome. Physical characteristics physical presentation. In all cases of fertility in individuals
are variable, as shown in these two patients. Puberty with Klinefelter or Turner syndrome, mosaicism is sus-
generally occurs at the normal time, but the testes remain pected even if not proved. As previously described, indi-
small. As hyalinization of the seminiferous tubules increases,
vidual cells with multiple X chromosomes undergo X
androgen production does not increase for proper pubertal
development, leading to abnormal body proportions; sparse inactivation of all but one X chromosome per cell, but
or absent facial, axillary, pubic, and body hair; female all are active until inactivation occurs. As in nonmosaic
distribution of adipose tissue; gynecomastia; decreased males, increasing numbers of Barr bodies are correlated
libido; decreased muscle mass and strength; and with decreasing IQ.
osteoporosis. The pituitary-gonadal system appears to Mitotic nondisjunction explains the following self-
function well until puberty, when testosterone production contradictory finding: individuals affected with Klinefelter
diminishes and gonadotropins increase. Hyalinization leads
syndrome are born more often to older women, yet a
to a decrease in inhibin B levels, and follicle-stimulating and
luteinizing hormone levels become greatly increased. maternal-age effect cannot be demonstrated for Turner
(Courtesy of Dr. Mark Stephen, Madigan General Hospital.) syndrome. If both disorders were to stem from a meiotic
nondisjunction event during the formation of the mother’s
because of decreased androgens, and testosterone therapy gametes, then both disorders should be subject to the
will reduce this risk. Similar to women with Turner syndrome, same strong influence of the mother’s age. This has led
these men have an increased risk of autoimmune disorders to the conclusion that the vast majority of Turner cases
such as diabetes, thyroid disorders, and systemic lupus arise from chromosomal errors that occur in a cell of the
erythematosus. early embryo by mitotic nondisjunction rather than in a
maternal or paternal germ cell by meiotic nondisjunction.
PHARMACOLOGY If this is the case, it is likely that most, if not all, live-
born 45,X individuals are mosaics. This implies that the
Androgen Replacement extraordinarily high in utero lethality of the 45,X condi-
Various drugs and routes of administration can treat tion reflects that those 45,X conceptuses lack a second
androgen deficiency. Methyltestosterone, oxymetholone, and cell line. In support of this view, the extent of mosaicism
fluoxymesterone may be given sublingually or orally, but is exceedingly high in females with Turner syndrome—as
have disadvantages such as erratic absorption and potential high as 75%.
for cholestatic jaundice and are less effective than It is important to determine the genotype in females with
intramuscular preparations. Testosterone propionate is a Turner syndrome to distinguish 45,X and 45,X/46,XX indi-
short-acting treatment. Testosterone enanthate and viduals from mosaic individuals with any cells containing a Y
cyclopentylpropionate are given intramuscularly and can
chromosome, such as 45X/46,XY. The presence of any Y chro-
cause virilization. Transdermal patches mimic normal diurnal
mosome material increases the risk of gonadoblastoma to
testosterone fluctuation.
Therapy is contraindicated in adolescents prior to greater than 95%. This can be avoided through correct iden-
epiphyseal fusion and in patients with prostate cancer. tification of mosaicism and subsequent removal of the streak
ovary and gonadal remnants.
 Disorders of Sex Steroidogenesis 201

Triple-X Female
BIOCHEMISTRY
The 47,XXX constitution occurs once in every 1000 newborn
females and accounts for about 50% of all females with Cholesterol
more than two X chromosomes. Similar to 47,XYY males, There are three sources of cholesterol for its many functions
most 47,XXX females have no apparent physical abnormali- in the body, such as the need for membranes and
ties and many are fertile. Evidently, the presence of an steroidogenesis. The liver is the major organ controlling
additional X chromosome does not impair fertility, and its cholesterol metabolism. Cholesterol may be made
extrahepatically and de novo in the liver from acetate, or it
presence is far less hazardous to the developing oocyte
may be received by the liver in the form of cholesterol-
than is the absence of an X chromosome in 45,X females.
enriched remnant chylomicrons from the intestine. It can
There is evidence that the developing oocytes of the 47,XXX also be taken up by the liver as plasma lipoprotein, primarily
female actually discard the additional X chromosomes by in the form of low-density lipoprotein (LDL). Once cholesterol
a mechanism known as selective disjunction. Thus, no XXY is synthesized or taken up by liver cells, it is exported by
or XXX offspring can be born to 47,XXX females. Children two mechanisms. Cholesterol is used to synthesize bile
born to 47,XXX females are either normal males or normal acids or it is included in bile as cholesterol and cholesteryl
females. esters. It is also exported to the blood in the form of
Clinically, triple-X females may appear as quiet infants very-low-density lipoprotein (VLDL).
who later have delayed motor, verbal, and emotional devel-
opment. However, very few of these females are distinguished
by these features in adulthood. Social problems may occur
at puberty, since these girls are tall for their age. As in ANATOMY & PHYSIOLOGY
Klinefelter males, the extremities of these females are longer
than normal, giving a greater lower body–to–upper body Gonads and Steroidogenesis
ratio. As a result of vertical growth, back problems and The ovarian follicle is composed of theca cells, granulosa
scoliosis may develop. Intellectually, there may be learning cells, and the primary oocyte. 3β-Hydroxysteroid
disabilities and intelligence may be slightly lower than that dehydrogenase (3β-HSD) is stimulated by gonadotrophs.
of siblings or a control group, but overall intelligence is Theca cells lack aromatase and produce androgens.
within the normal range. Several studies have highlighted Granulosa cells lack 17α-hydroxylase and produce
the importance of environment and social interaction for estrogen—primarily estradiol in the proliferative phase and
the normal development of these females. progesterone in the luteal phase.
The primary male steroids are testosterone,
dihydroxytestosterone, and estradiol. DHT and estradiol are
produced by the testes and by peripheral conversion (80%
is from peripheral conversion). Leydig cells produce more
●●● DISORDERS OF than 95% of testosterone and secrete estradiol, estrone,
SEX STEROIDOGENESIS pregnenolone, progesterone, 17α-hydroxypregnenolone,
and 17α-hydroxyprogesterone. Target cells convert
In addition to several similarities in the genes required testosterone to DHT with 5α-reductase.
for their development, steroidogenesis occurs in both the
adrenal gland and the reproductive system. Beyond the
impact of chromosomal anomalies and gene mutations
affecting the establishment of sex, steroidogenesis is critical Ovotesticular Disorder of
for proper development and function of gonads. Many
Sexual Development
disorders in this pathway result from specific gene muta-
tions that affect sex—as either a primary or a secondary Ovotesticular DSD is the presence of both ovarian and tes-
consequence—through development of abnormal gonads ticular tissue in an individual. The most common karyotype
and adrenals. is 46,XX, but 46,XX/46,XY and 46,XY may also occur. The
Steroid hormones, with the exception of retinoic acid, most common presentation of gonadal tissues is in an ovo-
are synthesized from cholesterol in the gonads, in adrenal testis, a gonad containing both tissues. Other presentations
cortex, and peripherally in such sites as skin, fat, brain, and include a testis that may be present on one side and an
placenta. Sex steroids include androgens, estrogens, and ovary on the other side, ovarian and testicular tissue on one
progestins. Mineralocorticoids, such as aldosterone, and glu- side and an ovary or testis on the other side, and an ovotestis
cocorticoids, such as cortisol, also are steroids and require or testis on one side along with an ovary in its normal
some of the same enzymes as sex steroid synthesis. The position.
effects of mutations in these latter pathways may have broad Genital ducts generally reflect the gonad that is present. In
and complex ramifications due to mineralcorticoid insuffi- individuals with an ovary on one side and a testis on the
ciency. A few examples of genetic disorders of sex steroido- other, müllerian derivatives develop on one side and wolffian
genesis that result in anomalies of sexual determination and derivatives on the other. In the presence of an ovotestis,
differentiation are discussed below. genital ducts are usually müllerian derivatives. External
 202 Disorders of Sexual Differentiation and Development

ANATOMY
Hypospadias
Hypospadias can occur anywhere along the urethral groove
when fusion of the urethral fold stops proximal to the tip of
the glans penis. It occurs in 1 in 100 to 1 in 200 boys.
Conditions associated with hypospadias are the following:
■ Inadequate testosterone produced by testes—

enlargement and development of genital tubercle and

scrotal swellings does not occur properly
■ Lack of androgen receptors
■ Lack of 5α-reductase
■ Congenital adrenal hyperplasia

Figure 11-7. 46,XX/46,XY infant with palpable gonad. From earlier discussions, it is plausible to consider that
(Courtesy of Tarek Bisat, MD, Mercer University School of translocation of an SRY allele might result in 46,XX ovotes-
Medicine.)
ticular DSD. While most of these individuals are SRY nega-
tive, 10% of individuals studied demonstrated SRY expression
in the ovotestis. Involvement of other genes have not been
Box 11-4. CLINICAL FEATURES OF identified; however, it is not difficult to hypothesize that
OVOTESTICULAR DSD mutations in other differentiation genes might perturb devel-
opment. The 46,XX/46,XY individual is usually a chimera
Genitalia are ambiguous. resulting from double fertilization. These individuals have two
Cryptorchidism is frequent. populations of cells.
Ovotestis may be located in labioscrotal fold.
Testis, ovary, or ovotestis is present.
Menses may occur.
Spermatogenesis is rare.
Breast development and virilization occur at puberty. ANATOMY & EMBRYOLOGY
Chimera
A chimera, or a person with two genetically distinct cell
genitalia may be ambiguous, male, or female (Fig. 11-7). If a types, may occur by one of three mechanisms:
■ Anastomosis of placental vessels
penis is present, hypospadias and cryptorchidism are not
■ Introduction of cell line artificially, such as with a
uncommon, but generally gonads or an ovotestis are palpable
in the labioscrotal fold (see Fig. 11-7). Breast development transfusion or transplantation
■ Fusion of two zygotes
and menses may occur at puberty. For 46,XX individuals,
pregnancy has been reported, but there is only one report of
a 46,XY individual fathering a child (Box 11-4).
46,XY Disorder of Sexual Development
ANATOMY & EMBRYOLOGY Enzyme Deficiencies
Testosterone, like other steroids, is synthesized from
Genital Ducts cholesterol in a series of steps requiring the steroidogenic
Mesonephric and paramesonephric ducts are important for enzymes P450scc (cholesterol side-chain cleavage enzyme),
establishing male and female reproductive systems. 3β-hydroxysteroid dehydrogenase (3β-HSD), CYP17 (17α-
Mesonephric ducts establish the male reproductive system. hydroxylase/c17,20-lyase), and 17β-hydroxysteroid dehydro-
The proximal portion becomes convoluted to form the genase (17β-HSD). Male and female embryos have the same
epididymis, and the remainder forms the ductus deferens androgen receptors; differences between males and females
and ejaculatory duct. result from differences in levels of androgens.
The paramesonephric ducts establish the female 17β-HSD plays an important role in the balance of
reproductive system. These ducts develop lateral to the gonadal steroids. It maintains a balance between active 17β-
gonads and mesonephric ducts. The mesonephric ducts
hydroxysteroids and relatively inactive 17-ketosteroids.
degenerate due to lack of testosterone. The cranial portions
Reducing the 17-ketosteroids is the last step leading to tes-
of the paramesonephric ducts form the uterine tubes and
the caudal portions fuse to form the uterovaginal tosterone in Leydig cells and estradiol in granulosa cells.
primordium, which forms the uterus and vagina. Oxidation of 17-ketosteroids occurs predominantly in the
periphery and is the balancing force.
 Disorders of Sex Steroidogenesis 203

17α-Hydroxylase 17β-Hydroxysteroid dehydrogenase

No sex hormones or cortisol No testosterone
Elevated production of mineralocorticoids DHEA Elevated estrogens from androstenedione
causes sodium and fluid retention leading sulfate Increased FSH and LH
to hypertension Phenotypically female
Phenotypically female but unable to mature Virilization at puberty

Cholesterol
Rate-limiting
step
17α-Hydroxy-
Pregnenolone DHEA Androstenediol
pregnenolone

17α-Hydroxy-
Progesterone Androstenedione Testosterone DHT
progesterone

Aromatase
11-Deoxycortico- 11-Deoxycortisol Elevated testosterone
sterone Aromatase and androstenedione
Elevated testosterone Masculization of female 5α-Reductase
and androstenedione genitalia Female genitalia
Corticosterone Cortisol Masculization of female Pubertal failure Partial virilization at puberty
genitalia Elevated testosterone to
Pubertal failure DHT ratio
17β-Estradiol (E2)
Aldosterone

Estrone (E1)

Estriol (E3)

Figure 11-8. Synthesis of androgens and estrogens. Enzyme mutations lead to a deficiency of androgens and estrogens.
Deficiency of 17α-hydroxylase (P450c17) shunts precursors into the aldosterone pathway. Deficiency of 17β-hydroxysteroid
dehydrogenase will shunt precursors to estrogens primarily, as will a deficiency of 5α-reductase. Deficiency of aromatase leads
to a deficiency of estrogens.

There are at least six isozymes of 17β-HSD in humans. Type and male genital ducts. Classic features include a clitoris-like,
3 17β-HSD is the most frequent cause of 46,XY DSD due hypospadiac phallus bound down in chorde, a bifid scrotum,
to defective testosterone biosynthesis (Fig. 11-8). At birth, a urogenital sinus opening onto the perineum, and a blind
46,XY males with a 17β-HSD mutation generally appear vaginal pouch opening into the urogenital sinus or the
female or have mildly ambiguous genitalia resulting from perineum (Box 11-5). At puberty, virilization occurs owing to
testosterone deficiency during differentiation. Although the increased testosterone even though there is an inability
having female external genitalia, these males have testes and to convert it to DHT.
typical male derivatives of the wolffian ducts: epididymes, vas
deferens, seminal vesicles, and ejaculatory ducts. As testos-
terone levels increase at puberty, these males experience
marked virilization with clitoral hypertrophy and breast Box 11-5. FEATURES OF 5α-REDUCTASE
enlargement. DEFICIENCY (46,XY DSD)
5α-Reductase is required for the conversion of testosterone
to dihydrotestosterone (DHT) (see Fig. 11-8). There are two External genitalia appear female at birth.
isozymes of this gene. Type 1 is expressed postnatally, primar- Testes are extra-abdominal, usually inguinal.
Hypospadiac microphallus with bifid scrotum is present.
ily in the liver and skin. Type 2 is expressed pre- and postna-
There is a blind vaginal pouch.
tally, predominantly in prostate, internal genital structures
It is sometimes referred to as “penis at 12 syndrome.”
derived from wolffian ducts, genital skin, and liver. 46,XY It is strongly suspected with normal blood testosterone level
individuals with mutations in this gene are males presenting but elevated T:DHT ratio.
with feminization or ambiguous genitalia along with testes
 204 Disorders of Sexual Differentiation and Development

As an X-linked gene, 5α-reductase deficiency may also

occur in 46,XX females, but this primarily requires a homo- BIOCHEMISTRY
zygous condition, or mutations in both alleles, for presenta-
tion. In these females, the external genitalia are normal Androgen Receptor
because female genitalia do not require testosterone or DHT The androgen receptor is a nuclear hormone receptor
to develop normally. Postpubertal development is normal. expressed from a gene consisting of eight exons and
Menarche is delayed, but fertility is normal. The effects of this organized like other steroid hormone receptors. The gene
mutation in females are seen at the sites particularly sensitive produces two mRNAs via differential splicing. Androgen
receptors bind testosterone and regulate expression of
to DHT; development of axillary and pubic hair is decreased
genes involved in the development of male secondary sex
and body hair is absent. Not surprisingly, type 2 5α-reductase
characteristics. The receptor has ligand-binding domains
is the predominant form of 5α-reductase found in hair that regulate the receptor’s DNA binding and transcription
follicles. activation functions. The major transcription activation
17α-Hydroxylase (P450c17) is a bifunctional enzyme. function of the receptor is located in the NH2-terminal
Unlike 17β-HSD and 5α-reductase, mutations in this gene domain, which is the hormone-binding domain. The DNA
have consequences beyond those of androgens and estrogen. binding domain has two zinc clusters.
Mutations affect P450c17 and c17,20-lyase activities by Mutations in these receptors fail to bind androgen or fail
reducing the production of cortisol, androgen, and estrogen to bind DNA effectively. The most severe mutations in this
and thus cause a corresponding increase in the production gene present clinically as androgen insensitivity syndrome,
of certain mineralocorticoids (see Fig. 11-8). A severe defi- characterized in 46,XY males by a lack of male secondary
sex characteristics and the presence of female secondary
ciency impairs androgen synthesis, causing the appearance
sex characteristics. Testosterone is present, but cells cannot
of female external genitalia in XY males. No uterus is
respond to it. Another type of mutation of the androgen
present because Sertoli cells produce AMH. Sexual infantil- receptor is present in Kennedy disease, a neuromuscular
ism is observed in XX females at puberty. In this case, disease causing spinal and bulbar muscular atrophy and
the uterus is hypoplastic. Adrenocorticotropic hormone characterized by proximal limb and facial muscle wasting. It
(ACTH) levels are elevated owing to the decrease in cortisol is caused by expansions of a CAG repeat.
production, further exacerbating excess mineralocorticoid
production. This leads to hypertension and hypokalemia.
Less severe mutations have less effect on the presenting
phenotype. Adrenal hyperplasia can occur with severe
deficiency.
In the absence of receptor function, resistance to andro-
gens during early development prevents masculinization of
Androgen Insensitivity Syndrome the external genitalia and development of wolffian ducts.
Androgens require a receptor to mediate function. Gener- Sertoli cells secrete AMH, and müllerian structures degener-
ally speaking, testosterone enters target cells and is con- ate. In this scenario, newborns have female genitalia and a
verted to dihydrotestosterone by 5α-reductase (see Fig. blind vaginal pouch. Testes are present either in the abdomen
11-8). DHT binds to the intracellular androgen receptor, or in the inguinal canal. At puberty, breasts develop in
also known as the DHT receptor, and a cascade of events response to increased LH and subsequent increases in tes-
is initiated that leads to the binding of specific hormone tosterone and androstenediol that is converted peripherally
response elements to DNA and ultimately causes the andro- to estradiol. LH is increased as a result of the ineffective
gen action. Disorders may occur if a mutation affects any response of the hypothalamus to testosterone at mutated
step of DHT binding to the appropriate receptor; this surface receptors.
includes mutations leading up to the production of DHT,
or at any point affecting binding or affecting androgen
action after binding occurs. Examples of both will be
46,XX Disorder of Sexual Development
described.
The androgen receptor itself may have mutations that affect
with Maternal Virilization
binding of DHT. In these cases, target cells are incapable of Aromatase Deficiency
responding to the presence of testosterone, and thus the Cytochrome P-450 aromatase catalyzes the aromatization
effect is an appearance of no testosterone. Previously known of androgens to their respective estrogenic derivative. In
as testicular feminization, androgen insensitivity syndrome addition to the testis and ovary, aromatase is found in skin,
(AIS) is an X-linked disorder occurring 1 in 20,000 births fat, brain, and placenta. Its expression is dependent on tissue-
and in which more than 300 mutations have been described. specific promoters. In ovarian granulosa cells, follicle-
Over 200 of these occur in exons 2 to 8. These 46,XY males stimulating hormone (FSH) and cAMP induce aromatase.
respond to androgen variably, and phenotypes may range In fibroblasts, it is induced by glucocorticoids and certain
from a normal male phenotype with a small phallus to a lymphokines. Mutations in the coding part of the gene will
female phenotype with female or ambiguous genitalia. Clini- alter the activity of aromatase, whereas those in the 5′
cally, AIS has complete, partial, and mild forms. In milder regulatory region will affect expression in a tissue-specific
presentations, males may be fertile. manner.
 Disorders of Combined Adrenocortical and Sex Steroidogenesis 205

Pathogenesis of a mutation in aromatase may result from

ANATOMY & PHYSIOLOGY
an accumulation behind the block or from estrogen depletion.
Androgen excess can masculinize the external female genita- Adrenal Cortex
lia or prematurely virilize the male external genitalia during
The adrenal cortex comprises three zones having different
fetal development. Similarly an affected fetus can cause
roles in steroidogenesis: zona glomerulosa, zona fasciculata,
virilization of the mother during pregnancy. and zona reticularis. The zona glomerulosa produces
DSD in females may also occur from defects in adrenocorti- aldosterone, the main mineralocorticoid, but lacks
cal pathway enzymes, as discussed below. In these females, 17α-hydroxylase and 17,20-lyase and thus cannot
virilization occurs at birth even in the presence of ovaries. synthesize cortisol and androgens. Cortisol, the main
glucocorticoid, is produced in the zona fasciculata and zona
reticularis. The zona reticularis also produces androgens
and small amounts of estrogens. The latter two zones
●●● DISORDERS OF COMBINED (fasciculata and reticularis) cannot produce aldosterone
ADRENOCORTICAL AND because they lack the 11β-hydroxylase enzyme.
SEX STEROIDOGENESIS Cortisol is considered a glucocorticoid because it was
originally recognized that it could increase plasma glucose
Deficiencies in enzymes of adrenocortical steroidogenesis
levels. Aldosterone promotes salt and water retention by the
may have severe consequences reflecting the absolute require- kidney and is therefore a mineralocorticoid. Cortisol and
ment for these end products. While agenesis or dysgenesis of aldosterone are similar in structure, but aldosterone has no
gonads has certain undesired outcomes for individuals, glucocorticoid activity. Glucocorticoids have many actions
adrenal dysgenesis is a far more critical and potentially life- beyond glucose regulation, including immunosuppressant
threatening event. In these cases, sex steroidogenesis is and anti-inflammatory actions, effects on protein and fat
secondarily affected. These defects comprise a group known metabolism, behavioral effects on the central nervous
as congenital adrenal hyperplasias (Fig. 11-9). system, and effects on calcium and bone metabolism.

Cholesterol DHEA sulfate

Rate-limiting
step

Pregnenolone 17α-Hydroxypregnenolone DHEA Androstenediol

3β-Hydroxysteroid dehydrogenase
No glucocorticoids, mineralocorticoids, androgens, estrogens
Salt excretion in urine
Early death

Progesterone 17α-Hydroxyprogesterone Androstenedione Testosterone DHT

21-Hydroxylase
Most common form of CAH
Usually a partial deficiency
ACTH elevated, causing an increased shift to sex hormones
and masculinization 17β-Estradiol (E2)

11-Deoxycorticosterone 11-Deoxycortisol

11β-Hydroxylase
Decreased cortisol, aldosterone, corticosterone
Increased deoxycorticosterone leading to fluid retention and hypertension Estrone (E1)
Masculinization

Corticosterone Cortisol Estriol (E3)

Aldosterone

Figure 11-9. Steroidogenesis pathway. Mutations affecting enzymes primarily responsible for the production of
mineralocorticoids and cortisol can have secondary effects on sex steroid production. Substrates that accumulate are shunted
to the right. The mineralocorticoid pathway occurs in the zona glomerulosa and the cortisol and androgen pathways occur
predominantly in the zona fasciculata of the adrenal gland.
 206 Disorders of Sexual Differentiation and Development

explain masculinization or virilization of some affected

PHYSIOLOGY 46,XX females.

Evaluating the Adrenal Cortex

There are three major tests to consider when evaluating the
21-Hydroxylase Deficiency
function of the adrenal cortex. Abnormalities in the cortex 21-Hydroxylase (P450c21) is responsible for conversion of
are reflected by overproduction or underproduction of progesterone to deoxycorticosterone (DOC) in the aldo-
hormones, and therefore these tests are designed to sterone biosynthetic pathway (see Fig. 11-9) and 17α-
stimulate or suppress production.
hydroxyprogesterone to 11-deoxycortisol in the cortisol
■ The ACTH stimulation test is the most reliable screening
pathway. The precursors and substrates that accumulate
test for decreased adrenal production. Synthetic ACTH
(250 μg) is administered, and serum steroids are measured
behind these blocks are directed into the androgen bio-
45 and 60 minutes later. The normal cortisol response by synthesis pathway. Deficiency of this enzyme is the most
the adrenal gland to this challenge is 20 μg/dL. common cause of congenital adrenal hyperplasia (CAH) and
■ The corticotropin-releasing hormone (CRH) challenge is is responsible for at least 90% to 95% of cases. Depending
useful to distinguish between ACTH-dependent and on the population, the incidence of this disorder ranges from
ACTH-independent hypercortisolism. For this procedure, 1 in 280 among the Yupik Alaska Natives to 1 in 28,000
1 μg/kg ACTH is administered, and ACTH levels are among Chinese (Table 11-2). About two thirds of affected
measured between 3 and 30 minutes. patients have the “salt-wasting” form resulting from seriously
■ The dexamethasone suppression test is used to test for
deficient aldosterone as well as cortisol. If untreated, these
hyperfunction of the adrenal cortex but is plagued by false
infants die in early infancy of hyponatremia, hyperkalemia,
negatives and false positives. However, it has some value
hypovolemia, and acidosis. Females are more likely to be
for evaluating mineralocorticoid excess. One procedure
provides for the administration of 0.5 mg of
diagnosed and treated early because they have ambiguous
dexamethasone by mouth every 6 hours for 2 days. genitalia or masculinization of genitalia (Fig. 11-10). They
To distinguish the various forms of congenital adrenal may have clitoromegaly and labioscrotal fusion with a phallic
hyperplasia, specific steroid substrates that accumulate in urethra. Ultrasonograms will demonstrate a uterus.
response to decreased enzyme activity are measured.

PHYSIOLOGY
Salt Wasting
Adrenal hyperplasia occurs owing to the feedback control
of the hypothalamus and pituitary. In the absence of sufficient Salt wasting can accompany two forms of congenital
adrenal hyperplasia: 21-hydroxylase deficiency and
products, such as cortisol and sex steroids, ACTH and gonad-
3β-hydroxysteroid dehydrogenase deficiency. Since the
otropins stimulate the adrenals to produce more products,
former is more common, salt wasting is seen most often
and thus cellular hyperplasia occurs. An important consider- with this form. Severe salt wasting will occur in about 75% of
ation for each of the following enzymes is the mutation itself. infants with severe 21-hydroxylase deficiency. This deficiency
Recall that the location and extent of change in the DNA can leads to decreased cortisol and aldosterone synthesis.
be demonstrated as either a negligible or mild effect or as a Reduced aldosterone, which functions normally to stimulate
serious effect due to little or no enzyme production. the kidney to reabsorb Na+ and water and enhance K+
secretion, will cause electrolyte and fluid loss due to
hyponatremia and hyperkalemia, which lead to acidosis,
3β-Hydroxysteroid dehydration, and vascular collapse if not corrected.
Dehydrogenase Deficiency The decrease in circulating volume also causes an
increase in renin, which activates the renin-angiotensin
system through stimulation of the baroreceptor reflex and a
3β-Hydroxysteroid dehydrogenase is expressed as two highly
decrease in renal arteriole pressure. Angiotensinogen is
homologous genes (HSD3B1, HSD3B2) and identified as cleaved into angiotensin I, which is then converted to
type I and type II. Type I gene expression is found predomi- angiotensin II. This octapeptide is unable to stimulate the
nantly in the placenta and the peripheral tissues of skin and adrenal cortex directly, or indirectly through stimulation of
mammary gland. Type II gene expression occurs mainly in the the anterior pituitary and release of ACTH to effect
adrenal glands and gonads. As expected, a mutation in the aldosterone production. Therefore, the net result is life
type II gene will interfere with mineralocorticoid, glucocor- threatening unless reversed.
ticoid, progesterone, androgen, and estrogen synthesis (see The situation is different with 11β-hydroxylase deficiency
Fig. 11-9). If a mutation is severe, salt-losing crises, hypomas- (CYP11B1 deficiency). In these infants, the lack of cortisol
culinized external genitalia in males (46,XY DSD), and clito- causes an increase in ACTH resulting in increased
aldosterone. This increases Na+ reabsorption and K+
romegaly at birth in females (46,XX DSD) may occur along
secretion by the kidney, leading to “salt retention” rather
with adrenal hyperplasia. Obviously, milder mutations, char-
than “salt wasting.” Directly related to the enzyme deficiency
acterized by the type and position of the mutation affecting is an increase in 11-deoxycortisol. This and the excess
the protein’s function, may not display the broad spectrum 11-deoxycorticosterone contribute to hypertension.
of phenotypes. Type I mutations are less problematic but may
 Disorders of Combined Adrenocortical and Sex Steroidogenesis 207

continues after birth, resulting in rapid bone growth and

TABLE 11-2. Incidence of Congenital Adrenal maturation (Fig. 11-11).
Hyperplasia The hallmark of CAH is inadequate production of gluco-
corticoids. Patients with mild CAH are frequently unable to
LOCATION FREQUENCY mount sufficient stress responses to trauma and infection.
One in 100 persons of all ethnic backgrounds is affected with
Alaska, Yupik Eskimo population 1 in 280 a milder mutation; however, the incidence is significantly
France and Italy 1 in 11,000 increased among Hispanics and Ashkenazi Jews.
La Reunion, France 1 in 2100
Scotland 1 in 17,000 Mild CAH is much more common than the classic form.
New Zealand 1 in 14,500 Men and women with mild CAH may have normal height
Japan 1 in 15,800 compared with the general population, yet shortened when
China 1 in 28,000 compared with their parents. Glucocorticoid precursors accu-
Data from Pang SY, Wallace MA, Hofman L, et al. Worldwide experience in
mulate and are converted to androgenic steroids, causing
newborn screening for classical congenital adrenal hyperplasia due to shortened stature, early puberty, severe acne, and virilization
21-hydroxylase deficiency. Pediatrics 1988;81:866–874. and infertility in females. Mineralocorticoid synthesis can
also be affected, resulting in electrolyte disturbances, hypo-
tension, and syncope. Some people with mild CAH can
mount limited glucocorticoid stress responses and are thus
never recognized as having the disorder. Others, however,
have frequent illnesses and decompensate when challenged
by common infections or minor trauma.
Males with congenital adrenal hyperplasia have an increased
incidence of testicular adrenal rest tumors (TARTs), which are
benign hyperplasias rather than true tumors. These “tumors”
originate from adrenal precursor cells that fail to migrate
completely to the adrenal gland during development and
remain part of the developing testis. As adrenal tissue, angio-
tensin II and ACTH receptors are located on the cell surface
and respond to elevated ACTH levels just as the zona fascicu-
lata cells do in the development of CAH. TARTs can cause
compression on seminiferous tubules and aspermia. There-
fore, proper adjustment of corticosteroid suppression therapy
in males with CAH can cause regression of TART.

11β-Hydroxylase Deficiency
11β-Hydroxylase (CYP11B1) converts deoxycorticosterone
(DOC) to corticosterone and 11-deoxycortisol to cortisol
Figure 11-10. Congenital adrenal hyperplasia. This 46,XY (see Fig. 11-9). A deficiency of this enzyme is the second
child was raised as a female. (Courtesy of Roberta Sonnino, most common cause of congenital adrenal hyperplasia. Most
MD, University of Minnesota School of Medicine.) mutations completely abolish the enzyme activity, although
the clinical presentation may be variable. The resulting corti-
Impaired cortisol synthesis due to 21-hydroxylase defi- sol deficiency leads to an increase in ACTH and a subsequent
ciency leads to increased ACTH levels and increased levels of increase in precursors that are diverted to the androgen syn-
adrenal androgen precursors and androgen secretion. Ele- thesis pathway. Females may have ambiguous or masculinized
vated androgen levels before 12 weeks of gestation lead to external genitalia at birth and become virilized in early child-
labioscrotal fusion and clitoral enlargement, whereas after 12 hood. In cases of ambiguity, there is clitoral enlargement and
weeks of gestation only clitoromegaly is induced. Virilization labial fusion (Fig. 11-12). Virilization varies from mild to

Continued
Before 12 weeks After 12 weeks virilization

Figure 11-11. Elevated androgens

and development. Exposure to high
levels of androgens during fetal Labioscrotal fusion Rapid bone growth
Clitoral enlargement Clitoral enlargement and maturation
development affects fetal development
in a time-dependent manner.
 208 Disorders of Sexual Differentiation and Development

KEY CONCEPTS
■ The development of gonads, kidneys, and adrenals have
common origins.
■ Disorders of sexual development may occur from chromosomal
nondisjunction, gene mutations, changes in gene expression, or
translocations.
■ The steroidogenesis pathway is important for mineralocorticoids,
cortisol, and androgens and sex steroids.
■ Defects in the steroidogenesis pathway are determined by
increased levels of substrates and decreased products.
■ Increased products are shunted with other enzymes, leading to
pathophysiology.
■ Defects in the steroidogenesis pathway resulting in salt wasting
are critical to identify at birth or shortly thereafter.
■ A very common form of adrenal hyperplasia may present as a
milder, later onset disease.

Figure 11-12. Ambiguous genitalia. (Courtesy of Tarek

Bisat, MD, Mercer University School of Medicine.)

●●● QUESTIONS
1. A 15-year-old female is evaluated for delayed
puberty. Physical examination reveals normal female
severe. Male infants have normal external genitalia, as with genitalia. Imaging studies reveal undeveloped streak
21-hydroxylase deficiency, but virilize prematurely. Both gonads and the presence of normal müllerian struc-
DOC and 11β-deoxycortisol are increased in blood, but tures. Chromosomal analysis reveals a 46,XY kar-
11-deoxycortisol has little activity; it is the action of DOC yotype. Which of the following best explains this
that provides the mineralocorticoid actions. Should an indi- finding?
vidual have a late onset of CYP11B1 deficiency, it is most A. Deletion of DAX
likely the result of a less pronounced mutation that allowed B. Deletion of XIST
adequate expression during earlier years. C. Deletion of DSS
The CYP11B1 gene functions primarily in mitochondria in D. Lyonization
the zona fasciculata of the adrenal cortex. A related gene, E. SRY mutation
CYP11B2, functions only in the zona glomerulosa. These two Answer. E
genes represent a gene duplication of CYP11B1 and are adja-
cent to each other. They are 95% homologous in exons and Explanation: SRY is required for expression of the testis-
determining factor, and therefore mutations in this gene on
90% homologous in introns. The function of CYP11B2 is the
the Y chromosome affect sex determination. Mutations with
conversion of corticosterone to aldosterone, and therefore a
the greatest effect occur in the HMG region of the protein,
mutation would cause an aldosterone crisis but not the
which is needed for the protein to bind the minor groove of
cortisol crisis that can occur with CYP11B1 deficiency. The
the DNA helix, inducing a conformational change for other
enzyme catalyzes three reactions in mitochondria: an gene transcription. DSS is a region on the X chromosome
11β-hydroxylase activity, an 18-hydroxylase activity, and an and can also result in sex reversal but when expression is
18-oxidase activity. These activities are regulated by angio- unbalanced by SRY expression; deleting DSS, which also
tensin II and potassium. Individuals with CYP11B2 mutations implies deletion of DAX1, will not create increased expres-
are clinically characterized by hypertension, variable hyper- sion and affect sexual development. Duplication of DAX will
aldosteronism, and increased levels of the abnormal adrenal cause sex reversal to a 46,XY female. Lyonization is not
steroids 18-oxocortisol and 18-hydroxycortisol. appropriately applied to this case because only one X chro-
CYP11B1 deficiency occurs in 1 in 100,000 live births. mosome is present. X-inactivated specific transcript (XIST)
However, in some populations the incidence is higher. For expresses RNAs that assist in maintaining an inactivated
example, in Israel, the incidence is 1 in 5000 births among state of one X chromosome in a 46,XX female.
Sephardic Jews. While uncommon in European women, this
deficiency is reportedly responsible for 15% of congenital Additional Self-assessment Questions can be Accessed
adrenal hyperplasia in Muslim and Jewish women. at www.StudentConsult.com
Population Genetics 12
and Medicine
CONTENTS interest in genetics, but the problem fascinated him as a
mathematical one. The solution ranks as one of the most
fundamental theorems in population genetics. As fate would
HISTORICAL PERSPECTIVE
have it, Hardy’s formula was arrived at independently in
the same year (1908) by a physician, Wilhelm Weinberg,
HARDY-WEINBERG EQUILIBRIUM
and the well-known equation bears both their names.
Basic Algebraic Formula
Application of Hardy-Weinberg Theorem
Estimating the Frequency of Heterozygotes ●●● HARDY-WEINBERG EQUILIBRIUM
Significance of the Heterozygote
X-Linked Loci Basic Algebraic Formula
CONSANGUINITY AND RECESSIVE INHERITANCE Assume there are only two possible alleles, A and a, at a
particular locus on an autosome. In addition, assume the
General Aspects of Consanguinity frequency of the A allele in the population is designated “p,”
ASSORTATIVE MATING AND INBREEDING and “q” is the frequency of the a allele. Under these con-
ditions, p + q = 1, since this is the totality of these alleles
DNA TECHNOLOGY AND CLINICAL DIAGNOSIS in an individual within a population. The probability of
bringing two gametes bearing the A allele together is simply
p × p = p2. The chance of obtaining the aa genotype is q2, and
the chance of obtaining Aa is 2pq. The “2” in 2pq derives
from the fact that there are two ways of forming the hetero-
zygote, since each allele can be contributed to the zygote
Prior chapters have focused on variations within DNA mol- either through the egg or through the sperm.
ecules that lead to disease. Oftentimes, specific variations are The simplest case for understanding gene expression is the
identified within particular populations at a greater frequency gene with only two alleles; however, as discussed in great
than in other populations. Many examples have been given detail in prior chapters, any change in DNA at a locus results
in which groups such as the Ashkenazi Jews, whites of north- in a new allele, making the possibilities seemingly endless.
ern European ancestry, or black populations have a propen- Though Hardy and Weinberg developed this formula prior to
sity for specific disorders. Understanding DNA variations by a proper appreciation of alleles, only two alleles are consid-
combining the study of populations with advancing molecular ered at a time and the formula still has applicability.
diagnostics is leading to a better understanding of gene action An important factor that influences the genetic composi-
and the development of disease. tion of a population is the system of mating among individu-
als. The simplest scheme of breeding activity in a population
is referred to as random mating (or panmixia), wherein any
●●● HISTORICAL PERSPECTIVE one individual has an equal chance of pairing with any other
At the turn of the 20th century, an intriguing question was individual. Random mating does not imply promiscuity; it
posed to the English geneticist R. C. Punnett. He was asked simply means that those who choose each other as mating
to explain the prevalence of blue eyes in humans in view partners do not do so on the basis of similarity or dissimilar-
of the acknowledged fact that the blue-eyed condition was ity in a given trait or gene.
a recessive characteristic. Would the dominant brown-eyed The absence of preferential mating in a population has
trait in time supplant the blue-eyed state in the human interesting consequences. The gametes in a panmictic popula-
population? The answer was not self-evident, and Punnett tion are mixed at random. Each gamete carries either A or a.
sought out his colleague Godfrey H. Hardy, an astute math- To predict the outcome of the random mixing of gametes, the
ematician at Cambridge University. Hardy had only a passing “Punnett square” is used (Fig. 12-1). This matrix essentially
 210 Population Genetics and Medicine

Egg Box 12-1. DIFFERENCES THAT DEVELOP WITHIN

A a
AND BETWEEN GENE POOLS

Meiotic assortment of DNA to gametes

Random assortment
A AA Aa
Recombination
Development and persistence of mutations
Sperm Failure of repair mechanisms
Development of a selective advantage—mutation favors
a Aa aa survival
Development of a selective disadvantage—mutation does
not favor survival of gene function

Figure 12-1. The distribution of genotypes in the next

generation: p2:AA 2pq:Aa q2:aa.
population became segregated or separated from the larger
population, subpopulations developed. Just as different popu-
brings into play the multiplication rule of probability: the lations may have developed unique mutations, subpopula-
chance that two independent events will occur together is the tions may have developed a specific genetic complement due
product of their chances of occurring separately. The Punnett to unique mutations that differ from other subpopulations or
square checkerboard displayed in Figure 12-1 reveals the the population as a whole. In both cases, the gene pool is
outcome of all possible combinations. limited by the DNA variation contributed by individuals
The distribution of genotypes in the next generation of a through mating. These differences among gene pools, which
randomly breeding population is p2:AA 2pq:Aa q2:aa. The are directly affected by the DNA sequences available, develop
algebraic expansion of the binomial equation (p + q)2 is through variations in meiotic reassortment and mutations
reflected in this distribution, p2:2pq:q2. The Hardy-Weinberg (Box 12-1). These changes may confer a selective advantage
theorem states that the proportions of AA, Aa, and aa geno- or disadvantage to the gene and the individual or have a
types, as well as the proportions of A and a alleles, will neutral effect. Of course, only those changes that are neutral
remain constant from generation to generation, provided that or that confer a selective advantage are important for the
the bearers of the three genotypes have equal opportunities survival of the population.
of producing offspring in a large, randomly mating popula- An understanding of Hardy-Weinberg equilibrium provides
tion. If blue-eyed persons are equally fertile as brown-eyed a basis for recognizing the forces that permit a change in gene
individuals and leave equal numbers of offspring each genera- frequencies. A few of the more obvious factors that prevent
tion, then blue-eyed persons as well as brown-eyed individu- a natural population from attaining stationary equilibrium are
als will persist in the population with the same frequency (1) mutation, (2) natural selection, (3) chance events in a
from one generation to the next. small population (genetic drift), and (4) migration. In any
population, the more individuals contributing to the gene
pool (i.e., the larger the population), the more stable the allele
Application of
frequencies within the population and the more difficult it
Hardy-Weinberg Theorem will be for frequencies to change. Conversely, small popula-
It may seem ironic that the Hardy-Weinberg theorem is tions may have fluctuations in frequencies often; in some
entirely theoretical. The following set of underlying assump- cases this can even occur between generations. The factors or
tions can scarcely be fulfilled in any natural population. It is forces affecting gene pools can profoundly modify the gene
implicitly assumed that there is the absence of recurring frequencies in natural populations. In essence, the Hardy-
mutations, any degree of preferential mating, differential mor- Weinberg theorem represents the cornerstone of population
tality or fertility, immigration or emigration of individuals, studies, since deviations from Hardy-Weinberg expectations
and fluctuations in gene frequencies due to sheer chance. But direct attention to the forces that disturb, or upset, the theo-
therein lies the significance of the Hardy-Weinberg theorem. retical equilibrium. The Hardy-Weinberg theorem is exceed-
In revealing the conditions under which changes in gene ingly useful for providing an estimate of (1) the frequencies
frequencies cannot occur, it brings to light the possible forces of heterozygous carriers of deleterious recessive alleles and
that could cause a change in the genetic composition of a (2) the risk of bearing offspring with detrimental recessive
population: mutations, preferential matings, mortality, infer- disorders in various marriages (Table 12-1).
tility, immigration and emigration, and chance fluctuations.
A population is a group of individuals living within a
Estimating the Frequency
defined geographic area. Historically, it may have been easier
to define a population because of the tendency for groups of
of Heterozygotes
individuals to remain in a location for long periods of time, Contrary to popular opinion, heterozygotes of a rare reces-
represented by centuries in some cases. As groups within a sive abnormality are rather common instead of comparatively
 Hardy-Weinberg Equilibrium 211

TABLE 12-1. Frequencies of Homozygotes and Heterozygous Carriers

FREQUENCY OF FREQUENCY OF HETEROZYGOUS RATIO OF CARRIERS

DISEASE HOMOZYGOTES CARRIERS (AA) TO HOMOZYGOTES

Sickle cell anemia 1 in 500 1 in 10 50 : 1

Cystic fibrosis 1 in 2500 1 in 25 100 : 1
Tay-Sachs disease 1 in 6000 1 in 40 150 : 1
Albinism 1 in 20,000 1 in 70 286 : 1
Phenylketonuria 1 in 25,000 1 in 80 313 : 1
Acatalasia 1 in 50,000 1 in 110 455 : 1
Alkaptonuria 1 in 1,000,000 1 in 500 2000 : 1

rare. Recessive albinism may be used as an illustration. The albino individuals (aa) in a given generation will come
frequency of albinos is about 1 in 20,000 in human popula- from normally pigmented heterozygous parents.
tions. When the frequency of the homozygous recessive (q2) Detrimental recessive alleles in a population are unques-
trait is known, the frequency of the recessive allele (q) can tionably harbored mostly in the heterozygous state. As shown
be calculated, as follows: in Table 12-1, the frequency of heterozygous carriers is many
times greater than the frequency of homozygous individuals
1 afflicted with a trait. Thus, an extremely rare disorder such
q2 = = 0.00005
20, 000 as alkaptonuria (blackening of urine) occurs in 1 in 1 million
persons. One in 500 persons, however, carries this detrimen-
q = 0.00005 = 0.007 tal allele in the hidden state. There are 2000 times as many
= about 1/140 or 1 in 140 (frequency of recessive allele) genetic carriers of alkaptonuria as there are individuals
affected with this defect. For another recessive trait, cystic
The heterozygotes are represented by 2pq in the Hardy- fibrosis, 1 in 2500 individuals is affected with this homozy-
Weinberg formula. Accordingly, the frequency of heterozy- gous trait. One in 25 persons is a carrier of cystic fibrosis.
gous carriers of albinism can be calculated as follows: In modern genetic counseling programs, an important con-
sideration has been the development of simple, inexpensive
q = 0.007 means of detecting heterozygous carriers of inherited disor-
ders. Molecular diagnostic techniques have increased the
p+q =1 number of carrier screening programs available for at-risk
populations.
p = 1 − 0.007 = 0.993

Therefore:
BIOCHEMISTRY
2pq = 2(0.993 × 0.007) = 0.014 Natural Selection for a Heterozygote:
= about 1/ 70 (frequency of heterozygous carriers) Sickle Cell Trait
Sickle cell trait is a heterozygous condition for a specific
Thus, although 1 person in 20,000 is an albino (recessive mutation in hemoglobin that is called hemoglobin S (Hb S).
homozygote), about 1 person in 70 is a heterozygous carrier— The occurrence of this mutation overlaps the distribution of
or, there are 285 times as many carriers as affected individu- malaria in Africa and is an example of a heterozygous
als. The rarity of a recessive disorder does not signify a condition that conveys a survival advantage in areas where
comparable rarity of heterozygous carriers. In fact, when the malaria is endemic. Sickle trait cells, those with only one
frequency of the recessive gene is extremely low, nearly all mutant allele, generally undergo little sickling at normal O2
the recessive genes are in the heterozygous state. tension. Though some sickling will occur as O2 tension
lowers, it is not as much as in sickle cell anemia with two
mutant alleles and twice as much mutant hemoglobin.
Significance of the Heterozygote Plasmodium falciparum requires normal intracellular K+
concentration. With lower O2 tension in the cell, potassium
When the frequency of a detrimental recessive allele becomes
diffuses out. P. falciparum grows poorly in the lower than
very low, most affected offspring (aa) will come from mating normal O2 tension occurring in Hb S cells and dies because
of two heterozygous carriers (Aa). For example, in the of the inadequate O2 and reduced K+.
human population, the vast majority (>99%) of newly arising
 212 Population Genetics and Medicine

X-Linked Loci Egg

In the above discussions, the genes and alleles considered A a

were autosomal genes with two possible alleles, A and a. For
X chromosome loci, the principles are similar but the male
Sperm a Aa aa
gamete may or may not carry an X chromosome (Fig. 12-2).
Since there is only one X chromosome in males, the genotype
frequency of any allele on that X chromosome is equal to the
allele frequency. In females, alleles may be distributed as A
p2AA: 2pqAa: q2aa, and thus a heterozygous carrier is deter-
mined just as with an autosomal trait. For example, if the Egg
frequencies of two X-linked alleles are 0.3 (A) and 0.7 (a),
then for a female offspring, the probability of carrying both A a
alleles (Aa) is
Sperm A AA Aa
2pq = 2(0.3 × 0.7) = 0.42

whereas the probability that a male offspring will carry either

B
allele is 0.3 or 0.7 for the A allele or a allele, respectively.
For recently arising mutations on the X chromosome, there Figure 12-2. X-linked loci. A, The sperm carries the a allele
on the X chromosome, and two offspring have an equal
may be a difference in allele frequencies between males and
chance of being Aa or aa from an Aa mother. B, The sperm
females. However, with each successive generation, differ- carries the A allele on the X chromosome, and two offspring
ences in frequencies between males and females becomes less have an equal chance of being AA or Aa from an Aa mother.
until equilibrium is approached. Allele frequencies will
approach equilibrium or be in equilibrium for those alleles
that have been in the gene pool for many generations. The TABLE 12-2. Alleles Shared by Consanguineous
expression of a recessive allele will occur at a higher fre- Matings
quency in males than in females just as implied above. In
females, the frequency of expression is q2. The X-linked form PROPORTION OF
of color blindness affects 1 in 20 white males, so q = 0.05. MATING SHARED ALLELES
The expected frequency in females is q2 = (0.05)2 = 0.0025
or 1 in 400 females. Parent-offspring 1/2
Brother-sister 1/2
Half-sibs 1/4
Uncle-niece, aunt-nephew 1/4
BIOCHEMISTRY First cousins 1/8
Half-first cousins 1/16
Lyonization of Ornithine Transcarbamylase (OTC) Second cousins 1/32
Deficiency in Females Third cousins 1/128

Another example of X chromosome genes and the role of

lyonization is ornithine transcarbamylase (OTC) deficiency,
the most common disorder of the urea cycle. This enzyme, alleles than individuals chosen at random from the general
part of a mitochondrial matrix, catalyzes the conversion of population (Table 12-2). If a recessive trait is extremely rare,
ornithine and carbamyl phosphate to citrulline. It is the the chance is very small that unrelated mating partners would
second step in the urea cycle. both harbor the same defective allele. The mating of close
As an X-linked disorder, OTC deficiency can be fatal in relatives, however, increases the risk that both partners have
newborn males owing to hyperammonemia. In females, received the same defective allele through some common
however, expression is variable because of lyonization. As ancestor. Therefore, the frequency of occurrence of a reces-
might be surmised, penetrance is 100% in males (with a sive disorder increases at the expense of the frequency of
severity index of 50), whereas it is only 20% in females and heterozygotes in the population.
the severity is low.
With increasing rarity of a recessive gene, it becomes more
and more unlikely that unrelated parents will carry the same
recessive allele. With an exceedingly rare recessive disorder,
●●● CONSANGUINITY AND the expectation is that most affected children will come from
cousin marriages. Human geneticists often infer that a reces-
RECESSIVE INHERITANCE
sive allele transmits a rare disorder when the incidence of
Offspring afflicted with a rare recessive disorder tend to arise consanguineous marriages is high. Thus, the finding that
more often from consanguineous unions than from marriages Toulouse-Lautrec’s parents were first cousins is the basis for
of unrelated persons. Close relatives share more of the same the popular view that the French painter was afflicted with
 Consanguinity and Recessive Inheritance 213

TABLE 12-3. Risk of PKU Offspring in Various Mating Relationships

MATING PARTNERS
THEORETICAL
FREQUENCY OF
A B
AFFECTED CHANCES OF
CHILDREN IF AFFECTED
Chances of Chances of BOTH PARENTS CHILDREN FROM
Carrier Status Carrying Allele Carrier Status Carrying Allele WERE CARRIERS SUCH A MATING

Unknown 1 in 80 Unknown 1 in 80 1 in 4 1 in 25,600

Unknown 1 in 80 Normal sibling of 2 in 3 1 in 4 1 in 480
phenylketonuric
Unknown 1 in 80 Parent of 1 1 in 4 1 in 320
phenylketonuric
Unknown 1 in 80 Phenylketonuric 1 1 in 2 1 in 160
Normal sibling of 2 in 3 Normal sibling of 2 in 3 1 in 4 1 in 9
phenylketonuric phenylketonuric
Normal sibling of 2 in 3 Parent of 1 1 in 4 1 in 6
phenylketonuric phenylketonuric
Normal sibling of 2 in 3 Phenylketonuric 1 1 in 2 1 in 3
phenylketonuric
Parent of 1 Parent of 1 1 in 2 1 in 2
phenylketonuric phenylketonuric
Parent of 1 Phenylketonuric 1 1 1
phenylketonuric
Phenylketonuric 1 Phenylketonuric 1 1 1

pycnodysostosis, characterized by short stature and a narrow

lower jaw, which is governed by a rare recessive gene, rather I
than as had been formerly thought, with achondroplasia, 1 2
which is determined by a dominant gene. Both demonstrate
similar phenotypes, but in the absence of laboratory data and
with the knowledge that the parents were related, the choice II
of an autosomal recessive disorder is a better probable 1 2 3 4
diagnosis. (Aa) (Aa)
One of the first indications that phenylketonuria (PKU) ? ?
III
is controlled by a recessive allele was the relatively high 1
percentage of first-cousin marriages among parents of
affected children. Data from the United States, England, Figure 12-3. An example of the effects of consanguinity.
and Norway indicated that, for this trait, the parents are
often close relatives. Individuals suffering from phenylke-
tonuria have peculiarities of posture and make jerky, or from such a marriage would be 1 in 9. Calculations of the
convulsive, movements. Untreated phenylketonuric patients risk of PKU in offspring from different types of marriages are
are mentally retarded, usually so severely that they are shown in Table 12-3.
institutionalized (see Chapter 4). The offspring of consanguineous marriages are said to be
The number of heterozygous carriers of a detrimental “inbred.” Thus, the expectation is that more genetic disorders
recessive PKU allele is about 1 in 80. Thus, the probability resulting from recessive traits will occur in these individuals.
that two carriers from the general population will marry is 1 However, many recessive genetic disorders occur within the
in 6400 (80 × 80). If both marriage partners are carriers, then general population that are not the result of consanguinity. In
theoretically 1 of every 4 children will be afflicted with PKU. fact, most result from the chance event of heterozygous car-
The first probability figure (1 in 6400) multiplied by the riers mating and unknowingly passing an affected allele to an
second probability figure (1 in 4) gives the total chance of 1 offspring.
in 25,600 for affected children from two normal persons
who marry at random from the general population. This prob-
ability is increased enormously if two normal individuals
General Aspects of Consanguinity
marry, both of whom had heterozygous carrier parents. The One way to show the effects of consanguinity is to consider
marital partners would each have a two-thirds chance of a situation in which a widow marries her late husband’s
being carriers themselves, and the risk of affected children brother. As Figure 12-3 shows, a child with phenylketonuria
 214 Population Genetics and Medicine

(III-1) was born in the first marriage of the man (II-1) and the ●●● ASSORTATIVE MATING
woman (II-2). The man dies, and his brother (II-3) feels
obliged, as required by certain religious laws, to marry the
AND INBREEDING
widow and assume responsibility for the family. The couple In some circumstances, mates are selected for characteristics
may ask the question: What are the chances that the second shared or not shared in common. These situations are referred
marriage will produce a child with PKU? Alternatively, what to as positive assortative mating and negative assortative
would be the chances of a phenylketonuric offspring if the mating, respectively. For those characteristics that are geneti-
widow were to marry instead an unrelated man (II-4) with cally determined, an increase in homozygosity will be
no family history of PKU? observed. This is not the same as inbreeding. Inbreeding
As Figure 12-3 suggests, the widow (II-2) and the late involves all loci on all chromosomes with a resulting increase
husband (II-1) are each heterozygous. The late husband’s in homozygosity at many loci; assortative mating affects only
brother (II-3) is phenotypically normal, but the probability is those characteristics that are similar. Examples are matings
1 in 2 that he harbors the recessive allele for phenylketonuria that occur between individuals with similar clinical conditions
(see Chapter 13, Recurrence Risk Assessment). This recessive such as dwarfism or congenital deafness.
allele was transmitted by one of his parents, either I-1 or I-2.
Both parents might have been heterozygous, but since the
trait is rare, it is more probable that only one parent was a
carrier.
NEUROSCIENCE
Individual probabilities may be categorized as follows:
● Chance that the widow (II-2) is a carrier = 1
Positive Assortative Mating
● Chance that the late husband’s brother (II-3) is a carrier
Positive assortative mating examples include selection of
= 1/2 mates for the following:
● Chance for a homozygous recessive child = 1/4
■ Intellectual ability
Thus, the total chance that a child of this marriage will be ■ Athletic talent
afflicted with PKU is ■ Physical characteristics
■ Parental income

1 × 1/ 2 × 1/ 4 = 1/ 8 ■ Place of birth
■ Home town

If the widow marries someone other than her late hus-

band’s brother, the probability that the unrelated man (II-4)
is a carrier is equal to the frequency with which carriers
occur in the general population. As noted earlier, 1 in 80 ●●● DNA TECHNOLOGY AND
persons is a carrier of the recessive allele for phenylketon-
CLINICAL DIAGNOSIS
uria. Hence, the chance that a man picked at random from
the general population happens to be heterozygous for PKU Prior to the development of DNA technology and applied
is 1 in 80. In this circumstance, the individual events are diagnostic capabilities, physicians were limited in their capac-
as follows: ity to determine whether an expectant mother might deliver
● Chance that the widow (II-2) is a carrier = 1 a child with a serious birth defect, such as Tay-Sachs disease.
● Chance that the unrelated man (II-4) is a carrier = This fatal disease takes its lethal toll by the age of 3 to
1/80 4 years. There are no known survivors, and there is no
● Chance for a homozygous recessive child = 1/4 cure. For the most part, physicians in the past referred
Thus, the total chance that the child will be afflicted with prospective parents with a family history of a severe con-
PKU is genital disorder to specialists in genetic counseling. Even
then, the prospective parents could be informed only of
1 × 1/80 × 1/ 4 = 1/ 320 the statistical probability that they would have a child with
a particular genetic disorder. Using statistical information
It should be evident that, if the widow marries her late comparable to that shown in Table 12-3, parents could be
husband’s brother, the likelihood of having a homozygous given no assurance that the actual outcome of the preg-
recessive child is increased 40-fold from 1 in 320 to 1 in nancy would be favorable. Many high-risk couples in the
8. Clearly, two siblings (in this case, two brothers) have a past avoided pregnancy rather than hazard the birth of an
greater chance of inheriting the same recessive gene from affected child. Today, this situation has been altered dra-
a common ancestor (in this case, one of their parents) than matically by the availability of molecular techniques that
do any two individuals selected at random in a general reveal a disorder in the fetus or in preimplantation embryos.
population. As a generalization, the farther removed two This approach to detecting genetic disorders, referred to
individuals are from a common ancestor, the smaller the as prenatal or preimplantation diagnosis, is now available
likelihood that both individuals will receive the same alleles to prospective parents for many single-gene disorders (see
from that ancestor. Chapter 13).
 Dna Technology and Clinical Diagnosis 215

NEUROSCIENCE TABLE 12-4. Mutation Detection Rates Using a

Panel of 25 Alleles*
Tay-Sachs Disease
Tay-Sachs disease is an autosomal recessive metabolic POPULATION DETECTION FREQUENCY
disease distinguished by a deficiency of hexosaminidase A,
resulting in the accumulation of gangliosides in neurons, Ashkenazi Jewish 95%
axons, and glial cells. Neuropathy occurs in these North American 80–85%
individuals because of demyelination, gliosis, and neuronal Spanish 75%
loss. In the early phase of the disease, changes are seen by Blacks 64%
magnetic resonance spectroscopy in the basal ganglia and Mexican 58%
Colombian 46%
thalamus. In the later phase of the disease, changes are
Venezuelan 33%
demonstrated by advanced brain atrophy and diffuse white
Other groups Variable
matter lesions of the basal ganglia, thalamus, and cortical
layer of the cerebrum. *Recommended by the American College of Medical Genetics for routine
screening and carrier testing.

Similarly, suspected genetic disorders at most stages of 80% of cystic fibrosis results from a single mutation in the
development and detection can be evaluated with develop- CFTR gene. Different allele frequencies vary with different
ment of proper diagnostic tools for gene evaluation. Impor- populations. Using a panel to screen for the most common
tant to clinical application, these tools are most effective mutations in a single population may not adequately identify
when applied to single-gene disorders. For complex multifac- cystic fibrosis in other populations (Table 12-4). However, this
torial disease, DNA diagnostics is not recommended. same information may provide better diagnostic screening
within specific populations because of the propensity of spe-
cific mutations to occur within a population. It may not
BIOCHEMISTRY remove all doubt about a result, and false negatives may still
occur, but increased confidence occurs when specific muta-
Diagnosis of Sickle Cell Disease tions are known to occur. Likewise, for disorders with a high
A single nucleotide change in the normal β-globin allele incidence of spontaneous mutations, identifying the molecu-
produces a change in the amino acid conformation. lar defect with molecular techniques provides a tool for
■ An A-to-T mutation in the sixth codon results in a glutamic
testing a proband’s offspring. Even with the advent of and
acid–to-valine substitution. increased reliance on molecular DNA techniques, the Hardy-
■ The A-to-T mutation changes the CTGAGG enzyme
Weinberg theorem remains a powerful component of risk
recognition site, and a diagnostic test can be designed
analysis and family counseling.
with this information.
■ Mutation replaces a charged glutamic acid with an

uncharged valine that affects structure.

■ Testing may be done prenatally by amniocentesis or

chorionic villus sampling.

Parents
KEY CONCEPTS
■ Two commonly used genetic probability equations are: p + q =
1 and p2 + 2pq + q2 = 1. Variations in allele (q) and disease (q2)
Children frequencies are determined with these equations.
1 2 3 ■ These equations are most often used with autosomal recessive
GTG disorders because these are readily recognized as being q2
Beta-S (aa). For autosomal dominant disorders, it can be difficult to
GAG distinguish between homozygous (p2) and heterozygous (2pq)
Beta-A individuals.
■ For X-linked recessive disorders, q is the disease and allele fre-
quency in males. For X-linked disorders in females, the allele and
Limitations to the use of DNA diagnostics for single-gene disease frequencies may not be the same.
disorders occur when there are many alleles for a particular ■ Recessive disorders are often present in offspring from related
gene. The larger the gene, the more opportunities for muta- individuals. The more deleterious genes in common between a
tions to occur and the less likely an individual can be tested male and female, the more likely two deleterious alleles will occur
for all alleles. An example is cystic fibrosis, for which over in the offspring.
1500 mutations have been described (see Chapter 9). About
 216 Population Genetics and Medicine

●●● QUESTIONS A. 100%

B. 50%
1. A study of the long QT syndrome (Romano-Ward C. 25%
syndrome) in a Utah community demonstrated a D. 1%
founder affect with an incidence of 1 in 5000. What E. 0%
is the frequency of unaffected individuals in this
Answer. C
population?
Explanation: This is an autosomal recessive presentation.
A. 0.998
The parents of children with rare disorders are unaware
B. 0.985
that they are heterozygous carriers of the mutated alleles
C. 0.199
that are inherited by the children until the disease or dis-
D. 0.014
order presents. Because each gamete represents an inde-
E. 0.002
pendent assortment of alleles, each is an independent
Answer. A event. There is a 25% chance of a recessive allele being
Explanation: Applying the Hardy-Weinberg equation to fre- inherited from each parent at the same locus. If the ques-
quencies almost always deals with autosomal recessive tion were worded differently and asked “What is the prob-
disorders rather than autosomal dominant disorders. The ability of having any three affected children in the same
reason is that, when someone has a recessive disorder, the family,” the answer would be 0.25 × 0.25 × 0.25, or the
genotype is known to be aa. With a dominant disorder, it is product of each individual probability.
often difficult to distinguish between a homozygote and a 4. Among a community of individuals, type O blood
heterozygote. Therefore, aa = a2 = 1/5000 = 0.014 and A = does not exist even though it is the most common
0.985. It is inappropriate to say that only homozygotes are blood group among all populations. In this popula-
unaffected, so 2pq must be considered; 2pq = 2(0.014 × tion, which of the following best describes the force
0.985) = 0.28. AA + Aa = 0.9852 + 0.028 = 0.97 + 0.028 = that allowed the O antigen alleles to disappear in
0.998. this population?
2. A 5-year-old male is hospitalized with anemia, jaun- A. Chance fluctuations
dice, and cholelithiasis. Laboratory testing reveals B. Decreased immigration
hemolysis and sickle cell anemia, which occurs at C. Decreased mortality
a frequency of 1 in 500 individuals. Which of the D. Increased genetic drift
following represents the frequency of sickle cell E. Increased infertility
carriers in the same population?
Answer. B
A. 0.002
Explanation: The application of the Hardy-Weinberg
B. 0.045
theorem has several assumptions for equilibrium to occur.
C. 0.086
While these assumptions are not maintained in real popula-
D. 0.5
tions, they do provide an explanation when observations do
E. 0.955
not meet theoretical expectations. If, when comparing any
Answer. C two populations, something occurs at a greater or lesser
Explanation: The frequency of sickle cell anemia is 1/500 frequency than expected from looking at another popula-
(q2), so q = 1/22.4 (approximately) or 0.045. According to tion, there must be a reason. These forces that alter the
the Hardy-Weinberg equation, the frequency of the hetero- observations are mutations, preferential matings (as in con-
zygous carrier is 2pq or 2(0.045)(0.955) = 0.086. sanguineous matings), mortality, infertility, immigration/
emigration, and chance fluctuations. Each of these can be
3. A couple has two children with a rare disorder and the premise upon which a particular frequency might be
is concerned about an unborn child developing the altered.
same disorder. What is the probability of the third
child developing this disorder? Additional Self-assessment Questions can be Accessed
at www.StudentConsult.com

?
Modern Molecular 13
Medicine
CONTENTS biology that is dictated by a unique set of alleles. As more is
revealed about how these alleles interact with each other and
the environment, specific treatments will be customized fol-
TOOLS OF MOLECULAR MEDICINE
lowing genetic diagnosis. In this chapter, modern medicine in
Revised Diagnostic Paradigm its appropriate molecular context is discussed with a focus on
Molecular Genetic Techniques three essential areas: (1) the tools of molecular medicine;
Mutation Detection (2) the role of genetics in diagnosis, screening, and counseling;
GENETIC TESTING AND SCREENING
and (3) molecular genetic approaches to treatment.

Fundamentals of Genetic Testing

Genetic Screening
Prenatal Genetic Testing and Preimplantation Genetic ●●● TOOLS OF
Diagnosis MOLECULAR MEDICINE
GENETIC COUNSELING AND RECURRENCE RISK Determining the complete DNA sequence of Homo sapiens
ESTIMATION has provided a template for understanding molecular defects
that underlie human infirmities. By elucidating the approxi-
Genetic Counseling
mately 23,000 genes that make up each person, the ability to
Recurrence Risk Assessment
see pathologic states as integrated entireties rather than snap-
GENETIC APPROACHES TO TREATMENT shots of a single altered biochemical pathway is unfolding. In
other words, modern biomedical scientists can take a
Gene Therapy
Gene Replacement Versus Gene Silencing
“systems-based” approach to the molecular basis of disease.
Pharmacogenomics and Individualized Medicine From a genetics point of view, this underscores a transition
from focusing on single-gene etiologies with biological and
NEW DIAGNOSTIC APPROACHES clinical manifestations toward medical genomics and an
Microarray Analysis understanding of how multiple genes simultaneously interact
to precipitate disease.
SUMMARY Today, physicians and biomedical scientists use data extrap-
olated, or “mined,” from the Human Genome Project as the
foundation for further understanding of molecular and cel-
lular processes. For example, considerable effort has been
invested in characterizing the function of proteins expressed
from the human genome and how these gene products inter-
Over the last 15 years, the relationship between genetics and act with each other, a study known as proteomics. Likewise,
physicians has undergone a radical change. Whereas genetics since many diseases result from the dysregulation of gene
was once an explanation for inheritance patterns, it is now expression, transcriptomics, or the integrated study of global
recognized that essentially all disease has a genetic basis. The gene expression and regulation, has been vitalized. Other
Human Genome Project coupled with an explosion of tech- integrated areas of study such as pharmacogenomics, metab-
nological advances has permitted elucidation of genetic olomics, and nutrigenomics have emerged; all feature the
mechanisms for common and rare disorders that range from current ability to see the larger picture regarding genes and
simple single-gene etiologies to complex polygenic or multi- proteins, including expression and interaction.
factorial disease susceptibilities. Because a genetic signature The knowledge base of molecular genetics has grown hand-
typifies disease, individualized genetic profiles can be obtained in-hand with the technologies that created the rapidly expand-
and the age of “personalized medicine” becomes possible. ing knowledge base. The challenge for the modern physician
Such a concept recognizes that each individual has a certain and medical geneticist is to apply the evolving genetic
 218 Modern Molecular Medicine

Patient interview
Family history
Pedigree analysis

Physical examination

Preliminary diagnosis

Laboratory testing Genetic testing

Blood work Test for known mutations
Metabolic indicators or chromosomal defects
Biochemical assays New mutation discovery

Figure 13-1. Diagnostic algorithm.

Molecular diagnostics is changing the Biomedical research Biomedical counseling
approach to diagnosis and treatment of New mutation discovery Diagnosis confirmation
Therapeutics Prognosis indicators
disease. Beyond standard laboratory Pharmacologic choices
Genotype/phenotype
tests, genetic testing is providing correlations Reproductive decisions
unique patient information leading to Genetic databases Environmental risks
personalized treatment regimens as Recurrence risks
well as family and patient information Education and support
that until recently was limited.

paradigms and investigative and diagnostic methodologies to physical examination, and supportive laboratory tests, a diag-
clinical practice. To do so requires an understanding of the nosis can typically be made. In the post–Human Genome
tools and approaches used in molecular medicine. These Project era, however, the diagnosis may be considered pre-
include the diagnostic paradigm, molecular genetic tech- liminary in some cases, with confirmation based on DNA-
niques used for diagnosis and prognosis, and an understand- based analysis or genetic testing. The association of a known
ing of the relevance of medical genetics to the modern health mutational or chromosomal variant with a disease phenotype
care system. confirms the diagnosis and may even provide prognostic
value if significant genotype-phenotype correlations are
documented. The importance of this portion of the modern
Revised Diagnostic Paradigm diagnostic paradigm is evidenced by the dramatic increase
For many years, a typical generalized paradigm for initial in genetic testing laboratories over the last 10 to 15 years,
patient-physician interaction has consisted of three central accompanied by the acceptance of insurance companies that
elements. First, the physician interviews the patient, paying molecular genetic diagnostics provides a high degree of accu-
special attention to the initial complaint. Questions generally racy and specificity.
focus on signs and symptoms and quality-of-life issues. A In the absence of a known pathogenic genetic variant, a
physical examination ensues, followed by the third element, physician may feel unsure of a clinical diagnosis. In this case,
laboratory testing, to assess either the functionality of organ it may be that the patient harbors a new, previously unchar-
systems or biochemical parameters in bodily fluids; typically acterized mutation. Such patients and families are the founda-
these include tests of blood, urine, and cerebrospinal fluid. tion for the discovery aspects of modern biomedicine. Hence,
Such inputs lead to a clinical diagnosis and an appropriate it is important that today’s physician realize the vital role of
standard of care. public and private research efforts to the biomedical enter-
In the current age of molecular medicine, however, addi- prise. By means of appropriate dialogue and approved
tional steps are added to this paradigm (Fig. 13-1). The first informed consent, the physician-patient axis can collaborate
part of the revised paradigm remains as described with one with medical geneticists and biochemists to discover new
important special emphasis. Given the genetic basis of most disease-causing mutations (Box 13-1). This, in turn, promotes
disease and the recent progress made in understanding robust genetic databases, new diagnostic capacity, and the
genetic predisposition, it is essential that physicians pay close possibility of novel or enhanced genotype-phenotype asso-
attention to family history and perform detailed pedigree ciations of interest to patient care.
analyses. Observations made here can provide important Finally, with the explosion of genetic information available
diagnostic clues. to the physician comes a responsibility to accurately convey
The remainder of the diagnostic algorithm requires addi- the implications of genetic findings to the patient. This indi-
tional input. Following the family history/patient interview, cates that genetic counseling should be added to the
 Tools of Molecular Medicine 219

Box 13-1. INFORMED CONSENT FOR HUMAN Nucleic Acid Visualization

SUBJECTS RESEARCH* Patient cells are necessary to initiate molecular genetic analy-
sis. Typically, patient material is a blood sample, biopsy speci-
Components of Informed Consent men, and amniotic fluid or chorionic villus sample. Using
Risks to subjects are minimized. blood as an example, total genomic DNA is extracted and the
Procedures are used that are consistent with sound DNA is visualized or prepared for subsequent analysis by a
research design and that do not unnecessarily expose number of techniques, including restriction fragment length
subjects to risk. polymorphism (RFLP) and Southern blotting, DNA amplifica-
Whenever appropriate, procedures are used that are already tion using the polymerase chain reaction (PCR), and DNA
being performed on the subjects for diagnostic or
sequence analysis.
treatment purposes.
Risks to subjects are reasonable in relation to anticipated
benefits. RFLP and Southern Blot Analysis
Only those risks and benefits that may result from the Southern blotting is a method to visualize DNA of interest.
research (as distinguished from risks and benefits of There are three fundamental elements to the Southern blot
therapies subjects would receive even if not participating procedure: (1) fragmentation of the DNA, (2) electrophoretic
in the research) are considered. separation of the DNA on the basis of fragment size, and
Long-range effects of applying knowledge gained in the (3) identification and visualization of informative fragments.
research are not considered. Fragmentation typically occurs through the use of restriction
Selection of subjects is equitable. endonucleases, also referred to as restriction enzymes.
There may be special problems with vulnerable populations: Restriction endonucleases are enzymes of bacterial origin
children, prisoners, pregnant women, mentally disabled
that bind to the DNA at specific sites and cleave both strands
persons, or economically or educationally disadvantaged
of the DNA. The DNA recognition sites for restriction
persons.
Informed consent will be sought from each prospective enzymes are typically palindromic sequences 4 to 8 bases
subject or the subject’s legally authorized representative. in length, and the double-stranded breaks occur within or
Informed consent will be appropriately documented. adjacent to the recognition sites. For example, the restriction
When appropriate, the research plan makes adequate enzyme EcoRI recognizes and binds to the six-nucleotide
provision for monitoring the data collected to ensure the sequence of 5′-GAATTC-3′. In this case, the enzyme nicks
safety of subjects. the phosphodiester bond between the G and the A on both
When appropriate, there are adequate provisions to protect strands, generating two DNA fragments with so-called sticky
the privacy of subjects and to maintain the confidentiality ends (Table 13-1). Because a 6-base-pair sequence such as
of data. GAATTC is encountered frequently in the human genome,
*Requires approval of an Institutional Review Board to provide certain
treatment of genomic DNA with the EcoRI enzyme gener-
safeguards to the participants. (Data from U.S. Department of Health and ates roughly 1 million DNA fragments. A single-base change
Human Services, Office for Human Research Protections, 45 CFR §46.111.) in a restriction enzyme recognition site prevents an endo-
nuclease from binding to the DNA, resulting in no cleavage
of the DNA. Conversely, a single-base change may create a
paradigm. Whether done by the physician or by professional new recognition site where none existed prior to the base
genetic counselors, this medical service conveys an essential change. Different individuals in the population harbor a
service to the patient because it can describe confirmation of variety of benign point mutations that alter restriction
diagnosis (refer to Table 7-4), prognostic indicators, risk enzyme recognition sites, thus resulting in a different col-
assessment for the patient and relatives, information critical lection of fragments. Such variation in DNA fragment sizes
for reproductive decisions and prenatal genetic testing, and obtained by restriction endonuclease digestion is visualized
even direct disease management (Box 13-2). New essentials by gel electrophoresis and Southern blotting; this procedure
in the generalized diagnostic paradigm are added to the core is termed restriction fragment length polymorphism, or RFLP,
essentials of patient interview and family history, physical analysis (Fig. 13-2).
examination, and laboratory testing. These include molecular For gel electrophoresis, DNA fragments are separated
genetic diagnostics, collaboration with biomedical scientists, through a porous matrix by the application of an electrical
and genetic counseling. current. Fragments migrate to the positive pole by virtue of
the inherent negative charge of DNA molecules. Smaller frag-
ments migrate faster than larger fragments, thereby resolving
Molecular Genetic Techniques in the gel on the basis of size. The fragments are visualized
Physicians should be able to interpret fundamental genetic by the addition of a dye molecule that intercalates between
data just as they interpret biochemical data, ECGs, or respi- the DNA base pairs and fluoresces upon illumination with
rometry results. To do so, an understanding of basic molecular UV light. Fluorescent dyes with greater sensitivity for staining
genetic methodologies is necessary. Below, these fundamental small quantities of DNA and of less toxicity have replaced
approaches and applications to molecular diagnostics are earlier toxic dyes such as ethidium bromide. Often, however,
described. a particular fragment of diagnostic value is of interest, but it
 220 Modern Molecular Medicine

Box 13-2. THE HEALTH INSURANCE PORTABILITY AND ACCOUNTABILITY ACT (HIPAA) PRIVACY RULE*

Elements of an Authorization
Core Elements Notice of the covered entity’s ability or inability to condition
The name(s) or other specific identification of person(s) or treatment, payment, enrollment, or eligibility for benefits
class of persons authorized to make the requested use or on the Authorization, including research-related treatment,
disclosure. and, if applicable, consequences of refusing to sign the
The name(s) or other specific identification of the Authorization.
person(s) or class of persons who may use the PHI or The potential for the PHI to be re-disclosed by the recipient
to whom the covered entity may make the requested and no longer protected by the Privacy Rule. This
disclosure. statement does not require an analysis of risk for
Description of each purpose of the requested use or re-disclosure but may be a general statement that the
disclosure. Researchers should note that this element Privacy Rule may no longer protect health information.
must be research study specific, not for future unspecified Optional Elements: Examples that may be relevant to the
research. recipient of protected health information.
Authorization expiration date or event that relates to the Your health information will be used or disclosed when
individual or to the purpose of the use or disclosure (the required by law.
terms “end of the research study” or “none” Your health information may be shared with a public health
may be used for research, including for the creation and authority that is authorized by law to collect or receive
maintenance of a research database or such information for the purpose of preventing or
repository). controlling disease, injury, or disability, and conducting
Signature of the individual and date. If the Authorization public health surveillance, investigations or interventions.
is signed by an individual’s personal representative, a No publication or public presentation about the research
description of the representative’s authority to act for the described above will reveal your identity without another
individual. authorization from you.
Required Elements If all information that does or can identify you is removed
The individual’s right to revoke his/her Authorization in from your health information, the remaining information
writing and either (1) the exceptions to the right to revoke will no longer be subject to this authorization and may be
and a description of how the individual may revoke used or disclosed for other purposes.
Authorization or (2) reference to the corresponding If you revoke this Authorization, you may no longer be
section(s) of the covered entity’s Notice of Privacy allowed to participate in the research described in this
Practices. Authorization.

*Provides comprehensive federal protection for the privacy of personal health information (PHI). Research organizations and researchers may or may not be
covered by the HIPAA Privacy Rule. (Data from U.S. Department of Health and Human Services, Office for Human Research Protections, 45 CFR §164.508.)

TABLE 13-1. Examples of Commonly Used Restriction Endonucleases

ENZYME SOURCE TYPE OF PRODUCT RECOGNITION SEQUENCE* FRAGMENT ENDS

AluI Arthrobacter luteus Blunt ends AG↓CT 5′ AG CT 3′

TC↑GA 3′ TC GA 5′
EcoRI Escherichia coli 5′ Overhang G↓AATTC 5′ G AATTC 3′
strain R CTTAA↑G 3′ CTTAA G 5′
PstI Providencia stuatii 3′ Overhang CTGCA↓G 5′ CTGCA G 3′
G↑ACGTC 3′ G ACGTC 5′
MnlI Moraxella Nonpalindromic sequence CCTCNNNNNNN↓ 5′ CCTC(N7) 3′
nonliquefaciens GGAGNNNNNN↑N 3′ GGAG(N6) N 5′

*Arrows show cleavage sites of sequence; N, any nucleic acid.

is impossible to identify an individual fragment with many restriction endonuclease digestion or small, synthetic DNA
fragments resolved in the gel. The ability to visualize single oligonucleotides.
DNA fragments requires the addition of a DNA probe that A gene-specific probe is not hybridized to its complement
hybridizes specifically to the fragment of interest. A DNA in an agarose gel owing to the difficulty of working with the
probe is typically single stranded and complementary to the gel matrix itself. Instead, the fragmented DNA is transferred
gene or region of DNA interest. It is labeled with a fluorescent to a dry filter and the probe is added. This is the essence of
or radioactive tag so that the probe-target complex can be the Southern blot procedure (see Fig. 13-2). Specifically, fol-
detected. Probes can be DNA fragments purified from lowing gel electrophoresis, the DNA strands are denatured
 Tools of Molecular Medicine 221

complement restriction fragment that enables the probe to

A B C specifically anneal only to its complement. Visualization of a
DNA fragment of interest is performed by laser-facilitated
detection of the fluorescent probe or by exposure of the filter
to x-ray film in the case of a radioactive probe. Thus, by
Blood samples means of a combination of restriction endonuclease digestion,
gel electrophoresis, and Southern blotting, visualizing a DNA
fragment of interest is accomplished.

Digest DNA with Polymerase Chain Reaction

restriction enzyme The introduction of polymerase chain reaction (PCR) has
revolutionized DNA-based diagnostics. The rapid, inexpen-
A B C sive amplification of specific DNA sequences made possible
: with PCR has tremendously enabled both preparative and
analytic procedures. PCR is the in vitro enzymatic amplifica-
tion of a short (up to 5- to 6-kb) DNA sequence. Amplifica-
tion can be initiated with even a single DNA molecule and
Separate by gel
electrophoresis
produces millions of copies in a period of a few hours. There
are four essential components to PCR: two deoxyoligonucle-
otide primers, a thermostable DNA polymerase, target DNA,
and nucleotides. The primers add specificity to the amplifica-
tion by defining the flanking regions of DNA sequences to be
; amplified. Primers are “designed” to reflect the complemen-
tary nucleotide sequence of the target. These are usually 15
Denature DNA to 30 nucleotides in length and synthesized by an automated
process. Primers bind to targets in a 5′-3′ direction on each
strand, and amplification occurs between them.
Blot onto membrane Amplification occurs during a series of denaturing, heating,
and cooling phases that are repeated numerous times. These
serve to dissociate double-stranded DNA, allow primers to
Add radioactive or
anneal, and facilitate new strand extension. The polymerase
nonradioactive probe
used in this procedure must be thermostable at high tempera-
tures, a feature not characteristic of enzymes. The best known
of the special thermostable polymerases was isolated from
Thermus aquaticus. Designated DNA taq polymerase, it with-
Expose to film
stands repeated cycles of 95°C or greater.
During denaturation, the target DNA in the reaction is
heated to 95°C, rendering the target DNA single-stranded by
breaking the hydrogen bonds between the two strands. The
A reaction is then cooled, typically to 45° to 65°C, to permit
annealing of the single-stranded primers to complementary
B
sequences in the target DNA. Finally, the temperature is
C increased to 70° to 75°C to allow extension or synthesis of
the new DNA most efficiently. During the extension phase,
DNA polymerase uses the free 3′-OH group on the primers
Figure 13-2. RFLP and Southern blotting. Radioactively to synthesize new DNA. This three-step, or three-phase, cycle
labeled probes are used less frequently since the advent of is repeated with the newly synthesized DNA strands serving
nonradioactive methods to label probes. as templates for additional strand formation. Early in the
process, the original genomic DNA is diluted out and the
ends of the newly synthesized DNA strands become entirely
with a strong base while still in the gel. The single-stranded defined by the primers. Overall, this cycle is repeated 20 to
DNA is then transferred to a nylon or nitrocellulose mem- 35 times and the number of DNA copies doubles with each
brane support by capillary action; sometimes application of cycle, thus resulting in millions of copies of specific, primer-
a vacuum facilitates transfer. At this point, the denatured directed DNA fragments (Fig. 13-3). It should be noted that
DNA is “fixed” to the membrane and a probe is applied the thermostable DNA polymerase, an excess of primers, and
under conditions that favor the reestablishment of DNA the formation of newly synthesized DNA fragments that serve
duplexes. It is the Watson-Crick hydrogen bond base-pairing as templates for subsequent cycles permit the iterative cycles
between the single-strand probe and its single-stranded of amplification.
 222 Modern Molecular Medicine

1 TABLE 13-2. Comparison of PCR and RFLP

2
Denaturation CHARACTERISTIC RFLP PCR
Hybridization of strand-specific primers
to their target sequences
Time requirement Days Hours
1
Amount of DNA Micrograms to Picograms or
2 milligrams less
Polymerase reaction required
Cost May be high with Rarely needs
radioactivity radioactivity
1 usage and and
3
4 disposal; therefore
2
less costly if less
Renewed denaturation nonradioactive expensive
Renewed hybridization of strand-specific probes used
primers Sensitivity Less sensitive More sensitive
1 to small to small
quantities quantities
3 of DNA or copy
numbers
2 of DNA
4

Renewed polymerase reaction

1 regions of genes are sequenced to detect a disease-specific

5 mutation. Because the human genome sequence is known,
6 PCR primers can be designed to amplify specific DNA for
3
2 7 sequencing. Since DNA sequencing is greatly facilitated by
abundant target DNA, only one application of PCR amplifi-
4
cation of a specific DNA substrate is generally needed for
8
sequencing.
The most common method of DNA sequencing is the
Sanger dideoxynucleotide chain-terminating technique (Fig.
20–30 reaction cycles of denaturation, primer
annealing, polymerase reaction 13-4). Here, heat-denatured, single-stranded template, such
as PCR products, is added to each of four tubes. Each tube
contains a mixture of the four nucleotides (A, G, C, and T),
which acts as a substrate for DNA polymerase. Each tube also
contains one of the four chain-terminating dideoxyribonucle-
106- to 107-fold amplification of fragment
otides (ddA, ddG, ddC, or ddT). Hence the “A” tube contains
flanked by primer binding sites
a mixture of the four normal radioactively labeled nucleo-
Figure 13-3. Polymerase chain reaction. tides (dA, dG, dT, and dC) plus the chain-terminating ddA; a
similar mixture of dNTPs to ddNTPs is found in the “G,” “C,”
and “T” tubes. The incorporation of any dideoxynucleotide
prohibits further DNA polymerization because these lack the
Later, application of the PCR in molecular genetic diagnos- 3′-OH group required by DNA polymerase to add the next
tics will be described. It should be apparent, however, that nucleotide. The addition of DNA polymerase and oligonucle-
PCR has two direct uses. First, it can provide abundant sub- otide primers, which are required to anneal to the target and
strate for mutation detection strategies. Second, it can be a initiate DNA polymerization via the free 3′-OH, to each reac-
stand-alone diagnostic tool for detecting changes in DNA tion tube initiates DNA synthesis. However, the chains are
length, such as small insertions or deletions. In either case, terminated at different positions owing to the random inser-
PCR has greatly facilitated the era of genetic medicine by tion of a dideoxynucleotide instead of a normal deoxynucleo-
providing a rapid and inexpensive tool to biomedical scien- tide. Stated differently, when a ddG is inserted by the DNA
tists working on both diagnostic and research procedures polymerase instead of a dG, synthesis stops at that point. In
(Table 13-2). the “G” tube reaction, for example, some chains will be ter-
minated at the initial G encountered by the DNA polymerase,
DNA Sequence Analysis while other chains will be halted at other G positions of the
In the post–Human Genome Project era, “unknown” regions chain. In the “G” tube, the ratio of dGTP/ddGTP is such that
of the chromosome are no longer sequenced to find new there will be a number of chains that have terminated at each
disease-causing mutations. More typically, genes or certain G position along the template.
 Tools of Molecular Medicine 223

G T C A T A G G T G A C

C A G T A T C C A C T G

G T C A T A G G T G A C

C A G
dTTP
; dATP
dCTP
dGTP

;ddTTP ;ddCTP ;ddATP ;ddGTP

CAGT A T CCAC T CAGT A T CCAC CAGT A T CCA CAGT A T CCAC TG

CAGT A T CAGT A T CC CAGT A CAG

G
CAGT CAGT A T C CA
T A
C
C

T C A G

G
T
C
A
C
C
T
A
T
A

440 1520 1600 1680 1760 1840 1920 2000

C G G C G T T G C T C C GT C A G AC T T TC G TC C A T T G C G G A A G A T T C C C T A C T G
120 130 140 150
944

708

472

236

B 0

Figure 13-4. DNA sequencing. A, Sequencing is diagrammed using radioactively or non-radioactively labeled deoxynucleotides.
DNA fragments of varying lengths are produced by the dideoxynucleotides and separated on a polyacrylamide gel. The sequence
is read from the top to the bottom of the gel. B, Four different fluorescent labels are used to tag the primer that is used in
dideoxynucleotide sequencing reactions, as shown in part A. The reaction products are then pooled and separated by capillary
electrophoresis. A laser-induced fluorescence detector monitors the signals in four spectral channels, which are plotted as four
colors corresponding to each base in the ladder. (Courtesy of NBII Program administered by the Biological Informatics Office of
the U.S. Geological Survey.)
224 Modern Molecular Medicine

The four reactions are separated electrophoretically to

BIOCHEMISTRY produce a visual “ladder” of DNA fragments. Labeling either
the DNA sequencing primers or the individual nucleotides
Ribonucleotides with a radioactive or fluorescent tag enables this visualiza-
The fundamental concept of the dideoxynucleotide chain tion. In automated sequencing, the ddNTPs are labeled with
terminating technique is that some deoxyribonucleotides fluorescent dyes that are detected by a scanner. Because the
lack an OH at the 3′ position of the sugar. For those gel lane containing “A” (from the “A” tube), “T,” and so
deoxyribonucleotides in which this occurs, called forth is known, it is a straightforward matter to read the
dideoxyribonucleotides, a phosphodiester bond cannot form
sequencing ladder.
with a 5′ H and chain elongation stops. These ddNTPs can
DNA sequencing was once done by polyacrylamide gel
be labeled with a radioactive or nonradioactive tag for
visualization of fragments incorporating them, or rather of
electrophoresis, but more recently this is being replaced by
the fragments that terminated elongation at that point. capillary electrophoresis (see Fig. 13-4B). This technique adds
automation to the process while introducing several highly
O: O: regarded features. DNA separation occurs in a capillary with
J J J J J J J
J J J J J J J

:O JPKO :O JPKO a diameter of 25 to 100 μm, which provides a high ratio

of surface area to volume that acts to dissipate heat pro-
O O duced. This feature allows the use of higher electrical fields,
:O JPKO :O JPKO which decreases time of separation and increases resolution
of DNA.
O O
:O JPKO :O JPKO
Mutation Detection
O Base O Base
The true basis of molecular diagnostics is the detection
CH2 CH2 of specific disease-causing mutations. Here, the most common
O O methods associated with genetic variation that are employed
in laboratories for the detection of common diseases
H H H H are discussed, including point mutations, deletions, and
H H H H
trinucleotide repeat expansions.
OH OH OH H
Ribonucleoside Deoxyribonucleoside
triphosphate triphosphate
Point Mutation Detection
(NTP) (dNTP) Mutation-specific RFLPs
In some single-gene disorders, the causal mutation is a point
O: mutation that alters a restriction endonuclease recognition
J J J J J J J

:OJPKO site. This type of mutation can either abolish an existing rec-
ognition site or create a novel site. When this happens, it
O permits a mutation-specific test that can be used for diagnos-
:O JPKO tic purposes. Perhaps the best example of a mutation-specific
RFLP test is found in sickle cell anemia. As described in
O
Chapter 6, sickle cell anemia results from an A-to-T missense
:O JPKO mutation and the substitution of a valine for a glutamate at
Base
the sixth amino acid of the β-globin polypeptide. This base
O
change affects the 5′-CCTNAGG-3′ (where N can be any
CH2 nucleotide) recognition site for the MstII restriction endo-
O nuclease because the central A is replaced by a T, rendering
a similar but different 5′-CCTNTGG-3′ sequence. MstII does
H H not recognize or cleave this altered DNA sequence. Hence,
H H
sickle cell anemia patients differ from the normal population
H H
by the loss of this particular restriction site, resulting in an
Dideoxyribonucleoside
RFLP for sickle cell anemia. In the laboratory, this is recog-
triphosphate
(ddNTP) nized by means of agarose gel electrophoresis in which normal
individuals have two smaller DNA fragments that correspond
to the affected individual’s single, longer DNA fragment (Fig.
13-5). While specific and sensitive, this methodology is rela-
tively rare in practice because the majority of point mutations
do not alter a restriction endonuclease recognition site. As
seen in Chapter 6 and suggested earlier in this chapter, PCR
can also be used to amplify a fragment of interest for restric-
tion analysis. The fragment sizes may differ between genomic
 Tools of Molecular Medicine 225

Normal
5 6 7 Codon number sequence T C G A T A G C
CCT–GAG–GAG Hemoglobin A Homoduplex
CCT–GTG–GAG Hemoglobin S A G C T A T C G
Normal oligo-
CCT–NAG–G Mst II site nucleotides
A G C T A T C G
A
Mutant Heteroduplex
sequence T C G T A G C
Mst II Probe Mst II Mst II
T
Globin gene
A
Normal
1.15 kb
sequence T C G T A G C
B 1.35 kb Heteroduplex
A G C A A T C G
Mutant oligo-
nucleotides
A G C A A T C G
Mutant Homoduplex
sequence T C G T T A G C
A

Homozygous Heterozygous Homozygous

1.35 kb normal mutant
:
1.15 kb
AA AS SS
C

Figure 13-5. RFLP analysis of the β-globin gene and sickle

cell anemia. A, An A-to-T point mutation occurs in codon 6.
B, The mutation eliminates a recognition site for MstII.
Individuals with sickle cell trait are heterozygous and have
1.15-kb and 1.35-kb fragments. Individuals with sickle
cell anemia have two longer 1.35-kb fragments. B
;

C, Electrophoretic gel demonstrating DNA fragments

for AA, AS, and SS individuals. Figure 13-6. Allele-specific oligonucleotide testing. A, Short
oligonucleotides are designed to reflect a normal sequence
or a mutated sequence. These allele-specific oligonucleotides
will then bind only if there is perfect complementation to
DNA versus PCR-generated DNA and a probe may be either denatured normal or mutated alleles, forming a
required to visualize a fragment in genomic DNA, but the homoduplex. Heteroduplexes do not form stable binding.
B, ASOs for both the normal and mutated sequences can be
results still demonstrate the effect of the mutation.
used together, and the resulting homoduplexes are separated
by gel electrophoresis.
Allele-specific Oligonucleotides
Thousands of single-base changes are associated with human
disease. This has necessitated the development of other
methods to identify individual point mutations because is a perfect complement to a mutant DNA sequence. For
mutation-specific RFLPs are relatively rare. One of these example, an ASO can be synthesized that hybridizes only to
methods is allele-specific oligonucleotide (ASO) hybridiza- the portion of the β-globin gene that contains the sickle cell
tion (Fig. 13-6). For this, a DNA synthesizer creates short, anemia mutation. Such an oligonucleotide does not bind to
synthetic oligonucleotides that are an exact complement of normal DNA because it is mismatched at the base altered in
the normal DNA sequence. These, in turn, are fluorescently sickle cell anemia. A mutation-specific ASO, then, can be used
or radioactively labeled and used to probe patient or control to screen patients, family members, and even members of
DNA in a process similar to the Southern blot probing. During the general population for the presence of the sickle cell
the hybridization process, these short oligonucleotides bind mutation.
only to a perfect sequence complement but not to any
sequence with even a single mismatched base. Thus, the oli- DNA Sequencing and Mutation Detection
gonucleotides bind only normal DNAs, making it simple to In special cases when a patient is clearly suffering from a
distinguish between normal DNA and mutant DNA. Con- particular disease but does not harbor known genetic variants
versely, a short oligonucleotide may also be synthesized that associated with the disease, the candidate gene may be
 226 Modern Molecular Medicine

subjected to complete or partial automated DNA sequence molecular genetics will grow more comprehensive as addi-
analysis. DNA sequencing is much more labor intensive and tional insights are made regarding genetic predisposition to
time consuming than the above methods if not automated, disease and the role of genetics in pharmaceutical efficacy.
but it is an excellent approach for the discovery of new or
rare mutations. This technique is so valuable that increased
technological developments have reduced the time and cost ●●● GENETIC TESTING
of sequencing; it is not unthinkable that samples currently AND SCREENING
referred to a research laboratory for evaluation will be
sequenced in a diagnostic laboratory in the near future.
Fundamentals of Genetic Testing
Currently, DNA-based tests exist for roughly 1500 diseases
Deletion Detection with about 900 in use in genetic laboratories. Clearly, genetic
Very small deletions, typically single-base deletions that testing for disease mutations and predispositions will have a
cause disease, may be screened for by means of the same greater impact on medicine as more genes are identified and
methodologies employed for point mutations. Larger dele- associated with pathology. DNA-based testing must have ana-
tions, however, require a different approach. PCR is ideal for lytical and clinical validity and utility. Analytical (laboratory)
detecting deletions in the 100-bp to 4-kb range, since primer validity, or efficiency, refers to the ability of a genetic test to
pairs amplify both a normal and mutated allele containing accurately indicate the genetic condition or genotype. It is
a deletion. Following PCR, DNA fragments are separated described by two elements—sensitivity and specificity (Table
by electrophoresis and compared with molecular weight 13-3). Sensitivity is a measure of how effective a particular
standards. Deleted alleles are obviously not as large as the test is at identifying the mutation or genotype of interest; it
nondeleted forms and are easily visualized. is also the detection rate for true-positive samples. Specificity
indicates how often a test identifies those samples that do not
Trinucleotide Repeat Expansion Detection have the mutation, which is the detection rate for true-
As described in Chapter 8, expansions of trinucleotide negative samples. To have value to the physician, gene tests
sequences are a relatively common genetic mechanism for must also have clinical validity or predictive value, defined
neurologic disease. Fragile X syndrome, Huntington disease, by the ability of a particular assay to detect disease. Positive
myotonic dystrophy, spinobulbar muscular atrophy, and predictive value reflects the percentage of all positive tests
Friedreich ataxia are all examples of disease caused by that are true positives; the converse is true for a negative
expanding trinucleotide repeats. This class of mutation poses predictive value. Again, sensitivity and specificity are rele-
a special challenge for diagnostics, as illustrated by fragile X vant. Here, clinical sensitivity refers to the proportion of
syndrome. Fragile X is due to an expansion of a CGG tri-
nucleotide in the 5′-untranslated region of the FMR1 gene. TABLE 13-3. Diagnostic Value of Tests Defined
Three classes of FMR1-associated CGG expansions are rec- by Sensitivity, Specificity, Predictive
ognized in the population: normal chromosomes contain Value, and Efficiency
between 5 and 50 repeats, premutation chromosomes contain
between 50 and 200 repeats, and full mutation chromosomes RESULTS OF A DIAGNOSTIC TEST
(affected individuals) feature 200 to 2000 CGG repeats. Dis-
tinguishing between these three allelic repeats seems, intui- PATIENT Test Positive Test Negative
tively, an ideal task for PCR. In practice, however, this is true
for normal and premutation alleles; PCR primers flank the Disease present True positive False negative
site of the CGG expansion and amplify the alleles. The same (TP) (FN)
is not always true for the full mutation because full mutation Disease absent False positive True negative
expansions can reach 6 kb in length, exceeding the size geno- (FP) (TN)
typing can be done on the basis of PCR alone. Thus, Southern
blotting is typically performed when fragile X syndrome is TP
Sensitivity (%) = × 100
suspected. In this case, a probe is used that hybridizes proxi- TP + FN
TN
mal to the expanded region. In this way, very large expansions Specificity (%) = × 100
FP + TN
can be detected. The dividing line between use of PCR and TP
Southern blotting to size the repeat tract falls between 70 and Positive predictive = × 100
value (%) TP + FP
100 repeats. Southern blots cannot identify a precise repeat
TN
size at the normal and low premutation ranges, but PCR is Negative predictive = × 100
value (%) FN + TN
unable to correctly identify repeat tracts over 70 to 100 units.
TP + TN
It is important to note that DNA diagnostics pervades the Test efficiency = × 100
(percentage of TP + TN + FP + FN
entirety of the health care system today. A large number of
laboratories utilizing genetic techniques now exist and stand times the test
provides a correct
as a testament to the ubiquity of genetic defects in medicine.
answer per total
Even in rural or remote settings, samples are sent to a labora- number)
tory for DNA analysis. The association between medicine and
 Genetic Testing and Screening 227

people who have a particular disease that tested positive for and presymptomatic diagnosis and intervention. Many of the
a causal mutation while clinical specificity indicates the pro- disorders identified in the newborn have been discussed else-
portion of people who do not have a particular disorder and where. Many do not present in the newborn with an obvious
have tested negative for the associated gene mutation. Both disease phenotype, and a highly specific and sensitive genetic
sensitivity and specificity are measured by comparing the test is ideal for early detection of disease genotypes. Certain
genetic test results with those obtained from a separate, defin- other disorders such as sickle cell anemia and congenital
itive diagnostic test, such as a biochemical assay. The clinical adrenal hyperplasia seem to be excellent candidates for
utility of a DNA-based test refers to its ability to be useful population-based newborn screening. However, because of a
to the clinician. In other words, a genetic test is useful only lowered incidence or suboptimal treatment paradigms even
if it clearly provides value to the diagnosis, prognosis, treat- with early intervention, such diseases are not included in
ment, or prevention of a disease. some newborn screening programs. Occasionally, newborn
DNA-based tests have two overarching utilities: genetic genetic screens are performed in certain regions of the world
testing and genetic screening. Genetic testing generally refers where they have a high incidence. For example, cystic fibrosis
to the confirmation or establishment of a diagnosis within a is one of the most common autosomal recessive diseases
patient or family that has a clear disease phenotype and found in people of Northern and Western European origin.
family history. In this instance, a gene test is usually an effort In these populations, the incidence of cystic fibrosis ranges
focused on an individual or family and on a single gene. from 1 in 2000 to 1 in 2500. Hence, several of these countries
Genetic screening, in contrast, is a population-based approach have instituted a DNA-based newborn screening procedure
that attempts to identify presymptomatic individuals in the to detect affected infants. Because there is no effective dietary
population who harbor disease-associated genetic variants. or pharmacologic treatment for cystic fibrosis, the expectation
Genetic screening includes such important concepts as het- is that early prophylactic treatment, such as physical therapy
erozygote carrier identification, newborn screening, prenatal and antibiotic administration, will improve the quality of life.
diagnostics, and presymptomatic testing for late-onset disor- A similar approach and rationale are being utilized in Africa
ders or disease predisposition. Hence, DNA-based screens for the early detection of sickle cell anemia.
have a significant public health component, whereas genetic
testing typically refers to an individual or family. Since genetic Heterozygote Carrier Screening
testing is a fairly straightforward concept, the following As disease databases grow and DNA-based assays continue
discussion centers on the principles of genetic screens. to be developed and optimized, the feasibility of carrier
screening for autosomal recessive diseases in adults has
increased. Here, the purpose is not to identify individuals at
Genetic Screening risk for disease development but rather individuals who are
In general, the feasibility and utility of genetic screening in a at risk for passing on deleterious alleles to their children.
population is greatly enhanced when certain criteria are met Hence, this type of genetic screen seeks to inform and educate
in three general areas: disease characteristics, diagnostic test healthy individuals regarding reproductive risks. Such screens
features, and the capacity of the health care system. Diseases are most effective when coupled with genetic counseling and
subject to genetic screens should be reasonably common, well prenatal diagnostic options.
characterized, severe enough to warrant population screening, Heterozygote screening makes the most sense when an
and treatable or preventable.The genetic test itself should have autosomal recessive disease is present at a relatively high
laboratory and clinical validity and be inexpensive and rapid frequency in the population. Owing to the high incidence of
enough to promote widespread use (see Table 4-2). Finally, the recessive disorders in certain subpopulations, carrier screen-
health care system itself should be prepared for the treatment ing is therefore most effective among particular ethnic groups
of the disease, including providing accessible therapeutic that harbor a high deleterious allele frequency (see Chapter
resources, counseling services, and educational programs. 12). Tay-Sachs disease is perhaps the most striking example
It is important to note that not all genetic screens are tests of this in the Ashkenazi Jewish population. Tay-Sachs disease
for mutations. Biochemical tests are available that can indi- (see Chapter 8) is a severe autosomal recessive neurologic
cate whether a particular gene is defective. Examples include disease that results in childhood death. It occurs commonly
the Guthrie test for hyperphenylalanine levels and the cre- in the Ashkenazi Jewish population, where the frequency of
atine kinase assay for Duchenne muscular dystrophy. heterozygotes is approximately 1 in 30, reflecting the high
However, PCR-based, mutation-specific assays are being risk in this subpopulation and suggesting that carrier testing
increasingly utilized, since they are typically rapid, noninva- has significant value to individuals of reproductive age. In
sive, inexpensive, and accurate. Examples of DNA-based fact, genetic testing for Tay-Sachs in the Ashkenazi Jewish
genetic screening include cystic fibrosis and sickle cell anemia. population has been a resounding success. Since 1970, such
testing performed either by biochemical assay or direct DNA
Newborn Genetic Screening testing has resulted in a 90% decrease in the incidence of
Genetic testing in the newborn represents a tremendous this disease in North America in this subpopulation. The
opportunity for disease intervention, provided the above majority of individuals born with Tay-Sachs disease now are
criteria are met. In newborn screening, it is particularly rel- of non–Ashkenazi Jewish ancestry. This decrease in incidence
evant that a disorder be prevented or ameliorated by early more accurately represents the combined effects of education,
 228 Modern Molecular Medicine

heterozygote genetic screening, and prenatal genetic diagno-

sis. Thus, the story of Tay-Sachs disease in the North ●●● GENETIC COUNSELING AND
American Ashkenazi Jewish population is a testament to the RECURRENCE RISK ESTIMATION
power and role of genetics in public health, presymptomatic
Genetic Counseling
diagnosis, and disease prevention.
The field of genetic counseling has emerged as an important
health care resource as a result of the identification of disease-
Prenatal Genetic Testing and
associated genes. The utility of the counselor is illustrated by
Preimplantation Genetic Diagnosis considering a couple who have had a child with a certain
Many methods are at the disposal of the physician to diag- disease. Among many questions faced by the physician and
nose genetic maladies before birth. These include direct team are, “Why did this happen?” “What is the prognosis?”
examination of fetal cells by chorionic villus sampling (CVS) and “Will this happen to other children that we may have?”
or amniocentesis, biomarker assays such as the maternal Equally likely is the scenario in which a consultand, who is
blood serum screen (triple screen), and ultrasonography. an individual seeking or being referred to a genetic counselor,
Direct DNA-based testing and analysis is permitted by amnio- has learned that a family member has been diagnosed with
centesis and CVS, and the combination of the above tech- a genetic disorder and wants to know the risk for developing
niques usually permits an accurate diagnosis. the abnormality or transmitting it to offspring. Other reasons
Although the above approach to prenatal testing is effica- for genetic counseling include infertility, teratogen exposure,
cious, it provides limited options for parents, since a diagnosis prenatal diagnosis with advanced maternal age, interpretation
is made after implantation. Preimplantation genetic diagnosis of newborn genetic screens, or a diagnosis of genetic disease.
(PGD) in combination with assisted reproductive technolo- These questions and scenarios are common and central to
gies, such as in vitro fertilization (IVF), on the other hand, many patients and to the health care system in general. Thus,
permits the implantation of only “normal” embryos. Typi- genetic counseling is an essential component in the health
cally, PGD is of interest to a couple at increased risk for care system and must be considered a core part of any
transmitting a harmful or predisposing allele to their off- modern medical genetic enterprise.
spring. PGD is initiated by the biopsy of a single cell from Genetic counseling is essentially a knowledge-based com-
the six- to eight-cell blastomere produced by IVF. At this munication and educational endeavor. The counselor estab-
point, blastomere DNA is analyzed for a particular genetic lishes that the patient understands the clinical diagnosis and
defect either by cytogenetic means or by direct mutation cognate prognosis and treatment, the genetic components of
detection. The latter is greatly facilitated by the use of PCR, the disorder (including inheritance pattern and risks), treat-
since DNA from the single cell can be exponentially ampli- ment, reproductive risks and options, and the availability of
fied. Only those embryos free of the genetic defect in question educational information and support groups. In providing
are implanted, virtually guaranteeing that the fetus will not information to at-risk patients or families, consultands are not
manifest the disorder of concern to the parents. guided to a particular option, but rather provided the context,
To date, PGD has been used to prevent Tay-Sachs disease, education, and information to make informed medical and
cystic fibrosis, Huntington disease, Duchenne muscular dys- reproductive decisions that are appropriate for personal and
trophy, β-thalassemia, early-onset Alzheimer disease, and sociocultural dictates. This “nondirective” approach may
many other severe disorders. Hundreds of babies have been actually run counter to the traditional view of medicine,
born using PGD coupled with IVF, and this number will where a biomedical team might suggest a course of action.
increase dramatically as technology improves coordinately Typically, a recommendation suggesting a course of action
with our understanding of the genetic basis of disease. seeks the prevention of further disease or amelioration of a
current disease state. However, genetic counseling seeks to
provide information. The counselor communicates essential
PATHOLOGY material in a manner that respects the integrity of the con-
sultand or patient to make the decision. This communication,
Prenatal Detection of Defects and Abnormalities
by the physician and the counselor, underscores the major
The triple screen has become the standard of prenatal care principles of medical ethics (Table 13-4).
for detecting neural tube defects and genetic abnormalities.
Maternal serum is used to analyze α-fetoprotein (AFP),
human chorionic gonadotropin (hCG), and unconjugated Recurrence Risk Assessment
estriol (E3).
An important function of the medical genetics community is
estimation of the recurrence risk for a disease within a par-
ANOMALY AFP HCG E3 ticular family. This is typically performed by a genetic coun-
selor; the information sought is ordinarily related to risks to
Neural tube defects ↑ Normal Normal close family members or offspring.
Trisomy 21 ↓ ↑ ↓↓ Accurate risk assessment relies on the essential elements
Trisomy 18 ↓ ↓ ↓
of accurate diagnosis, detailed family history and mode of
inheritance determination, knowledge of DNA or biochemical
 Genetic Approaches to Treatment 229

significant genetic component and can therefore cluster in

TABLE 13-4. Major Principles of Medical Ethics families. Theoretical probability is not used in this example
because nonsyndromic cleft lip and palate is not inherited in
PRINCIPLE DESCRIPTION a typical mendelian fashion. Instead, the risk to any particular
relative in a pedigree is calculated based on observations from
Respect for The patient has the right to make existing cleft lip and palate families.
autonomy informed decisions without coercion. Empirical risk calculation is valuable to the physician,
This is the basis for the practice of genetic counselor, and patient but has limitations. For example,
“informed consent.”
Beneficence Health care providers offer services that genetic heterogeneity may influence actual risk. In other
are beneficial to the patient. words, a single complex disease may have different genetic
Justice Health care providers fairly distribute bases, and each might have a cognate recurrence risk. Empiri-
care to patients. cal risks are typically calculated over a large population that
Nonmalfeasance The health care provider is committed
could encompass all of the genetic heterogeneity for a given
to protect patients from harm by
providing the best standard of care. disease. One way to potentially minimize such errors is to use
Dignity The patient is treated with dignity. families that match the patient’s demographic and sociocul-
Integrity Health care providers are truthful and tural characteristics. Ethnicity, geographic location, lifestyle,
honest with patients about conditions and socioeconomic class are all important to match, as closely
and treatments.
as possible, with the family under consideration. In the end,
an average empirical risk is provided to the physician and
patient, but this risk must be interpreted carefully as an
average estimate rather than a precise probability.
tests that quantify the defect, and appropriate genetic data-
bases that indicate carrier frequencies in the relevant popula-
tion. For disorders due to a single gene, the recurrence risk
●●● GENETIC APPROACHES
to family members is often calculated by careful pedigree
analysis and application of the rules of mendelian assortment
TO TREATMENT
of dominant, recessive, or X-linked alleles (see Chapter 12). As noted in Chapter 4, genetic disease can be treated by a
In actuality, however, pedigree analysis can be ambiguous variety of means. In addition to some of the treatments pre-
because of the complicating effects of incomplete penetrance, sented in Table 4-6, gene therapy has the potential to cure a
variable expressivity, unknown carrier status, late age of onset, genetic disease, where “cure” is a treatment alleviating
and genetic heterogeneity. In such cases, however, modified disease symptoms and restoring health. In the absence of
recurrence risks are estimated using bayesian analysis. cure, the next best option may be safe and effective drug
Bayesian analysis is a form of probability theory that administration. The emerging field of pharmacogenomics
attempts to determine relative recurrence risks based on pedi- facilitates this option. Below, the fundamentals and medical
gree analysis, phenotype, and/or laboratory test results alone. applicability of gene therapy and pharmacogenomics are
In other words, bayesian risk analysis incorporates additional discussed.
information beyond simple mendelian segregation into the
risk calculation. For a given question of interest such as, “Is
individual A a carrier of a deleterious mutation?”, bayesian
Gene Therapy
analysis considers the two alternative possibilities: individual Gene therapy is the introduction of a gene, or DNA sequence,
A is a carrier and individual A is not a carrier. It is not an into a cell to correct a genetic defect. Typically, it aims to
easy concept for those who do not use it regularly and will correct a defective allele or to replace an absent or deficient
not be discussed further here. gene product (“gene replacement”) by introducing a func-
tional allele. Such an approach, while frequently publicized
Empirical Risk as the ultimate cure for genetic disorders, is in its infancy.
As discussed, simple recurrence risk estimation for single- Nevertheless, recent advances in our understanding of the
gene disorders of known inheritance patterns is based on human genome have permitted significant progress in this
mendelian segregation of disease alleles. However, many dis- area, and more than 8000 gene therapy protocols for both
orders feature multifactorial or polygenic genetic mecha- inherited and noninherited disorders have been approved
nisms. As discussed in earlier chapters, these diseases are worldwide (Table 13-5).
characterized by a strong genetic component, with relatives To increase the probability of success, the disease under
typically showing a higher incidence of the disease relative consideration should be amenable to genetic therapy. Most
to the incidence in the general population. Recurrence risks protocols involve a compensation approach for recessive dis-
for complex diseases are therefore determined empirically, orders in which loss of function has occurred. In this context,
since the totality of genetic and environmental factors is often many of the autosomal recessive inborn errors of metabolism
unknown. In practice, empirical risks are determined by (see Chapter 4), such as phenylketonuria (PKU), are good
observations made in families that have the same disease. candidates for gene therapy, since restoration of as little as
As an example, nonsyndromic cleft lip and palate has a 10% of normal enzyme activity is sufficient to see clinical
 230 Modern Molecular Medicine

TABLE 13-5. Examples of Diseases for Which Somatic Cell Gene Therapy Protocols Are Being Tested

DISEASE TARGET CELL INSERTED GENE

Adenosine deaminase deficiency Lymphocytes, bone marrow stem cells Adenosine deaminase
Cystic fibrosis Bronchial epithelium CFTR
Duchenne muscular dystrophy Myoblasts Dystrophin
Familial hypercholesterolemia Hepatocytes LDL receptor
Fanconi anemia Hematopoietic stem cells FANCC
Gaucher disease Macrophages Glucocerebrosidase
Hemophilia B Hepatocytes, fibroblasts Factor IX

improvement. Other criteria for ideal candidates for gene harmful immunologic response, easily produced, and direct-
therapy include: able to the appropriate target cell, and it has a long half-life.
1. Single-gene defect Although no current vector system meets all these criteria,
2. Well-characterized sequence and regulatory elements viral vectors are easy to manipulate and can deliver DNA
3. Significant morbidity and mortality (i.e., there is a rea- to target cells and thus may be considered reasonable
sonable risk-benefit ratio) vectors. Three main classes of viral vectors are in use today:
4. Cells that are experimentally accessible and otherwise retroviruses, adenoviruses, and adeno-associated viruses
represent adequate target cells (Table 13-6).
Stem cells are ideal for many gene therapy procedures, Retroviral vectors are derived from RNA viruses that can
since an introduced gene may have high replicative potential integrate their genomes into the host cell chromosomal DNA
and be distributed in a large number of differentiated daugh- after making a DNA copy from their RNA genome using
ter cells. Bone marrow stem cells, in particular, provide an reverse transcriptase. This integration process is efficient and
excellent opportunity for hematologic disease treatment stable. These vectors are genetically engineered so that they
because they are relatively well characterized and can be are rendered incapable of infection by removal of most of the
cultured. Diseases such as thalassemia and sickle cell anemia, virus’s genome. For a given disease, the “corrected” gene of
and perhaps certain metabolic disorders, are good candidates choice replaces the removed viral DNA, and retroviruses are
for bone marrow therapies because marrow cells can be capable of containing up to 8 kb of exogenous DNA. This size,
altered to express an appropriate gene product for delivery while appreciable, limits the utility of retroviral vectors in that
to relevant tissues via the circulatory system. Typically, the cDNAs of large genes, such as the gene responsible for Duch-
transfer of genes into cells relies on either a viral or nonviral enne muscular dystrophy, cannot be inserted into these
vector. vectors. The inserted gene typically includes regulatory
sequences such as promoter elements and polyadenylation
Gene Transfer Procedures signals. Because the vectors cannot replicate, they must be
Theoretically, gene therapy can be applied to either somatic introduced into packaging cell lines. These cell lines have
or germline cells. However, because treating somatic cells been infected with a retrovirus that lacks the virion packaging
yields clinical improvement in the patient and because techni- signals. Such cells make many viral proteins, but no virus
cal and ethical concerns have limited protocols that alter the particles can be formed. An introduced vector (sometimes
genes of gametes, the much more common somatic cell gene termed a “helper virus”) contains the packaging proteins, and
therapy is discussed here. thus the combination of vector and packaging cell lines pro-
The goal of somatic cell gene therapy is to improve or vides all proteins necessary for the production of infectious
cure a genetic defect within a patient’s somatic cells. To do particles. These particles—containing the therapeutic DNA of
so, two general approaches may be utilized. First, an ex interest yet still incapable of replication—are used to transfect
vivo approach involves the isolation and culturing of cells the target cells. Following transfection, the introduced DNA
from the patient. A corrected or altered gene construct is inserts into the target cell’s genome and is expressed by
added to these cells, and the cells are reintroduced to the normal mechanisms.
patient. Second, an in vivo approach features the direct Retroviral vectors have two potential drawbacks. First, they
injection of a gene into a resident tissue or organ. In either only infect replicating cells and are therefore not a viable
case, the gene of interest requires a vector to facilitate its option for certain target cell types such as neurons or skeletal
journey to—and entry into—the appropriate target cell. muscle. Second, because retroviruses integrate into the host
genome randomly, the possibility exists for random inser-
Viral Vectors for Gene Transfer tional mutagenesis whereby a functional allele is inactivated
Vectors serve as the vehicle for entry of a gene into the or an inactive allele is activated, leading to consequences of
cell. A perfect vector is safe with no adverse reactions or this change.
 Genetic Approaches to Treatment 231

TABLE 13-6. Comparison among a Few Viral Vectors

VECTOR ADVANTAGE DISADVANTAGE

Retrovirus Long-term expression 7-kb insertion

Integrates into DNA May inactivate or activate another gene upon insertion
Adenovirus Does not integrate into host Short-term expression
30-kb insertion size Immunogenic
Adeno-associated virus Most commonly used Limited size inserts
Long-term expression
Not immunogenic
Lentivirus Infects nonproliferating cells Requires complex expression vector systems
Ex vivo delivery Toxic proteins
Integrates into DNA Concern that HIV will replicate
Not immunogenic Requires host DNA polymerase
Herpes simplex virus Neurotropic Cytotoxic
40- to 50-kb inserts Immunogenic

or an immune response. Other advantages of this vector are

IMMUNOLOGY & MICROBIOLOGY that they can infect a wide range of target cell types and are
capable of integrating into the host genome at a specific site
Retroviruses (chromosome 19q13.3-qter), permitting stable gene expres-
Retroviruses are single-stranded RNA (ssRNA) viruses that sion without pathogenicity. However, AAVs can integrate only
bind to cell surface receptors and enter the cell. RNA is 5 kb of DNA.
converted to DNA by reverse transcriptase and integrated
into the genome with integrase. A psi sequence (packaging
signal sequence) is located outside the viral genes and is
required for packaging. Therefore, elimination of this
IMMUNOLOGY & MICROBIOLOGY
sequence is preferred in order to prevent the retrovirus from
repackaging itself. A cell infected with a retrovirus that has a Adeno-Associated Viruses (AAVs)
mutated psi sequence will be able to produce viral proteins
important for viral structure but will not be able to package AAVs are ssDNA nonpathogenic parvoviruses. They require
the proteins. However, if another virus with an intact psi a helper virus, which usually is an adenovirus, to proliferate.
sequence and new genetic material in place of the structural Adenoviruses can be inactivated by heat, thereby reducing
genes (gag, pol, and env) coinfects the cell, the new genetic immunogenicity associated with other viral vectors.
material can be packaged with the proteins produced from AAVs are composed of two genes flanked by inverted
the virus with the defective psi sequence. terminal repeats (ITRs). The cap gene produces the capsid
structural proteins; the rep gene is important to replication,
gene expression of replication proteins, and integration
(it inhibits adenovirus replication). The ITR contains the
promoter region.

Adenoviruses are double-stranded DNA viruses that have

the important advantage of being able to infect a wide spec-
trum of cells, including nondividing cells. They also can At present, virtually all vector gene delivery systems suffer
harbor large pieces of DNA, up to 35 kb in length, and can from key deficiencies limiting the promise of gene therapy. In
be purified to produce high titers of infectious particles. general, these include transient or unregulated gene expres-
Adenoviruses do not integrate into the host chromosome, sion and the inability to easily access target cells or infect
which means that they are unlikely to insertionally activate significant numbers of target cells. Much effort is being put
or inactivate a proto-oncogene. On the other hand, the lack forth to better understand the biology of viral vectors, target
of integration renders adenovirus-mediated gene transfer cells, and the molecular interactions of these elements to
unstable and transient. Finally, adenoviruses can provoke a promote stable, safe, and efficient gene transfer.
significant immune response in the patient. This, coupled with
the need for repeated introduction of the adenoviral vector Nonviral Vectors for Gene Transfer
owing to transient viral gene expression, hallmarks the More recently, nonviral vectors have been considered for
primary disadvantage of adenovirus-mediated gene therapy. gene transfer (Table 13-7). These vehicles are presumably less
Adeno-associated viruses (AAVs) are parvoviruses that are toxic and easier to produce. At least four types of nonviral
common in the human population and do not provoke disease vectors are being investigated, including naked DNA, DNA
 232 Modern Molecular Medicine

encapsulated in a liposome (a lipid bilayer that can fuse with drug sensitivity has a genetic basis that is reflected in altered
cell membranes), protein-DNA conjugates that can be tar- erythrocyte metabolism. Another drug, succinylcholine, is
geted to particular cell receptors, and artificial chromosomes used during anesthesia to induce muscle paralysis. The extent
(Table 13-8). Artificial chromosomes may represent the future of paralysis may last from a few minutes to a few hours in
of gene therapy vectors. They are synthetic constructs that different individuals given the same dose. This difference
contain the minimal functional elements of a chromosome, results from the altered kinetics of pseudocholinesterase and
including centromere and telomeres. These constructs can is inherited in an autosomal recessive manner.
incorporate up to 10 Mb of DNA, meaning that very large
pieces of exogenous DNA can be introduced to a cell by this
method.
PHARMACOLOGY & PHYSIOLOGY
Gene Replacement Versus Uses of Succinylcholine
Gene Silencing Succinylcholine, also known as scoline or suxamethonium
chloride, is composed of two linked acetylcholine molecules.
Gene replacement techniques are unlikely to be efficacious
It imitates the action of acetylcholine at neuromuscular
for diseases occurring from either a dominant negative or junctions but causes prolonged opening of the
gain-of-function genetic mechanism. In these cases, the goal acetylcholinesterase receptor channels, leading to
becomes to silence or reduce the expression of the pathogenic depolarization of the membrane. Initially, repetitive muscle
gene. Three general approaches are being developed in this excitation and tremors occur, followed by relaxation
context. First, antisense oligonucleotide therapy involves the secondary to inactivation of Na+ channels. Succinylcholine
introduction of a short, synthetic DNA oligonucleotide that is degraded by pseudocholinesterase, rather than
is complementary to the abnormal mRNA into a cell. The acetylcholinesterase, but at a much slower rate.
oligonucleotide is considered antisense relative to the mRNA, Succinylcholine is used for short-term muscle relaxation
and binding of the antisense oligonucleotide to the target during anesthesia. It does not produce unconsciousness or
anesthesia itself but is used to produce sustained muscle
mRNA hinders or eliminates translation of that mRNA.
relaxation, or flaccid paralysis, after unconsciousness—a
The effect of this action is decreased expression of the
condition that is useful in certain types of surgery when it is
disease gene. important to prevent contraction and excitation of muscles.
Second, ribozyme therapy is being developed to disable
target mRNAs. Ribozymes are autocatalytic RNA molecules
that cleave RNA targets, including mRNA. It is possible to
produce ribozymes that are specific for a particular nucleo-
tide sequence context. This means that ribozymes can be PHARMACOLOGY & NEUROSCIENCE
introduced that cleave the mRNA of disease genes but not of Opioids
normal copies.
Opioids interact with receptors for three families of
Finally, RNA interference (RNAi) uses RNA molecules for
endogenous peptides: endorphins, enkephalins, and
posttranscriptional gene silencing (Fig. 13-7). Specifically,
dynorphins. There are three major classes (with several
small interfering RNAs (siRNAs) are used to block mRNAs subclasses) of opioid receptors in the central nervous
of disease genes. In a procedure similar to the DNA-based system: μ, κ, and δ. Opioids, in minute doses, excite
antisense approach described above, siRNAs are short RNA neurons in the periaqueductal gray matter and nucleus
oligonucleotides that are introduced into cells in the more reticularis paragigantocellularis. Descending pathways
stable form of double-stranded RNAs (dsRNAs). The dsRNAs from the midbrain have an inhibitory effect mediated by
are processed into the single-stranded siRNAs by a multisub- 5-hydroxytryptamine (5-HT), enkephalins, and noradrenaline
unit protein complex called the RNA-induced silencing on pain transmission from the dorsal horn. Opioids can also
complex (RISC). siRNAs can base-pair with mRNAs of a inhibit pain directly at the dorsal horn and inhibit peripheral
target gene and mediate either mRNA degradation or trans- nociceptive afferent neurons.
Morphine is an opioid that relieves pain by interfering with
lational arrest. siRNAs as small as nine nucleotides can attain
the sensation of pain. It interacts with δ and κ receptors
great target specificity. As with all gene therapy approaches,
weakly but is a powerful agonist at the μ opiate receptors in
delivery of siRNAs to the appropriate cell and stable, long- the central nervous system. Manifestations of morphine and
lasting expression of siRNAs are areas of major concern and heroin include analgesia, euphoria, respiratory depression,
research effort. and dependence.

Pharmacogenomics and
Individualized Medicine To better illustrate that genes determine drug effects,
Though most consider pharmacogenomics a recent applica- codeine is considered. This drug is one of many that are con-
tion of genetic understanding, the concepts are not that verted by members of the P-450 protein family to other
recent. Recall that some African Americans have an acute forms. In the liver, codeine is metabolized by CYP2D6 to
hemolytic crisis when given primaquine (see Chapter 6). This morphine. There are allelic variations in the CYP2D6 gene,
 Genetic Approaches to Treatment 233

TABLE 13-7. Comparison between Viral and TABLE 13-8. Comparison among Several
Nonviral Gene Delivery Nonviral Vectors

NONVIRAL VECTOR ADVANTAGE DISADVANTAGE

FEATURE VIRAL DELIVERY DELIVERY
“Naked” DNA Long-term Limited uptake
Size of insert Limited by viral Potentially vaccination protection
capsid limitless Liposomes Easy to vary Less efficient than
Host immune Potentially Minimized phospholipid viral vectors
response problematic Inexpensive Toxicity at high
Technical difficulty More difficult Easier Nonpathogenic dose
level Nonimmunogenic Not cell specific
Targetable to Possible Possible Polycation Cell-specific Targeting material
different cells conjugates design to nucleus is not
Gene transfer Reasonably efficient Inefficient Nonpathogenic specific
Fate of delivered Transient or Transient Limited expression
gene persistent Artificial Large Limited uptake
Stability in vivo Reasonable Not stable chromosome inserts—10 kb Limited expression
Nonimmunogenic
Does not
integrate

DNA

mRNA

Small interfering RNA

Ribozyme/
Antisense Antisense RNA
DNA enzyme
oligonucleotide

RISC

RNase H
RNase?

Translation
blocked
Protein

Figure 13-7. Examples of posttranscriptional gene silencing tools. Four common approaches for targeting mRNA to induce
posttranscriptional gene silencing and their corresponding mechanism of action. (Modified with permission from Kukreck J:
Antisense technologies. Eur J Biochem 2003;270:1628–1644.)

resulting in poor metabolism, extensive metabolism, and production of morphine. These latter individuals are more
ultrametabolism. In about 10% of the Caucasian population, likely to experience adverse effects and toxicity with
homozygosity occurs for alleles that are either nonfunctional any standard dose of codeine. The best scenario is those
or have very little function, and in these individuals codeine individuals who have extensive metabolism and gain pain
does not metabolize to morphine. There are also individuals relief without incurring risk for toxicity. Of course, once the
having ultrametabolism of codeine, resulting in an increased genotype is determined for an individual, physicians will
 234 Modern Molecular Medicine

recognize which patients require less codeine, the ultrametab- record. Once high-density SNP maps spanning the genome
olizers, and those who require a different drug, the poor are completed, the next step is correlating individual or
metabolizers. Since there are at least 29 polymorphisms for groups of SNPs with disease efficacy and adverse events. With
the CYP2D6 gene, this and other P-450 genes are attractive this genetic prescreen, drugs can be prescribed that are tai-
candidates for microarray analysis (see below). Determining lored not only to the disorder but also to the individual
individual genotypes for P-450 genes, responsible for clearing genetic signature, leading to more personalized or individual-
over half of the drugs introduced to the body, provides physi- ized medicine.
cians with powerful information to anticipate and avoid The potential benefits of pharmacogenomics are not limited
potentially lethal drug interactions. to individually tailored drug therapy. Other benefits include
The clinical utility of the Human Genome Project is also more accurate dosing information (determined by a person’s
apparent with the development of another tool to identify genetics rather than height and weight), rapid drug design,
and quantitate a high density of genetic polymorphisms. acceleration of drug discovery and approval, and overall
Single nucleotide polymorphisms, or SNPs, span populations decreased health care costs.
and racial and ethnic groups. Not all changes in nucleotides
are classified as SNPs; an SNP is defined as two or more
nucleotides represented at a particular site that are present
in at least 1% of the population. SNPs may occur outside a
●●● NEW DIAGNOSTIC APPROACHES
gene or have an effect on protein function, as do many point A key goal for medical geneticists is the development of rapid,
mutations. SNPs are classified as linked, causative, coding, accurate, and inexpensive methods for genetic diagnosis,
and noncoding (Fig. 13-8). prognosis, and disease susceptibility prediction. Many such
SNP maps are being developed in large genetic databases protocols, including mass spectroscopy and nanoparticle tech-
(Fig. 13-9). With this foundation, an individual’s SNP geno- nology, are under development. However, DNA microarray
type can be determined, although this can be a slow and analysis represents a viable diagnostic and prognostic tool
expensive process. The advent of DNA microarrays for DNA that is already of clinical value and is rapidly becoming an
sequencing and mutation detection, however, holds the industry standard in genetic medicine.
promise for making individualized SNP surveys rapid and
inexpensive. It is not inconceivable that, in the near future,
personal SNP profiles will be a standard part of the medical
Microarray Analysis
DNA-based diagnostic procedures have traditionally relied
on the analysis of a single gene or a small group of
genes. However, new technology permits the simultaneous
Linked SNPs
measurement of thousands of gene sequences or changes in
Outside gene
gene expression. This technology is termed DNA microarray
Also called indicative SNPs
May correlate to drug
analysis; it is also known as DNA array or gene chip analysis
response or certain disease (Fig. 13-10). This is a hybridization-based technique that
No effect on protein involves a nucleic acid probe hybridizing to a target nucleic
production or function acid. The targets are small pieces of DNA that are spotted
in grid-like fashion on a small glass, nylon, or plastic slide,
or “chip.” This DNA can be gene fragments, cDNAs, or syn-
Causative SNPs thesized oligonucleotides. Key to this technology is that the
Inside gene grid contains thousands of target DNAs, each with its own
May correlate with disease Regulatory
or response to treatment sequences position on the grid. Typical arrays contain 10,000 to 20,000
genes per chip, and these gene targets are “spotted” using
Noncoding SNP technology derived from the semiconductor microelectronics
Changes amount of protein
industry. To begin to see the utility of this methodology,
produced
Gene recall that the entire human genome contains approximately
Coding SNP 23,000 genes. The probe sequences are either DNA fragments
Changes amino acid sequence or cDNAs derived from a control (or normal) and experi-
mental (or disease) cell type. Probes are typically labeled
with a fluorescent tag such that, following hybridization with
Coding
region a microarray chip, probe-target hybrids are indicated by a
scanning detection system featuring lasers, cameras, and a
Protein
computer that can identify precisely which spots on the grid
mRNA yield a hybridization signal. The collection of positive hybrid-
ization signals on the grid results in a “molecular signature”
Figure 13-8. Classification of SNPs. Not all SNPs are that can be useful for the two current primary clinical uses
disease causing, but they can be informative in forming for microarray technology: gene expression profiling and
genotypic signatures. mutation detection.
 New Diagnostic Approaches 235

Distribution of SNPs Genetic Reference Physical Chromosome 15

map markers map ideogram
12 8 0
0.0 D15S128
p13
7 12 1 28.53 p12
14.7 D15S165
p11.2
62.68
p11.1
D15S126
q11.1
184 41 2 2 q11.2
q12
D15S117
q13
45.5
89 17 1 3 D15S153 q14
51.2 166.11
62 16 4 q15
q21.1
61.9 D15S131 206.95 q21.2
67 19 5 q21.3
68.8 229.45 q22.1
161 52 3 6 q22.2
D15S205 243.25
78.2 q22.3
64 24 1 7 q23
86.1 D15S127 q24
8 3 1 8 297.82
q25
98.9 D15S130 324.81 q26.1
337.35 q26.2
1 9 346.39 q26.3
110.4 D15S120
26 18 10
110.4 cM 360.78 cR

Confirmed SNPs Validated SNPs Candidate SNPs

Figure 13-9. SNP map of chromosome 15. Approximately 99.9% of DNA sequences are identical between individuals. Over
80% of the remaining 0.1% of sequences differ at single nucleotide polymorphisms (SNPs). (Redrawn with permission from NCI
Cancer Genome Anatomy Project. Available at: http://gai.nci.nih.gov/html-snp/imagemaps.html.)

Prepare microarray
Gene Expression Profiling
Prepare cDNA probe
The use of DNA microarrays is a method to study gene
"Normal" Tumor expression. For example, it may be of interest to compare
gene expression profiles for a particular tissue in normal and
diseased states. In this application, gene chips are made in
RT/PCR
which the target DNA typically consists of thousands of
Label with cDNAs. The probes may be cDNAs derived from the mRNAs
fluorescent dyes
from the normal and diseased tissue. Probe cDNAs from the
normal tissue would be labeled with a particular fluorescent
tag (red, for example), and cDNAs from diseased tissue would
Microarray on
glass, be labeled with a different fluorescent tag (green, for example).
Combine equal
nylon, or The “red” and “green” cDNA probes would be washed over
plastic chip the gene chip during the hybridization step. Encountering its
amounts
complementary target, a probe forms a hybrid, and this is
indicated as a “positive” hybridization by the scanner. Com-
Hybridize probe
to microarray Scan puter software calculates the relative amounts of the red and
green signal at each address or spot on the grid. Such a ratio
represents the relative amount of mRNA expression of that
Figure 13-10. Gene expression analysis using microarray gene in the normal and the diseased tissue. In other words, a
technology. Colors detected by the scanner reflect expression given address on the target grid may give a strong red signal
and level of expression in normal and tumor samples. indicating strong mRNA expression in normal tissue and
 236 Modern Molecular Medicine

T G A A A G A G A
C C T G T C C T
C

A SNP SNP SNP

Haplotype 1 C T G A C T A A G T A C C G A

Haplotype 2 C T G A C T A A G T A C C T A

Haplotype 3 C T G A C T A G G T A C C G A

Haplotype 4 C T G A C T A G G T A C C T A

Haplotype 5 C C G A C T A A G T A C C G A

Haplotype 6 C C G A C T A A G T A C C T A

Haplotype 7 C C G A C T A G G T A C C G A

Haplotype 8 C C G A C T A G G T A C C T A
B
Figure 13-11. Single nucleotide polymorphisms. A, Three 2-nucleotide changes (SNPs) shown for one of two complementary
DNA strands. B, Possible combinations of SNPs lead to eight possible haplotypes (23). A child inherits one haplotype from each
parent, yielding a SNP profile that may be associated with a particular phenotype.

weak expression in diseased tissue, a strong green signal Gene Chips and Mutation Detection
indicating high expression levels in the diseased tissue, a One of the first uses of microarray technology was the iden-
yellow signal indicating expression of the gene in both normal tification of point mutations. This type of array detects genetic
and diseased tissue, or a black signal indicating no expression variation among humans, as seen with SNPs. Microarrays can
in either tissue type. When all gene targets on the chip grid also detect specific disease-causing mutations, such as the
are considered, an expression signature for a given disease spectrum of mutations that cause PKU or cystic fibrosis. In
state is demonstrated. An illustration of this application is the latter example, the DNA targets on the array grid repre-
shown in Chapter 5 (see Fig. 5-26). sent pieces of DNA that contain all known or relevant varia-
In Chapter 5, comparative genomic hybridization (CGH) tions of a gene or genes; such variations may include either
was introduced as a cytogenetic technique that can be used benign polymorphisms or disease variants or both. This means
to demonstrate changes in chromosome content, as illustrated that adjacent addresses on the grid represent the same gene
with double minutes and heterogeneous staining regions (see but differ by as little as a single base pair. For example, a gene
Fig. 5-9). This same concept can be used with microarray chip to perform genetic diagnosis for phenylalanine hydroxy-
analysis by placing genomic DNAs on a slide or support lase (PAH)–associated hyperphenylalaninemia contains many
rather than cDNAs or DNAs as described above. The genomic oligonucleotide or cDNA targets representing all known
DNAs are selected clones, often bacterial artificial chromo- genetic variants of the PAH gene. Probes are generated by
some (BAC) DNA clones, representing the physical genomic isolating DNA from a patient, followed by PCR amplification
map. These microarrays are referred to as aCGH (array com- of the patient’s PAH gene that also incorporates a fluorescent
parative genomic hybridization). For each gene, the ratio of tag in the probe. The probe is then applied to the chip and
red to green fluorescence corresponds to the ratio of DNA hybridizes and fluoresces with only the grid address corre-
copy number of that gene in the tumor sample compared with sponding to the patient’s PAH mutation. In this way, mutation
the normal sample. Since each clone represents a known detection is rapid and comprehensive.
region of a chromosome and complex computer software will There is also clinical utility in the identification of SNP
identify the region of chromosomes with copy number altera- patterns. As discussed above, SNPs are benign single nucleo-
tions, this technique provides high resolution for detecting tide polymorphisms. Typically, SNPs are base changes without
chromosomal alterations, amplifications, deletions, and loss pathology. Several thousand SNPs have been identified in
and gain. humans, and more are being added to genetic databases every
 Questions 237

week. Accordingly, and using the above method, gene chips ●●● QUESTIONS
have been produced that can test individual genomes for the
presence or absence of the known SNPs. This produces an 1. The high rate of prostate cancer in African American
SNP signature and can indicate collections of SNPs, or hap- males prompted investigation of an at-home PSA
lotypes, that consistently occur together (Fig. 13-11). An indi- (prostate-specific antigen) screening kit with 12,000
vidual SNP or a SNP haplotype may be associated with male volunteers over the age of 45. Of these, 3360
individuals had PSA levels greater than 4.0 ng/mL,
certain disease states via genetic linkage. In this case, rapid
and in 8640 individuals, PSA levels were less than
and accurate microarray analysis reveals a haplotype or SNP
4.0 ng/mL. Each participant then consented to have
signature predicting disease predisposition. As discussed
a blood sample drawn to compare the results.
above, SNP profiling will also greatly enhance the ability to
use genetic signatures for the determination of drug choice,
as SNP genotypes will—for a given drug—predict efficacy PSA LEVELS CONFIRMED AT
and adverse reactions on an individual basis. 4.0 ng/mL OR GREATER
AT-HOME
●●● SUMMARY SCREENING KIT True False

Within the near future, most human genes will be character- Positive screening test 2400 960
ized regarding location in the genome, sequence, and func- (≥4.0 ng/mL)
tional identity.At the same time, molecular genetic technology Negative screening test 600 8040
will advance to a point where it will be routine to ascertain (<4.0 ng/mL)
an individual’s collection of disease and disease predisposition
alleles as well as benign SNPs. These data will permit a more
What is the sensitivity of the screening test?
precise form of medicine—one that is specific to an individu-
al’s unique genetic profile. Genetic predispositions will be A. 23%
identified, and DNA-based diagnostics will be routine. Knowl- B. 71%
edge of genetic risks will allow individual presymptomatic C. 80%
opportunities for disease intervention including lifestyle D. 87%
change. Intelligent and rational drug design will be the norm, E. 89%
since it will be based on integrated genetic and biochemical Answer. A
signatures. Gene therapy is emerging as a permanent curative Explanation: Sensitivity is the number of actual positives
approach in the treatment of both inherited and noninherited that are correctly identified. PSA levels tend to increase with
disease. Hence, it is unlikely to be an exaggeration to state that age, with normal values generally less than 4.0 ng/mL. In
today is the dawn of the new age of molecular medicine. this case, the question becomes what percentage of indi-
viduals who have PSA levels greater than 4.0 ng/mL were
also determined using the home testing kit to have an ele-
KEY CONCEPTS vated PSA. The true positives (TP) and false negatives (FN)
■ Molecular tools can be applied to almost any aspect of biomedi- are 2400 and 8040, respectively. To determine sensitivity:
cal research. Application to diagnostics is increasing. [TP/(TP + FN)] × 100 = 23%.
■ New applications of molecular techniques to diagnostics and
therapies are often in research protocols and clinical trials. Additional Self-assessment Questions can be Accessed
Informed consent for these studies differs from that given for at www.StudentConsult.com
treatment already approved and used in daily practice. Informed
consent is an ongoing process. Case Studies and Case Study Answers are available
■ HIPAA authorization for research protocols and clinical trials may online on Student Consult www.StudentConsult.com
be incorporated into informed consent documents.
■ Genetic screening is used for diseases that are reasonably
common, well characterized, severe, and treatable or
preventable.
■ Criteria for genetic screening include disease characteristics,
diagnostic test features, and health care system capacity.
■ Clinical tests must demonstrate acceptable sensitivity, specificity,
predictive value, and efficiency.
■ Genetic counseling is a knowledge-based interaction between
patients and health care providers.
■ Gene therapy targets somatic cells and not germ cells. There-
fore, a successful therapy is limited to the individual affected and
not offspring.
This page intentionally left blank
 239

Index
Note: Page numbers followed by f indicate Adenomatous polyposis, familial (FAP), 76t, Ambiguous genitalia, 207–208, 207f
figures; those followed by t indicate tables; 80–81, 80f Ameloblasts, 118b
and those followed by b indicate boxed Adenomatous polyposis coli (APC), 81b Amenorrhea, 196b
material. Adenosine (A), 1–2, 2b, 3f AMH (antimüllerian hormone), 17b,
Adenosine deaminase deficiency, 64t, 230t 194–195, 194f, 204
A S-Adenosylmethionine (SAM), 58, 58b, AMH gene, 194f
A (adenosine), 1–2, 2b, 3f 154–155 AMI (acute myocardial infarction), 160b
A-form DNA, 1b Adenoviruses, 172, 231, 231t Amino acid(s), 54b
AAT. See α1-Antitrypsin (AAT) Adoption studies, 148 essential, 54b
AAT gene, 172–173, 172t Adrenal gland nonessential, 54b
AAVs (adeno-associated viruses), 231, anatomy of, 205b plasma increase in, 58b
231b, 231t, e40 development of, 195b triplet coding of, 4, 6b
ABL oncogene, 25, 71t, 72–73, 72f–73f, e3 evaluation of, 206b urinary, 56b
ABO blood group, 32b, 93–94, 94t, 95f, steroidogenesis in, 205, 205f Amino acid motifs, 193b
113 Adrenal hyperplasia, congenital (CAH), Aminoacidopathies, 54–60
Bombay phenotype of, 95–96, 113 207–208, 207f, 207t, e34–e35, e35 of homocystine metabolism, 58–60, 59f
genotypes/phenotypes of, 93–94, 94t, 96, mild, 207 of phenylalanine metabolism, 54–57, 55f,
96t Adrenal hypoplasia congenita (AHC), 195 56t–57t
incompatibility in, 94t, 96–97 α-Adrenergic blockers, in long QT of tyrosine metabolism, 55f, 57–58
inheritance of, 96, 96t syndrome, 167 γ-Aminobutyric acid (GABA), 154b
molecular genetics of, 94–96 Adrenocortical steroidogenesis, disorders of, Amplification
soluble antigens of, 96 205–208, 205f, 207f–208f, 207t gene, 73–74, 75f
ABPs (androgen-binding proteins), 17b Adrenocorticotropic hormone (ACTH), 204 triplet repeat, 35–37, 37f
Acanthosis nigricans, 11, 124b, e25 Adrenocorticotropic hormone (ACTH) Amyloid β-peptide, 149
Acatalasia, 211t stimulation test, 206b Amyloid plaques, 148–149
Aceruloplasminemia, 188, e28 Adult hemoglobin A (Hb A), 32, 32b, 103, β-Amyloid precursor protein (APP) gene,
Acetyl-CoA carboxylase, 62 103f, 105f, 106t 149
Acetyl-CoA:α-glucosaminide AFP (α-fetoprotein), e3 Analytical validity, 226–227
N-acetyltransferase deficiency, 143f, Africans, iron overload among, 187b Anaphase
143t, 144 Aganglionic megacolon. See Hirschsprung of meiosis, 17
α1,3-N-Acetylgalactosamyl transferase, 32b disease of mitosis, 15, 16f
N-Acetylglucosamine-6-sulfatase deficiency, Agglutination reaction, 93 Anaphase lag, 21–22
143f, 143t, 144 AHC (adrenal hypoplasia congenita), 195 Andersen syndrome, 165t, 167
α-N-Acetylglucosaminidase deficiency, 143f, AHC gene, 194f Androgen-binding proteins (ABPs), 17b
143t, 144 AIS (androgen insensitivity syndrome), 204 Androgen insensitivity syndrome,
aCGH (array comparative genomic AKAP9 gene, 165t 204
hybridization), 236 Albinism, 29b, 31, 55f Androgen receptor, 204, 204b
Achondroplasia, 122–125, 123f, 132, e19 carrier for, 211t Androgen replacement, 200b
autosomal inheritance of, 29, 29b classic (oculocutaneous), 57, e9 Androgen synthesis, 202, 203f
clinical manifestations of, 29, 122, 123f Aldosterone, 205b Anemia
epidemiology of, 29, 122 Alkaptonuria, 51–52, 51f, 55f, 64 Fanconi, gene therapy in, 230t
FGFR3 gene in, 123–125, 124f–125f carrier for, 211, 211t hemolytic, 98–102, 98b
mitochondrial inheritance of, e4 Allele(s), 4, e2 erythrocyte hemoglobin defects and,
natural history of, 124–125 conservation of, 4 102–110, 103f–105f
prenatal diagnosis of, 125, 125f dominant, 28 erythrocyte membrane defects and,
vs. pycnodysostosis, 212–213 expression of, 53 98–100, 98f–99f, 99t–100t
Acidemia(s), organic, 61–63, 62f, 63t hemizygous, 32–33 erythrocyte metabolic defects and,
biotinidase deficiency as, 62–63 notation for, 28 100–102, 100b, 102t
maple syrup urine disease as, 61–62, 62f, null, 95–96, 173 fava bean–related, 102
63t–64t recessive, 28, 29t glucose-6-phosphate dehydrogenase
Acidosis, metabolic, 62b Allele frequency, 212 deficiency as, 100–102, 101b, 101f,
Acrocentric chromosome, 12, 14f Allele-specific oligonucleotide (ASO) 102t
Acrocephaly, 120 testing, 225, 225f hemoglobinopathies as, 102–104, 103f
ACTH (adrenocorticotropic hormone), 204 Allelic heterogeneity, 53, 170, e1 hereditary elliptocytosis as, 98,
ACTH (adrenocorticotropic hormone) Allelic variation, 28, 53. See also 98f–99f, 100t
stimulation test, 206b Mutation(s) hereditary pyropoikilocytosis as, 98f,
Actin, 175 Allopurinol, 136b 99–100, 100t
Action potential, cardiac, 166b Alport syndrome, 64t, 115t hereditary spherocytosis as, 98,
Activated protein C resistance, 112 Alternative splicing, 52b 98f–99f, 99t–100t
Acute chest syndrome (ACS), 106b Alu elements, 4, 4b primaquine-induced, 101–102
Acute lupus erythematosus, 11 AluI enzyme, 4b, 220t sickle cell anemia as, 104–107,
Acute myocardial infarction (AMI), 160b Alzheimer disease, 148–150, 158 104f–105f, 105t–106t, 106b, 107f.
Adeno-associated viruses (AAVs), 231, epigenetic studies in, 148 See also Sickle cell anemia
231b, 231t, e40 familial, 148–149 thalassemias as, 107–110, 108f, 109t
 240 Index

Anemia (Continued) Arachnodactyly, in Marfan syndrome, 119, BAX protein, 86b

sideroblastic, 59b 121f Bayesian analysis, 229
spur cell, 97b Arcus cornealis, 161, 163f, e25 Bazett’s formula, 166b
Anencephaly, 42 Aromatase deficiency, 204–205 BCAAs (branched-chain amino acids), 61,
Aneuploidy, 18 Array comparative genomic hybridization 62f
cause of, 18–22, 18f (aCGH), 236 BCL2 gene, 157, 157b
trisomy 21 as. See Down syndrome Artemin (ARTN), 182–183 BCL2 protein, 86b
Angelman syndrome, 7b, 26t, e7 Arthrochalasis, in Ehlers-Danlos syndrome, BCLX protein, 86b
clinical manifestations of, 39 120t BCR-ABL fusion gene, 25, 72–73, 72f–73f,
epigenetics of, 43 Artificial chromosome e3, e13
genetics of, 29b, 38, 39f bacterial, 164 BDNF (brain-derived neurotrophic factor),
uniparental disomy in, 38–39 for gene transfer, 233t 147
Angiokeratoma, 138, 139f ARTN (artemin), 182–183 Becker muscular dystrophy (BMD), 29b,
Angiokeratoma corporis diffusum, 138b Ascertainment bias, 148 125
Angiotensinogen, 206b Ashkenazi Jews Beckwith-Wiedemann syndrome, 181b
ANKB gene, 165t breast cancer in, 83, 85f Behavioral genetics, 147–148, 153f
Ankyrin, 97, 98f, 99t cystic fibrosis in, 170 Bell-shaped curve, 41, 41f
Antibodies, 93 Gaucher disease in, 141 Bence Jones proteins, e3
in type 1 diabetes mellitus, 46–47, 48f Niemann-Pick disease in, 142 Beneficence, 229t
Anticipation, 146 Tay-Sachs disease in, 138, 227–228 Bertram, Ewart, 22
Anticodons, 5 ASO (allele-specific oligonucleotide) testing, β-cells, 44b
Antigen(s), 93 225, 225f destruction of, 47, 48f
blood group, 93–97, 94t. See also ABO Association studies, 148 BH4 (tetrahydrobiopterin synthase), 55f
blood group; Rh blood group in bipolar disorder, 156, 157t Bile acid sequestrants, in familial
Antigen recognition, 47b Assortative mating, 214 hypercholesterolemia, 163, 163b
Antimüllerian hormone (AMH), 17b, Ataxia Bile acids, 163b
194–195, 194f, 204 fragile X–associated, 36 Biliary cancer, screening for, e38
Antiparallel orientation, of DNA, 2, 3f, 5 Friedreich, 29b, 36t Bilirubin, elevated levels of, 102, 102b
Antisense oligonucleotide therapy, 232 spinocerebellar, 29b, 36t BIM protein, 86b
Antitelomerase therapy, 89b, 90f, 90t Atherosclerosis, 160b–161b Biotin, 62
α1-Antitrypsin (AAT), 172 ATP7A gene, 187–188, 189t, e28 Biotin-unresponsive 3-methylcrotonyl-CoA
α1-Antitrypsin (AAT) deficiency, 172, 172t, ATP7A protein, 188, 189f carboxylase deficiency, 63t
e25 ATP7B gene, 187–189, 189t, e29 Biotinidase deficiency, 62–63, e10
ATT gene in, 172–173, 172t ATP7B protein, 188, 189f Bipolar disorders, 155–157, 155t–157t
cigarette smoking and, 173–174, ATT deficiency. See α1-Antitrypsin treatment of, 157, 157b
173f–174f deficiency Bivalent chromosome, 16–17
liver disease in, 175, e26 ATT gene, 172–173, 172t Bleeding disorders, 110–112, 110t
lung disease in, 173–174, 173f–174f Authorization, elements of, 220b Blood group antigens, 93–97. See also ABO
null allele in, 173 Autoimmunity, 46–47, 47b, 48f blood group
treatment of, 174–175, 174t Autonomy, 229t Blood-testis barrier, 17b
ZZ genotype in, 172t, 173, e26 Autosomal dominant inheritance, 28–30, Blood transfusion, in β-thalassemia, 110
α1-Antitrypsin (AAT) gene, 172–173, 172t 29b, 30f Bloom syndrome, 11
Aortic aneurysm, 121, 122f characteristics of, 29–30 BMD (Becker muscular dystrophy), 125
Aortic coarctation, 198b nonpenetrance in, 34–35, 34f BMP genes, 178t
Aortic regurgitation, in Marfan syndrome, Autosomal recessive inheritance, 29b, Bond
121, 122f 30–32, 31f, 31t hydrogen, 1
AP (apurinic/apyrimidinic) endonuclease, 8f characteristics of, 31–32 phosphodiester, 3b
APAF1 (apoptotic protease-activating factor consanguinity and, 32, 212–214 Bone marrow
1), 86b genotype in, 30, 31t erythropoietin-induced hypertrophy of,
APC (adenomatous polyposis coli), 81b Autosomes, 4, 12, 13f 109b
APC gene, 76t, 78t, e11 transplantation of, 141b
in colorectal cancer, 80, 80f B Bowlegs, 195b
in familial adenomatous polyposis, 81 B-form DNA, 1b bp (base pair), 1–2, 2f
APC-GSK complex, 81b B lymphocytes, 47b Brachycephaly, 120
Apgar score, e5 B-RAF oncogene, 71t Brachydactyly, 29b
Apoceruloplasmin, 187–188 Bacterial artificial chromosomes (BACs), Brain-derived neurotrophic factor (BDNF),
Apolipoprotein E (APOE), 149b 164, 233t 147
Apolipoprotein E (APOE) gene, 149–150, BAK, 86b Brain disease. See Neurologic disease(s)
149t, 158 Balanced rearrangement, 24–25 Branched-chain amino acids (BCAAs), 61,
Apoptosis, 86b Band 3, 97–98, 98f, 99t 62f
in bipolar disorder, 157 Barr, Murray, 22 Branched-chain keto acid decarboxylase,
Apoptotic genes, 76t, 85–88, 157b Barr body, 22–23, 33, 38b 61
in Li-Fraumeni syndrome, 85–86, 87f–88f Basal cell carcinoma, 9b BRCA1 gene, 76t
in prostate cancer, 86–88, 88f Basal nuclei (ganglia), 190b in breast cancer, 83, 85f
Apoptotic protease-activating factor 1 in schizophrenia, 153f in ovarian cancer, 83–85, 85f
(APAF1), 86b Base, nitrogenous, 1 BRCA2 gene, 76t
APP gene, 149 Base analogs, 7, 8b in breast cancer, 83, 85f
Appendicular skeleton, 123b Base excision repair, 8, 8f, 11 in ovarian cancer, 83–85, 85f
Apurinic/apyrimidinic (AP) endonuclease, Base pair (bp), 1–2, 2f BRCA1 protein, 83
8f BAX gene, 81, 85–86, 87f, e12 BRCA2 protein, 83
 Index 241

Breast cancer, 76t, 81–83, 82b Carrier (Continued) Chondrodysplasia, 115t

BRCA genes in, 83, 85f for cystic fibrosis, 169, 211t Christmas disease, 29b, 64t, 110–111, 110t,
classification of, 82b genetic screening for, 227–228 230t
familial, 82 heterozygous, 31 Chromatid, 2f, 12
invasive, 82b, 84f for phenylketonuria, 211t, 213–214, 213f diameter of, 1
in Klinefelter syndrome, 199 for sickle cell anemia, 106, 211t, 216, e37 in nondisjunction, 20–21
mRNA expression profiling of, 91f, 92 for Tay-Sachs disease, 211t, 227–228 sister, 12, 17
noninvasive, 82b, 84f Cataract, in galactosemia, 60f, 61, e8–e9 Chromatin, 1–2, 2f
risk factors for, 83b CATCH-22, 178b Chromosomal abnormalities
types of, 82b, 91f Catechol-O-methyltransferase (COMT), 58b, numerical, 6, 18–23, 18b, 18f
Brushfield spots, 18–19, 19f, e2 154–155, 155f structural, 6, 23, 23f
Buccal smear, 22–23 Catecholamines, 58b Chromosomal mosaicism, 39–40, 200
Burkitt lymphoma, 70–71, 72f β-Catenin, 81, 81b Chromosome(s), 4, 12–24
CAV3 gene, 165t acrocentric, 7, 12, 14f
C CBAVD (congenital bilateral absence of vas anatomy of, 14f
C (cytosine), 1–2, 2b, 3f deferens), 167, 167t, 170–171, 171b, artificial, 233t
CAAT box, 5 176 bacterial, 164
CACNA1C gene, 165t CBS (cystathionine β-synthase) deficiency, autosomal, 4, 12
Cadherins, 81b 58–60, 59f, e8–e9 balanced rearrangement of, 24–25
Café-au-lait spots, 35, 79–80, 79b, 133, CBS gene, 60 bivalent, 16–17
134f, e2, e31 CD4+ T lymphocytes, 47b classification of, 12, 13f
CAH (congenital adrenal hyperplasia), CD8+ T lymphocytes, 47b deletions of, 6–7, 7b, 25, 26t
207–208, 207f, 207t, e34–e35, e35 CDK4, 66f, 67, 68f duplications of, 26
mild, 207 CDK6, 67, 68f fluorescent in situ hybridization for, 14,
Calcification, in retinoblastoma, 77f, 79b CDK4 oncogene, 71t 15f
Calpain-10, 47–48 CDKN1A gene, 85–86, 87f fluorophore painting of, 14, 15f
Camptomelic dysplasia, 194, 196f CDKs (cyclin-dependent kinases), 14b, 67, G-banding of, 12–14, 13f
Cancer, 65–92 67b, 67f homologous, 4, 12, 16f, e2
breast. See Breast cancer Cell cycle, 14–15, 14b identification of, 12–14, 13f
colorectal, 76t, 80–81, 80f, 82f, e11 G1-to-S transition of, 67–68, 67f inversions of, 7, 7f, 26, 26f
cytogenetic alterations in, 91–92 genetic regulation of, 65–68, 66f–67f karyotype of, 12–14, 13f, 15f
DNA-based screening for, 91f, 92, e38 Cell division–controlling genes, 74–79, 76t long arm of, 12, 14f
gastric, 96 in neurofibromatosis, 79–80, 79b metacentric, 12, 14f
genetic paradigm for, 68, 70f, 80–81, 80f. in retinoblastoma, 68f, 74–79, 76t, metaphase, 3f, 12, 13f
See also Oncogenes; Tumor 77f–78f, 79b nomenclature for, 12–14, e3
suppressor genes Cell membranes, 97b numerical abnormalities of, 18–23, 18b,
heritable vs. sporadic, 78f, 78t, 79 Central dogma, 1, 4 18f
heterogeneity of, 91–92 Central hearing disorder, 117b organization of, 3–4
metastatic, 69b Centrioles, 15b Philadelphia, 25, 72–73, 72f–73f, e3, e13
monoclonality of, 68, 91–92 Centromere, 12, 13f Q-banding of, 12–14
mutations in, 68, 70f. See also Cephalic index (CI), 122f reciprocal translocation of, 23–24, 23f
Oncogenes; Tumor suppressor genes Ceramide, 142 robertsonian translocation of, 7, 24, 24f
ovarian, 76t, 83–85, 85f Cerebral infarction, 160b sex, 4, 12, 13f. See also X
pathology of, 69b Cerebro-oculo-facio-skeletal syndrome, 10t chromosome(s); Y chromosome
prostate, 76t, 86–88, 87b, 88f Cerebrosides, 136–137 alleles of, 28
screening for, 92 Ceruloplasmin, 188, 188b, e28 numerical abnormalities of, 19–20,
telomerase in, 88–89 CF. See Cystic fibrosis (CF) 22–23
types of, 69b CFTR (cystic fibrosis transmembrane short arm of, 12, 14f
Capillary electrophoresis, 223f, 224 regulator) gene, 53, 169–171, 170f, structural abnormalities of, 23, 23f
Carbohydrate metabolism disorder, 60–61, 170t sub-bands of, 14
60f–61f, 64t, e8–e9, e9–e10 therapeutic manipulation of, 172 submetacentric, 12, 14f
Carcinoembryonic antigen, e3 CFTR (cystic fibrosis transmembrane tetrad, 16–17
Carcinoma, 69b regulator) protein, 167–171 translocation of, 7, 23–25, 23f–24f
Cardiac action potential, 166b CGH (comparative genomic hybridization), Chromosome painting, 14, 15f
Cardiac disorder(s), 159–167 75f, 236 Chromosome rescue, e7
familial hypercholesterolemia as, Charcot-Marie-Tooth disease, 29t, e36 Chronic myeloid (myelogenous) leukemia
159–164, 162f–163f CHARGE association, 178b (CML), 72–73, 72f–73f, e13
long QT syndrome as, 164–167, 165f, 165t Chelation, 190, 190b BCR-ABL gene in, 25, 72–73, 72f–73f, e3
Cardiopulmonary disorder(s), 159–176 Chemical mutagens, 7, 8b pathology of, 25b
α1-antitrypsin deficiency as, 172, 172t, Cherry-red spot, 138, 138f phases of, 73, 74b
173f–174f, 174t Chiasmata, 16–17 treatment of, 73, 73b
cystic fibrosis as, 167–172, 167t, 168b, Child abuse, vs. osteogenesis imperfecta, Chronic progressive external
168f–170f, 170t–171t, 171b 118 ophthalmoplegia (CPEO), 130b,
familial hypercholesterolemia as, Chimera, 27, 202b 131–132
159–164, 162f–163f Chloride (Cl−) transport, in cystic fibrosis, Chronic rhinosinusitis (CRS), 171, e26
long QT syndrome as, 164–167, 165f, 165t 168–169, 169f Chylomicrons, 159t
Caretaker genes, 74 Cholesterol, 159t, 176 CI (cephalic index), 122f
Carrier metabolism of, 163b Cigarette smoking, α1-antitrypsin deficiency
for albinism, 211t pharmacologic reduction of, 163, 163b and, 173–174, 173f–174f
for alkaptonuria, 211, 211t synthesis of, 160, 161f, 201b Ciliary body, 120b
 242 Index

CIP/KIP inhibitors, 66f, 67–68, 68b Consultand, 228 Cystic fibrosis transmembrane regulator
Cirrhosis, in α1-antitrypsin deficiency, 175 Coombs test, 190b (CFTR) gene, 53, 169–171, 170f, 170t
CKIs (cyclin kinase inhibitors), 67–68, 68b Copper (Cu+) therapeutic manipulation of, 172
Cl− (chloride) transport, in cystic fibrosis, accumulation of. See Wilson disease Cystic fibrosis transmembrane regulator
168–169, 169f chelation of, 190 (CFTR) protein, 167–171
Cleft lip, 42 as cofactor, 187–188, 187b Cystinosis, 64t
Clinical utility, 226–227 deficiency of. See Menkes syndrome Cystinuria, 180–181, 191
Clinical validity, 226–227 metabolism of, 188, 189f classification of, 180–181, 181t
CML. See Chronic myeloid (myelogenous) requirement for, 187–188, 187b founder effect in, 181, 182f, 191
leukemia (CML) transport of, 188 treatment of, 181b
Coagulation, 111b Copper transporter 1 (CTR1) gene, 188 Cytochrome c oxidase, 187b, 188
deficiency of, 110–112, 110t Cornea Cytochrome P-450 3A4 system, 163b
excess of, 112, 112t Kayser-Fleischer ring of, 190, 190f, e29 Cytochrome P-450 aromatase, 204
Cockayne syndrome, 9, 10t, 11 opacity of, in Fabry disease, 139f Cytogenetics, 12
Codeine, 232–234 Corneal arcus, 161, 163f, e25 Cytosine (C), 1–2, 2b, 3f
Coding strand, 5, 5f Coronal suture, 120b
Codominant inheritance, 32, 106 Corticotropin-releasing hormone (CRH) D
Codons, 5, 6b challenge, 206b D-Amino acid oxidase activator (DAOA)
COL1A1 gene, 116–118, e17, e19 Cortisol, 205b gene, 154
COL1A2 gene, 116, 118 COX 17, 189f D-Amino acid oxidase (DAO) gene, 154,
COL2A1 gene, 116 CPEO (chronic progressive external 154b
COL3A1 gene, 116 ophthalmoplegia), 130b, 131–132 DAX1 gene, 194f, 195
COL5A1 gene, 119 CpG islands, 38b DCC (deleted in colon cancer) gene, 78t,
COL5A2 gene, 119 CPK (creatine phosphokinase), 128b, e17 80, 80f
Colchicine, 12 Craniosynostosis, 120b DCIS (ductal carcinoma in situ), 82b, 84f
Colestipol, 163b Creatine phosphokinase (CPK), 128b, e17 DCYTB gene, 184–185
Collagen, 115 CRH (corticotropin-releasing hormone) DDB2 gene, 9t–10t
genes for, 116 challenge, 206b ddNTP (dideoxyribonucleoside
structure of, 115, 116f Cri du chat syndrome, 7b, 25, 26t triphosphate), 224b
types of, 115, 115t Crossing-over, unequal, 36, 37f, e15 De novo mutation, 7, 40
Colorectal cancer, 76t, 80–81, 80f, 82f, e3, CRS (chronic rhinosinusitis), in cystic Deep vein thrombosis, 112, 112t
e11. See also Hereditary nonpolyposis fibrosis, 171 Deformation, 179b
colorectal cancer (HNPCC) CSA gene, 9, 10t Deletion syndromes, 7, 7b, 25, 26t
Comparative genomic hybridization (CGH), CSB gene, 9, 10t Dentatorubropallidoluysian atrophy
75f, 236 CT. See Connective tissue (CT) (DRPLA), 36t
Compound heterozygote, 161–162 CTR1 (copper transporter 1) gene, 188 Dentinogenesis imperfecta, 117, 118b,
COMT (catechol-O-methyltransferase), 58b, Cyclin(s), 14b, 66f–68f, 67, 67b 118f
154–155, 155f Cyclin-CDK complex, 67–68, 67f, 85–86 Deoxyribonucleic acid. See DNA
COMT gene, 154, 155f Cyclin D, 67, 67f–68f, 68b (deoxyribonucleic acid)
Concordance, 45 Cyclin-dependent kinases (CDKs), 14b, 67, Deoxyribose, 3b
Conductive hearing disorder, 117b 67b, 67f Dermatan sulfate, 142, 143f, 143t
Congenital adrenal hyperplasia (CAH), Cyclin kinase inhibitors (CKIs), 67–68, Dermatosparaxis, in Ehlers-Danlos
207–208, 207f, 207t, e32, e34–e35, 68b syndrome, 120t
e35 CYP11B1 gene, 207–208 Dermis, 115b
mild, 207 CYP2D6 gene, 232–234, e38–e39 Dexamethasone suppression test, 206b
Congenital bilateral absence of vas deferens Cystathionine β-synthase (CBS) deficiency, DHT (dihydroxytestosterone), 196b, 201b,
(CBAVD), 167, 167t, 170–171, 171b, 58–60, 59f, e8–e9 203f, 204
176 Cystic fibrosis (CF), 29b, 31, 167–172, Diabetes
Congenital disorder, 29 e25–e26 gestational, 49–50, e5
Congenital hypertrophy of retinal pigment allelic heterogeneity in, 170 maturity-onset, of young, 49, 49f, 49t,
epithelium, e2, e11, e29 carrier for, 169, 211t e6
Connective tissue (CT), 114 carrier screening in, 170–171, 171t, 215, Diabetes mellitus (DM), 44
collagen of, 115, 115t, 116f 215t, e36–e37 atherosclerosis risk in, 161
disorders of, 117–122, 117t chronic rhinosinusitis in, 171 bipolar disorder and, 157t
anatomic basis for, 114, 115t, 116f clinical features of, 167–168, 167t gender and, e5
differential considerations in, 122, 123t congenital bilateral absence of vas type 1 (insulin-dependent), 44–45
Ehlers-Danlos syndrome as, 119, 119f, deferens in, 167, 167t, 170–171 autoimmunity in, 45–47, 48f
120t differential diagnosis of, 168b family studies in, 45, 46t
Marfan syndrome as, 119–122, gene for, 53, 169–171, 170f, 170t HLA studies in, 46
121f–122f gene therapy for, 171–172, 230t molecular mimicry in, 47
osteogenesis imperfecta as, 117–118, incidence of, 162t, 169 sex-specific thresholds in, e5
117t, 118f infertility in, 168, 176 twin studies in, 45–46
types of, 115b inheritance of, 169 type 2 (non–insulin independent), 44–45,
Connexins, e8 lung function in, 167, 167t, 168b, 47–49
Conotruncal anomaly face syndrome, 178b 168f family studies in, 45
Consanguinity, 32, 212–214, 212t, 213f, e4 molecular pathophysiology of, insulin therapy in, 44b
general aspects of, 213–214 168–169, 169f obesity and, 47
Consent, informed, 219b, e39 newborn screening in, 227 risk assessment for, 48–49
Conservation, of alleles, 4 survival with, 169f screening for, e38
Constitutively expressed genes, 52 treatment of, 64t sex-specific thresholds in, e5
 Index 243

Diabetes mellitus (DM) (Continued) DNA (deoxyribonucleic acid) (Continued) Duchenne muscular dystrophy (DMD)
susceptibility genes in, 47–48 unique, 3–4 (Continued)
twin studies of, 45–47 Z form of, 1b genetics of, 125–128, 127f, e17
type 1B (type 1.5), 45 DNA glycosylases, in DNA repair, 8, 8f, 11 Gowers maneuver in, 126f
Diagnosis, 122, 123t DNA methyltransferase, 38b Ductal carcinoma, 82b
paradigm for, 218–219, 218f DNA microarray analysis, 234–237, 235f, Ductal carcinoma in situ (DCIS),
security of, 123t, e19 e39 82b, 84f
Diakinesis, 16–17 DNA repair, 8–10 Duodenal ulcers, 96
Dideoxynucleotide chain-terminating base excision, 8, 8f Duplication errors, 7, 26
technique, 222, 223f, 224b mismatch, 10–11, 10f, 35 Dust cells, 137b
Dideoxyribonucleoside triphosphate nucleotide excision, 8–9, 9f Dwarfism, short-limb. See Achondroplasia
(ddNTP), 224b proofreading, 11 Dysbetalipoproteinemia, 159t
Diet DNA repair genes, 76t, 80–85 Dysbindin, 154
galactose-free, 61 in breast cancer, 81–83, 85f Dysostosis multiplex, 144b
phenylalanine-free, 56–57, 63, e8 in colorectal cancer, 80–81, 80f, 82f Dysplasia, 179b
Differentially expressed genes, 52, in ovarian cancer, 83–85, 85f Dystrobrevin, 127f
53t DNA sequence analysis, 222–224, 223f Dystrobrevin-binding protein 1 (DTNBP1)
Differic transferrin, 186f Dolichocephaly, 120, 122f gene, 154
DiGeorge sequence, 178b Dominant allele, 28 Dystroglycan, 127f
DiGeorge syndrome, 7b Dominant effects, of recessive mutation, Dystrophin, 52b, 127–128, 127f
Dignity, 229t 29t retinal, e17
Dihydrobiopterin deficiency, 56 Dominant inheritance DZ (dizygotic) twins, 45b, 147–148
Dihydrobiopterin synthetase, in autosomal, 28–30, 29b, 30f
phenylketonuria, 55f, 56, 56t characteristics of, 29–30 E
Dihydropteridine reductase, in nonpenetrance in, 34–35, 34f ECE-1 gene, 183t
phenylketonuria, 55f, 56, 56t X-linked, 29b, 34, 34f ECM (extracellular matrix), 114, 114b
Dihydropyridine receptor, 175 Dopamine, 154–155, 155f EcoRI, 219, 220t
Dihydroxytestosterone (DHT), 196b, 201b, Dopamine β-hydroxylase, 187b, 188 Ectopia lentis, 59
203f, 204 Dopamine β-monohydroxylase, 188 EDN3 gene, 183t
Diploid cell, 16–17, 16f Dopamine D4 receptor, 156, e20 EDNRB gene, 183t
Diplotene, 16–17 Dosage compensation, 23, 33 Edwards syndrome, 21, 21f, 22t, e2
Discordance, 45 Dosage-sensitive sex reversal (DSS), 195 E2F transcription factor, 66f, 67, 68f
Disomy, 20–21 Double helix, 1–2, 2f–3f Efficiency, test, 226–227, 226t
uniparental, 38–39, e7 Double minutes (dmin), 73–74, 75f–76f Ehlers-Danlos syndrome, 115t, 119, 119f,
Dispermy, 27 Down syndrome, e6 120t, 132
Disruption, 179b Brushfield spots in, e2 Electron transport chain (ETC), 128–129
Divalent metal transporter 1 (FDMT1) causes of, 18–22, 18f Electrophoresis, 107b
gene, 184–185, 188 clinical features of, 18–19, 18b, 19f capillary, 223f, 224
Divalent metal transporter 1 (FDMT1) deletion errors in, 25 gel, 219–220, 221f, 224
protein, 184, 186f familial, 25 Elliptocytosis, hereditary, 98, 98f–99f,
Dizygotic (DZ) twins, 45b, 147–148 incidence of, 19 100t
DJ-1, 151t karyotype for, 18, 18f, 26–27 Emphysema, 173–174, 173f–174f
DM. See Diabetes mellitus (DM) maternal age and, 19, 19f Empirical risk, 229
DMD (Duchenne muscular dystrophy), 125, meiotic nondisjunction in, 19–21, 20f, “End replication problem”, 88
127f, 230t, e17, e36 25 Endonucleases, restriction, 219, 220t
dmin (double minutes), 73–74, 75f–76f mosaicism in, 21–22 ENDRB gene, 183t
DMT1 (divalent metal transporter 1) gene, nondisjunction in, 19–21, 20f, 25 Enhancer, 5
184–185, 188 paternal translocation in, 25 env gene, 69b
DNA (deoxyribonucleic acid) screening for, 19b Environmental factors, in multifactorial
A form of, 1b translocation in, 18f, 23–24, 24f, 27, e2 inheritance, 43–44
antiparallel orientation of, 2, 3f, 5 Down syndrome critical region, 25 Enzymes, 4
B form of, 1b DQA1 gene, 46 deficiencies of. See Inborn errors of
coding strand of, 5, 5f DQB1 gene, 46 metabolism
compaction of, 1, 2f–3f DRD4 (dopamine D4 receptor) gene, 156 restriction, 4b, 219, 220t
diameter of, 1 DRPLA (dentatorubropallidoluysian Epidermis, 115b
double helix of, 1–2, 2f–3f atrophy), 36t Epigenetics, 43–44, e1
5′-to-3′ orientation of, 5b, 5f Drugs studies of, 148
hairpin structure of, 35b acanthosis nigricans with, 124b EPO (erythropoietin), bone marrow
highly repetitive, 3–4, 6 long QT syndrome with, 166b, e25 hypertrophy with, 109b
methylation of, 37–38, 38b, 145b oxidative, glucose-6-phosphate Epstein-Barr virus, 71
middle repetitive, 3–4 dehydrogenase deficiency and, ERB-A oncogene, 71t
mitochondrial (mtDNA), 40–41, 128–130, 101b ERB-B oncogene, 71t
129f, e21 DSS (dosage-sensitive sex reversal), 195 ERCC1 gene, 9t
mutation of. See Mutation(s) DTNBP1 (dystrobrevin-binding protein) ERCC2 gene, 9t–10t
orientation of, 2, 3f, 5, 5b gene, 154 ERCC3 gene, 9t–10t
repair of, 8–10, 8f–10f Duchenne muscular dystrophy (DMD), ERCC4 gene, 9t–10t
slippage of, 35 29b, 125, e17, e36 ERCC5 gene, 9t–10t
structure of, 1–2, 2f–3f clinical presentation of, 125, 126f–127f ERE (estrogen response element), 164b
sugar of, 3b epidemiology of, 125 Erlenmeyer flask deformity, 140f
template strand of, 4–5, 5f gene therapy in, 230t Erythroblastosis fetalis, 97, 97f
 244 Index

Erythrocyte hemoglobin defect(s), 102–110, α-Fetoprotein (AFP), e3 G1 phase, 14, 14b, 65–66, 66f
103f–105f FGFR3 (fibroblast growth factor receptor-3) G2 phase, 14, 14b, 65–66, 66f
hemoglobinopathies as, 102–104, 103f gene, 124–125 G proteins, 69, 69b, 72f, 135b
sickle cell anemia as, 104–107, mutation in, e1 GA (glutaric acidemia), 63t
104f–105f, 105t–106t, 106b, 107f. FGFR (fibroblast growth factor receptor) GABA (γ-aminobutyric acid), 154b
See also Sickle cell anemia gene, 123, 124f–125f GAD1 (glutamic acid decarboxylase 1)
thalassemias as, 107–110, 108f, 109t FH. See Familial hypercholesterolemia (FH) gene, 154
Erythrocyte membrane, 97 Fibrillin, 119–120, e8 GADD45 (growth arrest and DNA damage
defects in, 98–100, 98f–99f, 99t–100t Fibroblast growth factor receptor-3 45), 85–86, 87f
lipids of, 97b (FGFR3) gene, 124–125, e1 gag gene, 69b
Erythrocyte metabolic defects, 100–102, Fibroblast growth factor receptors (FGFRs), GAGs (glycosaminoglycans), 142b
100b, 102t 123, 124f excess accumulation of, 142–144, 143f,
Erythromycin, in sickle cell anemia, 105b Fibrous plaque, 161b 143t–144t
Erythropoietin (EPO), bone marrow First polar body, 17 Gain-of-function mutation, 29t, 35, 70. See
hypertrophy with, 109b FISH (fluorescent in situ hybridization), 14, also Oncogenes
Essential amino acids, 54b 15f, 73, 76f Galactitol, 60f, 61
Estradiol, 201b, 203f Flexner-Wintersteiner rosettes, 79b Galactocerebrosidase, 142
Estrogen response element (ERE), 164b Fluorescent in situ hybridization (FISH), 14, Galactocerebrosidase deficiency, 137f, 142
ETC (electron transport chain), 128–129 15f, 73, 76f Galactocerebroside, 142
Ethics, 228, 229t Fluorochromes, 15f Galactokinase deficiency, 61
Euploidy, 18 Fluorometric assay, 56–57 Galactose, 60–61
Exon(s), 4–5, 5f Fluorophores, 14 Galactose-1-phosphate uridyltransferase,
alternative splicing of, 52b Fluoxymesterone, 200b 61, 61f, e9–e10, e37
mutation in, 6 FMR1 (fragile-X mental retardation 1) gene, Galactosemia, 29b, 60–61, 61f, e8–e9,
Exon shuffling, 160 145 e9–e10
Expressivity, 35 FMRP (fragile-X mental retardation cataract in, 60f
Extracellular matrix (ECM), 114, 114b protein), 145b frequency of, e37
Eye, 75b FMTC (familial medullary thyroid treatment of, 64t
ciliary body of, 120b carcinoma), 183 α-Galactosidase
Folate deficiency, 43b deficiency of, 138–139
F Folic acid, 43b recombinant, 139
Fabry disease, 64t, 137f, 138–139, 139f, Folic acid deficiency, 60 Galactosphingosine, 142b
e23 Follicle-stimulating hormone (FSH), 17b α1,3-Galactosyltransferase, 32b
Factor V Leiden, 112, 112t, e16 FOS oncogene, 71t Gamete, formation of, 17
Factor VIII deficiency. See Hemophilia A Founder effect, 83, 85f, 181, 182f, 191, e4 Gametogenesis, 17
Factor IX deficiency. See Hemophilia B FOXD1 gene, 178t γ-Aminobutyric acid, 154b
Familial adenomatous polyposis (FAP), 76t, Fractures, in osteogenesis imperfecta, 118, Gangliosides, 136–137, 138b
80–81, 80f e17 GAP (GTPase activating protein), 69,
Familial Alzheimer disease, 148–149 Fragile-X mental retardation 1 (FFMR1) 79–80
Familial hypercholesterolemia (FH), gene, 145 Gap 1 (G1) phase, 14, 14b, 65–66, 66f
159–164, 159t, 175–176, e25 Fragile-X mental retardation protein Gap 2 (G2) phase, 14, 14b, 65–66, 66f
gene therapy in, 230t (FMRP), 145b Garrod, Archibald, 51–52
genetics of, 161 Fragile X syndrome, 26, 29b, 35, 36t, Gastric cancer, 96
heterozygous, 159t, 161–162, 162f–163f 145–146, e24 Gastroenteritis, infantile, 169
homozygous, 162–163 clinical features of, 145–146, 146f, 146t Gastrointestinal disorders, 181–183. See
incidence of, 162t DNA diagnostics for, 226 also Hirschsprung disease
LDL metabolism in, 160 premutation in, 36 Gastrointestinal stromal tumor (GIST),
LDL receptor in, 160–161, 161f Southern blotting in, 226 e11
treatment of, 64t, 163–164 transmission of, 37 Gatekeeper genes, 74
Familial hypertriglyceridemia, 159t Fragile X–associated tremor/ataxia Gaucher cells, 137b, 140f
Familial incontinentia pigmenti, 50 syndrome (FXTAS), 36 Gaucher disease, 137f, 139–141, 139b,
Familial medullary thyroid carcinoma Frameshift mutation, 6–7, e1 140f, e22–e23
(FMTC), 183 Friedreich ataxia, 29b, 36t enzyme replacement in, 141
Family relationships, shared genes and, 42, Frontal bossing, 122, 123f, e15 gene therapy in, 230t
42t, 43f Frontal lobe, in schizophrenia, 153f type 1, 64t, 140–141, 140f, 140t
Family studies, in diabetes mellitus, 45, Fructosemia, 64t type 2, 140–141, 140t
46t FSH (follicle-stimulating hormone), 17b type 3, 140–141, 140t
Famine, 43 Fucosyltransferase, 32b, 94–96 Gaze palsy, supranuclear, vertical, 141b
Fanconi anemia, gene therapy in, 230t Fucosyltransferase 1 (FUT1) gene, 94 GBA gene, 141
Fanconi syndrome, 58b Fucosyltransferase 2 (FUT2) gene, 94 GC box, 5
FAP (familial adenomatous polyposis), 76t, Fusion gene, 25, e3 GCK (glucokinase) gene, 49, 49t
80–81, 80f FUT1 gene, 94 GDNF (glial cell line–derived neurotrophic
Favism, 102 FUT2 gene, 94 factor), 182–183, 183f, 183t
FBN1 gene, 119–122 FXTAS (fragile X–associated tremor/ataxia GDNF (glial cell line–derived neurotrophic
Ferritin, 184, 184b syndrome), 36 factor) gene, 178t, 183t
Ferroportin 1, 184–185 GDP (guanosine diphosphate), 69, 69b, 72f,
Ferrous iron (Fe++), 105b G 114b
Fetal hemoglobin (Hb F), 103, 103f, 106 G (guanine), 2b, 3f GEF (guanine nucleotide exchange factor),
malaria and, 101b G-banding, 12–14, 13f 69
in β-thalassemia, 109 G0 phase, 65–66 Gel electrophoresis, 219–220, 221f, 224
 Index 245

Gender GIST (gastrointestinal stromal tumor), e11 Guanine nucleotide exchange factor (GEF),
multifactorial inheritance and, 42–43 GLA gene, 138 69
X-linked inheritance and, 33–34 Glaucoma, e25 Guthrie test, 56–58, 57b
Gender development disorder(s), 192–201 Gleevec (imatinib mesylate), 73, 73b GVH (graft-versus-host) reaction, 141b
Klinefelter syndrome as, 23, 176, Glial cell line–derived neurotrophic factor
198–200, 198b, 199f–200f, e32 (GDNF), 182–183, 183f, 183t H
triple-X female as, 201 Glial cell line–derived neurotrophic factor H antigen, 32b, 94–96, 94t, 95f, 113
Turner syndrome as, 19–20, 23, 196–198, (GDNF) gene, 178t, 183t H-RAS oncogene, 71t
197b, e31–e32, e33 Globin, 32b, 102–103. See also Hairpin structure, 35b
XYY syndrome as, 200 Hemoglobin (Hb) HAMP gene, 184t
Gender differentiation, 192–201, 194f Globin genes, 103, 103f Haploid cell, 16–17, 16f
Gene(s). See also Chromosome(s) deletions of, 107–108, 108f, 110, e15 Haploinsufficiency, 197
cancer. See Oncogenes mutations in, 105–110, 105f, 106t, 107f, Haplotype, 98
constitutively expressed, 52 225f Hardy, Godfrey H., 209
differentially expressed, 52, 53t nomenclature for, 103 Hardy-Weinberg equilibrium/theorem,
enhancer of, 5 Globoid cell, 142, 142b 209–212
exon of, 4–6, 5f Globoid cell leukodystrophy, 142 application of, 210, 211t, 216
intron of, 4–5, 5f Globotriaosylceramide, 138 Hb. See Hemoglobin (Hb)
late-acting, 35 Glucocerebrosidase defect, 137f, 139–141, HBA1 gene, 103
nontranslated, 4 139b, 140f, e22–e23 HBA2 gene, 103
organization of, 4–5 Glucocerebrosides, 139 hCG (human chorionic gonadotropin),
promoter of, 5, 5f Glucocorticoids, 205b 195b, e5
RNA-specifying, 4 hypercholesterolemia with, 161 Hcy (homocysteine), 58, 59b–60b
structural, 4 Glucokinase, 49 HD. See Huntington disease (HD)
tissue-specific, 52, 53t Glucokinase (GCK) gene, 49, 49t HE (hereditary elliptocytosis), 98, 98f–99f,
triplet repeats of, 35–37, 36t, 37f Glucose 100t
unexpressed sequences of, 4–5, 5f formation of, 60–61, 61f Health Insurance Portability and
variability in, 4, 6–8, 28. See also in maturity-onset diabetes of the young, Accountability Act (HIPAA) Privacy
Mutation(s) 49, 49f Rule, 220b, e39
Gene amplification, 73–74, 75f Glucose-6-phosphate dehydrogenase Hearing loss, 117b
Gene chips, 236–237 (G6PD), 100 in long QT syndrome, 164
Gene expression gene for, 101 Height
constitutive, 52, 53t oxidative stress and, 101b, 101f in achondroplasia, 122
differential, 52, 53t Glucose-6-phosphate dehydrogenase distribution of, 41, 41f
variable, 53, e1 (G6PD) deficiency, 29b, 100–102, 102t, in Marfan syndrome, 120–121
Gene expression profiling, 91f, 92, 235–236 e14 positive assortative mating and, e36
Gene pool, 210, 210b favism and, 102 Heinz bodies, 100b, e14, e25
Gene silencing, 232, 233f primaquine sensitivity and, 101–102 Helix-loop-helix motif, 193b
Gene therapy, 229–232, 230t sex-linked inheritance of, 102 Helix-turn-helix motif, 193b
in α1-antitrypsin deficiency, 174, 174t Glutamic acid decarboxylase 1 (GAD1) Hematologic disorder(s), 93–113
in cystic fibrosis, 171–172 gene, 154 bleeding disorders as, 110–112, 110t
in familial hypercholesterolemia, 164 Glutaric acidemia (GA), 63t hemolytic anemia as, 98–102
nonviral vectors in, 172, 172b Glycine, 116b erythrocyte hemoglobin defects and,
viral vectors in, 171b, 230–231, 231t, Glycogen synthase kinase (GSK), 81b 102–110, 103f–105f
233t, e40 Glycophorins, 97 erythrocyte membrane defects and,
Gene transfer procedures, 230–232, 231t, Glycosaminoglycans (GAGs), 142b 98–100, 98f–99f, 99t–100t
233t excess accumulation of, 142–144, 143f, erythrocyte metabolic defects and,
Genetic anticipation, 37 143t–144t, e23 100–102, 100b, 102t
Genetic change, 6–8, 7b–8b, 7f Glycosphingolipids, 136–137, 138b glucose-6-phosphate dehydrogenase
Genetic code, 6b Glycosyltransferases, 32b deficiency as, 100–102, 101b, 101f,
Genetic counseling, 228 GM2 gangliosides, 137–138, 137f 102t
Genetic screening, 227–228 Goiter, familial, treatment of, 64t hemoglobinopathies as, 102–104, 103f
carrier, 227–228 Gonadal differentiation, 192–201, 194f hereditary elliptocytosis as, 98,
newborn, 227 Gottosman, Irving, 147 98f–99f, 100t
vs. testing, 227 Gowers maneuver, 126f hereditary pyropoikilocytosis as, 98f,
Genetic testing, 226–228 G6PD. See Glucose-6-phosphate 99–100, 100t
fundamentals of, 226–227, 226t dehydrogenase (G6PD) hereditary spherocytosis as, 98,
prenatal, 228 Graft-versus-host (GVH) reaction, 141b 98f–99f, 99t–100t
vs. screening, 227 Granulosa cells, 201b sickle cell anemia as, 104–107,
Genital ducts, 202b GRFA1 gene, 182 104f–105f, 105t–106t, 106b, 107f.
Genitalia, ambiguous, 207–208, 207f Growth arrest and DNA damage 45 See also Sickle cell anemia
Genomic imprinting, 29b, 37–39, 38f–39f (GADD45), 85–86, 87f thalassemias as, 107–110, 108f, 109t
Genotype, 28 Growth factor receptor defect, 122–125, hemophilia as, 110–112, 110t
in autosomal recessive inheritance, 30, 123f–125f thrombophilia as, 112
31t GSK (glycogen synthase kinase), 81b Heme, 32b, 105b, 185b
expressivity of, 35 GSPD gene, 101–102 Hemizygosity, 32–33
Genu varum, 123f, 195b GTP (guanosine triphosphate), 69, 69b, 72f, Hemochromatosis, 176, 183–187, e30
Germ-line mosaicism, 40, 40f 114b gender and, 187
Gestational diabetes, 49–50, e5 GTPase activating protein (GAP), 69, 79–80 genetics of, 184, 184t
Giemsa staining, 12–14, 13f Guanine (G), 1–2, 2b, 3f hereditary, 185, 187f
 246 Index

Hemochromatosis (Continued) Hepatic disorder(s), 183–190 HJV (hemojuvelin) gene, 184, 184t
incidence of, 162t hemochromatosis as, 64t, 162t, 183–187, HLAs (human leukocyte antigens), 46b
secondary, e28 184t, 187f, e30 in diabetes mellitus, 45–46
treatment of, 64t Menkes syndrome as, 64t, 187–188, HMG-CoA (3-hydroxy-3-methylglutaryl-
types of, 184t 189t, e28 CoA) lyase deficiency, 63t
Hemoglobin (Hb), 32b, 103–104 Wilson disease as, 64t, 188–190, 189t, HMP (hexose monophosphate) shunt, 100,
defects in, 102–110 190f, e28, e28–e29, e29 100b
hemoglobinopathies as, 102–104, 104f Hepatic nuclear factor-1α (HNF-1α), 49, HNF-1α (hepatic nuclear factor-1α), 49,
sickle cell anemia as, 104–107, 104f, 49f 49f
105t, e15. See also Sickle cell Hepatorenal tyrosinemia, 58 HNF1A gene, 49, 49t
anemia Hepcidin, 184–185, 186f, e27, e30 HNPCC (hereditary nonpolyposis
thalassemia as, 107–110, 108f, 109t Hephaestin, 184 colorectal cancer), 80–81, 80f, 82f,
classification of, 109t Hereditary elliptocytosis (HE), 98, 98f–99f, e12–e13, e12
identification of, 107, 107b, 107f 100t adenine glycosylase mutation in, 11
oxygen binding to, 105b Hereditary hemochromatosis (HH), 185, mismatch repair in, 10
Hemoglobin A (Hb A), 32, 32b, 103, 103f, 187f ovarian cancer and, 85
105f, 106t Hereditary nonpolyposis colorectal cancer hnRNA (heterogeneous nuclear RNA), 5
Hemoglobin AS (Hb AS), 101b, 106, 106t, (HNPCC), 80–81, 80f, 82f, e12–e13, Hodgkin lymphoma, 71b
211b e12 Homocysteine (Hcy), 58, 59b–60b
Hemoglobin (Hb) Barts, 107–108 adenine glycosylase in, 11 Homocysteine-cysteine, 59b
Hemoglobin C (Hb C) disease, 104f–105f, mismatch repair in, 10 Homocystine, 59b
105–106, 106t ovarian cancer and, 85 Homocystinuria, 58–60, 59f, e8
Hemoglobin F (Hb F), 103, 103f, 106 Hereditary pyropoikilocytosis (HP), 98f–99f, clinical manifestations of, 59
malaria and, 101b 99–100, 100t forms of, 59
in β-thalassemia, 109 Hereditary spherocytosis (HS), 98, 98f–99f, genetics of, 60
Hemoglobin (Hb) Gower 1, 103, 103f 99t–100t, e14 Homogeneously staining regions (HSRs),
Hemoglobin (Hb) Gower 2, 103, 103f Herpes simplex virus, 231t 73–74, 75f–76f
Hemoglobin H (Hb H) disease, 108, 109t Heterodisomy, 39 Homogentisic acid, 51–52, 51f
Hemoglobin (Hb) Portland, 103, 103f Heterogeneous nuclear RNA (hnRNA), 5 Homoplasmy, 40–41
Hemoglobin S (Hb S), 32, 32b, 103, Heteroplasmy, 40–41, 129–130, e18 Homozygosity, 28
105–106, 105f, 106t. See also Sickle Heterozygosity, 28 Homozygotes, frequency of, 211t
cell anemia loss of, 77–78, 78t Homozygous dominant expression, 28
Hemoglobinopathy, 102–104, 104f in colorectal cancer, 80 Homozygous recessive expression, 28,
Hemojuvelin (HJV) gene, 184, 184t in retinoblastoma, 77–78 30
Hemolysis, 98b Heterozygote(s) HPC1 gene, 76t, 78t, 87–88
erythropoietin secretion and, 109b compound, 161–162 HPC2 gene, 87–88
Hemolytic anemia, 98–102 frequency of, 210–211, 211t HPLC (high-performance liquid
erythrocyte hemoglobin defects and, screening of, 227–228 chromatography), 107b
102–110, 103f–105f significance of, 211 HPP (hereditary pyropoikilocytosis),
erythrocyte membrane defects and, Heterozygote advantage, 169 98f–99f, 99–100, 100t
98–100, 98f–99f, 99t–100t Heterozygous carrier, 31 HPRT (hypoxanthine-guanine
erythrocyte metabolic defects and, genetic screening for, 227–228 phosphoribosyltransferase), 136b,
100–102, 100b, 102t Hexosaminidase A, 138, 138f 136f
fava bean–related, 102 Hexosaminidase A deficiency, 137–138, HPRT (hypoxanthine-guanine
glucose-6-phosphate dehydrogenase 137f, 158 phosphoribosyltransferase) deficiency,
deficiency as, 100–102, 101b, 101f, Hexosaminidase B deficiency, 137–138, 29b, 135–136, 136f, e23
102t 137f, e22 HPRT1 gene, 135–136
hemoglobinopathies as, 102–104, 103f Hexose monophosphate (HMP) shunt, 100, HS (hereditary spherocytosis), 98, 98f–99f,
hereditary elliptocytosis as, 98, 98f–99f, 100b 99t–100t, e14
100t HFE gene, 184–185, 184t, 187, e30 3β-HSD (3β−hydroxysteroid dehydrogenase
hereditary pyropoikilocytosis as, 98f, HFE protein, 185, 186f deficiency), 206
99–100, 100t HH (hereditary hemochromatosis), 185, 17β-HSD (17β−hydroxysteroid
hereditary spherocytosis as, 98, 98f–99f, 187f dehydrogenase deficiency), 202–204,
99t–100t High-performance liquid chromatography 203f
primaquine-induced, 101–102 (HPLC), 107b HSD3B1 gene, 206
sickle cell anemia as, 104–107, 104f– Highly repetitive DNA, 3–4, 6 HSD3B2 gene, 206
105f, 105t–106t, 106b, 107f. See HIPAA (Health Insurance Portability and HSRs (homogeneously staining regions),
also Sickle cell anemia Accountability Act) Privacy Rule, 220b, 73–74, 75f–76f
thalassemias as, 107–110, 108f, 109t e39 HST oncogene, 71t
Hemolytic disease of the newborn, 96–97, Hippocampus, in schizophrenia, 153f hTER, 88, 89b, 90f, 90t
113 Hirschsprung disease, 177, 181–183, hTERT, 88, 89b, 90f, 90t
Hemophilia A, 29b, 33, 33f, 64t, 110–111, e27 HTT gene, 146–147
110t clinical manifestations of, 182 HTT protein, 147
Hemophilia B, 29b, 64t, 110–111, 110t epidemiology of, 182 Human chorionic gonadotropin (hCG),
gene therapy in, 230t genetics of, 182–183, 183f, 183t 195b, e5
Hemorrhagic stroke, 160b long-segment, 182, 182f Human-derived products, risks of, 175b
Heparan, 143t pathophysiology of, 181b Human Genome Project, 217, 234
Heparan N-sulfatase deficiency, 143f, 143t, short-segment, 182 Human leukocyte antigens (HLAs), 46b
144 Histiocytes, 137b in diabetes mellitus, 45–46
Heparan sulfate, 142, 143f, 143t Histones, 1 Hunter syndrome, 143–144, 143t, e22–e23
 Index 247

Huntingtin, 147 I Inheritance (Continued)

Huntington disease (HD), 36t, 146–147, ICD (implantable cardioverter-defibrillator), X-linked dominant, 34, 34f
147f, e20, e21–e22 in long QT syndrome, 167 X-linked recessive, 32–34, 33f
genetic anticipation in, 37 IDDM. See Insulin-dependent diabetes multifactorial, 28, 41, 41b
incidence of, 162t mellitus (IDDM) in diabetes mellitus, 44, 46t. See also
late-acting gene in, 35 IDL (intermediate-density lipoprotein), Diabetes mellitus
Hurler-Scheie syndrome, 142–143, 159t environmental factors in, 43–44
143t–144t IDUA gene, 142–143 epigenetic factors in, 43–44
Hurler syndrome, 142–143, 143t–144t Iduronate sulfatase deficiency, 143–144, gender and, 42–43
Hydatidiform molar pregnancy, e4–e5, 143f, 143t, e23 liability in, 42, 42t, 43f
e5 Iduronidase, 142–143 phenotypic distribution in, 41, 41f–42f
Hydrogen bond, 1 α-L-Iduronidase deficiency, 142–143, 143f, relatives’ risk in, 42, 43f
Hydrops fetalis, 108, 109t 143t nonmendelian, 29b, 35–41
3-Hydroxy-3-methylglutaryl-CoA IEF (isoelectric focusing), 107b genomic imprinting in, 37–39, 38f–39f
(HMG-CoA) lyase deficiency, 63t IGFBP3 gene, 85–86 mitochondrial, 40–41, 40f
Hydroxyl groups, 2, 3f Imatinib mesylate, 73, 73b mosaicism in, 39–40, 40f
11β-Hydroxylase deficiency, 205f, 206b, Immunoglobulin class switching, 52b triplet repeats in, 35–37, 36t, 37f
207–208, e34 Immunoglobulin genes, 70–71 unifactorial, 28
17α-Hydroxylase deficiency, 203f, 204 Implantable cardioverter-defibrillator (ICD), Initiator (inr), 5
21-Hydroxylase deficiency, 205f, 206–207, in long QT syndrome, 167 INK4A/ARF inhibitors, 66f, 67–68, 68b
206b, 207f, 207t, e34–e35 In situ breast cancer, 82b INK4a gene, 76t, 78t
p-Hydroxyphenylpyruvic acid oxidase, In vitro fertilization (IVF), 228 Insulin, 44b
57–58 Inactivation, skewed, 33–34 injection of, 44b
17-Hydroxyprogesterone, e35 Inactive-X hypothesis, 23 Insulin-dependent diabetes mellitus
Hydroxyproline, 115b Inborn errors of metabolism, 51–64 (IDDM), 44–45
3β-Hydroxysteroid dehydrogenase, 201b, aminoacidopathies as, 54–60 autoimmunity in, 46–47, 48f
205f, e35 biotinidase deficiency as, 62–63, e10 familial, 45, 46t
deficiency of, 206 cystinuria as, 180–181, 181t, 182f HLA studies in, 46
17β-Hydroxysteroid dehydrogenase, 202 galactosemia as, 60–61, 60f–61f, 64t, molecular mimicry in, 47
isozymes of, 203, 203f e8–e9, e9–e10 Insulin-producing β-cells, 44b
Hyperaminoaciduria, 58b historical perspective on, 51–52 destruction of, 47, 48f
Hyperargininemia, 58b homocystinuria as, 58–60, 59f, e8 Insulin sensitizers, 44b
Hyperbilirubinemia, 102 hyperhomocystinuria as, 58–60 Integrins, 114b
Hypercholesterolemia hyperphenylalaninemia as, 54–57, 55f, Integrity, 229t
familial, 159–164, 159t, 175–176, 56t–57t, e8 Intercalating agents, 7, 8b
e25 Lesch-Nyhan syndrome as, 29b, 135–136, Intermediate-density lipoprotein (IDL),
gene therapy in, 230t 136f, e23 159t
genetics of, 161 maple syrup urine disease as, 61–62, 62f, Interphase, 14, 65–66
heterozygous, 159t, 161–162, 63t–64t Intron, 4–5, 5f
162f–163f model of, 52–53, 52f Inversion mutation, 7, 7f, 26, 26f, e1
homozygous, 162–163 newborn screening for, 53–54, 54t, e8 Ionizing radiation, 7–8, 8b
LDL metabolism in, 160 organic acidemias as, 61–63, 62f IPF1 gene, 49t
LDL receptor in, 160–161, 161f treatment of, 63–64, 64t IRE (iron response element), 184b
treatment of, 163–164 tyrosinemia as, 57–58, 64t Iris, Brushfield spots of, 18–19, 19f, e2
incidence of, 162t Inbreeding, 212–214, 212t, 213f Iron
treatment of, 64t Incontinentia pigmenti, 50 absorption of, 185, 185b, 186f
Hyperelasticity, in Ehlers-Danlos syndrome, Individualized medicine, 232–234 metabolism of, 183–184, 184t
119, 119f Infantile gastroenteritis, 169 overload of, 110, 184, 187b. See also
Hyperglycemia, maternal, 50 Infarction Hemochromatosis
Hyperhomocystinuria, 58–60 cerebral, 160b transport of, 188
Hypermobility myocardial, 160b, 161 Iron regulator protein (IRP), 184b
in Ehlers-Danlos syndrome, 119, 120t Infection Iron response element (IRE), 184b
in Marfan syndrome, 121f in cystic fibrosis, 168b IRP (iron regulator protein), 184b
Hyperphenylalaninemia, 54–57, 55f, with human-derived products, 175b Ischemic stroke, 160b
56t–57t, e8 in sickle cell anemia, 105b Isodisomy, 39
Hypertriglyceridemia, 159t in type 1 diabetes mellitus, 46–47 Isoelectric focusing (IEF), 107b
Hypogonadotropic hypogonadism, 194f, Infertility, in cystic fibrosis, 168, 176 Isoleucine, 61, 62f
195 Inflammation, macrophages and, 174b Isovaleric acidemia, 63t
Hypomethioninemia, 60 Informed consent, 219b, e39 IVF (in vitro fertilization), 228
Hypophosphatemic rickets, 29b Infundibulopelvic ligament, 85b
Hypospadias, 202b J
Inheritance
Hypothyroidism mendelian, 28–35, 29b, 29t Jervell and Lange-Nielsen syndrome, 164,
atherosclerosis risk in, 161 autosomal dominant, 28–30, 29b, 30f 165t, 166–167
bipolar disorder and, 157t autosomal recessive, 30–32, 31f, 31t Joint hypermobility, 119, 121f
Hypoxanthine-guanine codominant, 32 Joubert syndrome, 181b
phosphoribosyltransferase (HPRT), consanguinity and, 32, 212–214, 212t, Justice, 229t
136b, 136f 213f
Hypoxanthine-guanine K
expressivity in, 35
phosphoribosyltransferase (HPRT) late-acting genes in, 35 K-RAS oncogene, 71t
deficiency, 29b, 135–136, 136f, e23 penetrance in, 34–35, 34f Kallmann syndrome, 176, e31–e32
 248 Index

Karyotype, 12–14, 13f, 15f Lesch-Nyhan syndrome, 29b, 135–136, Loss of heterozygosity (LOH) (Continued)
for Down syndrome, 18, 18f, 26–27 136f, e23 in familial adenomatous polyposis, 81
Karyotyping, spectral, 14, 15f Leucine, 61, 62f in neurofibromatosis type 1, 80
Kayser-Fleischer ring, 190, 190f, e29 Leucine-rich repeat, 151t Lovastatin, 163b
KCNE1 gene, 165t, 166–167 Leucine zipper motif, 193b Low-density lipoprotein (LDL), 159t
KCNE2 gene, 165t, 166–167 Leukemia, 69b metabolism of, 160–161, 161f
KCNH2 gene, 165t, 166 myeloid (myelogenous), chronic. See Low-density lipoprotein (LDL) receptor,
KCNJ2 gene, 165t, 167 Chronic myeloid (myelogenous) 160–161, 160f–161f, 175
KCNQ1 gene, 165t, 166–167, 176 leukemia (CML) Low-density lipoprotein receptor (LDLR)
Kearns-Sayre syndrome, 130b, 131–132, Leukocoria, 74, 77f gene, 160, 160f
e18 Leukodystrophy, globoid cell, 142 mutations in, 161, 162f
Kell antigen, 94t Lewy bodies, 150, 150b, e22 therapeutic manipulation of, 164
Kennedy disease, 29t, 36t Leydig cells, 199f, 201b LQT. See Long QT syndrome
Kernicterus, 102, 102b LHON (Leber hereditary optic neuropathy), Lumpectomy, 82b
α-Ketoacid decarboxylase, 61, 62f 29b, 41, 130 Lung transplantation, in cystic fibrosis, 171
Ketothiolase deficiency, 63t Li-Fraumeni syndrome, 76t, 85–86, 88f, 92, Lung volume reduction surgery, 175b
Kidney(s) e12 Lutheran antigen, 94t
agenesis of, 178, 178b, 178t, 179f Liability, 42, 43f Lymphocytes, 47b
disorders of, 177–181, 177b–178b Limb-girdle muscular dystrophy, type 1C, Lymphoma, 69b
duplication of, 177b 165t Burkitt, 70–71, 72f
ectopic, 177b Limbic system, in schizophrenia, 153f Hodgkin, 71b
horseshoe, 177b Lipids, membrane, 97b non-Hodgkin, 71b
malrotated, 177b Lipoprotein(s), 159b Lynch I syndrome, e11
multicystic dysplasia of, 178–180, 179f, intermediate-density, 159t Lynch II syndrome, e11
180t, 181b low-density, 159t Lyon, Mary, 23
stones of, 180 metabolism of, 160–161, 161f Lyon hypothesis, 33
Kinetochore, 66b very-low-density, 159t Lyonization, 23, 33–34, 198, 212b, e15
Kinky hair disease, 64t, 187–188, e28 Lipoprotein lipase deficiency, 159t Lysosomal storage disease(s), 136–144,
KIR2.1, 167 Liposomes, 233t 137f
KIT, e11 Lisch nodules, 135, 135f, e2, e20–e21 Fabry disease as, 64t, 137f, 138–139,
KIT oncogene, 71t, e11 Lithium, 157, 157b 139f, e23
Klinefelter syndrome, 176, 198–200, e32 Liver disease, 183–190 Gaucher disease as, 137f, 139–141, 139b,
breast cancer and, 199–200 in α1-antitrypsin deficiency, 175, e26 140f, 140t, 230t, e22–e23
clinical features of, 198–199, 198b, 200f hemochromatosis as, 64t, 162t, 183–187, Krabbe disease as, 137f, 142
epidemiology of, 198 184t, 187f, e30 mucopolysaccharidoses as, 142–144,
history of, 23 Menkes syndrome as, 64t, 187–188, 189t, 143t–144t, e23
Leydig cells in, 199f e28 Niemann-Pick disease as, 137f, 141–142
lyonization in, 198 Wilson disease as, 64t, 188–190, 189t, Sandhoff disease as, 137–138, 137f, e22
STS in, 198–199 190f, e28, e28–e29, e29 sphingolipidoses as, 136–142, 137f
Knudson, A. G., 74–75 Liver transplantation Tay-Sachs disease as, 137–138, 137f, 158
Knudson’s two-hit hypothesis, 74–75, 78f in α1-antitrypsin deficiency, 175 Lysosomes, 136
Krabbe disease, 137f, 142 in familial hypercholesterolemia, 163 Lysyl oxidase, 187b, 188
KSS (Kearns-Sayre syndrome), 130b, in maple syrup urine disease, 64
131–132, e18 Lobular carcinoma in situ (LCIS), 82b M
Kupffer cells, 137b Locus heterogeneity, e1 M-FISH (multiplex fluorescence in situ
Kyphoscoliosis, in Ehlers-Danlos syndrome, LOH (loss of heterozygosity), 77–78, 78t hybridization), 15f
120t in breast cancer, 83 M (mitosis) phase, 14–15, 14b, 16f, 65–66,
in colorectal cancer, 80 66f
L in familial adenomatous polyposis, 81 Machado-Joseph disease, 36t
Laboratory validity, 226–227 in neurofibromatosis type 1, 80 Macro-orchidism, 145
Lambdoid suture, 120b Long QT syndrome, 164–167, 176 Macrocephaly, 122, 123f
Landsteiner, Karl, 93 acquired, 166b Macrophages, 174b
Langerhans cells, 137b Andersen, 165t, 167 Macrosomia, 50
Late-acting genes, 35 drug-induced, 166b, e25 Macula, cherry-red spot of, 138, 138f
LCIS (lobular carcinoma in situ), 82b epidemiology of, 164 MAD proteins, 66b
LDL (low-density lipoprotein), 159t founder effect in, 216 Major histocompatibility complex (MHC),
metabolism of, 160–161, 161f genetics of, 164, 165t 46
LDL (low-density lipoprotein) receptor, Jervell and Lange-Nielsen, 164, 165t, Malaria
160, 160f, 176 166–167 glucose-6-phosphate dehydrogenase
LDLR gene, 160, 160f management of, 167 deficiency and, 100
mutations in, 161, 162f mortality in, 164 Hb F and, 101b
therapeutic manipulation of, 164 Romano-Ward, 164, 165t, 166, 216 hereditary elliptocytosis and, 98
Leber hereditary optic neuropathy (LHON), torsades de pointes in, 164, 165f primaquine for, 101–102
29b, 41, 130 Long-segment disease, in Hirschsprung sickle cell trait and, 101b
Leg, thrombosis of, 112b disease, 182, 182f Malformation, 179b
Leigh syndrome, e24 Loss-of-function mutation, 29t, 35. See also Mania, 155t–157t, 156–157
Lens, dislocation of, 59 Tumor suppressor genes Manic-depressive illness, 155–157,
Lenticular nucleus, 190b Loss of heterozygosity (LOH), 77–78, 78t 155t–157t
Lentivirus, 231t in breast cancer, 83 MAO (monoamine oxidase), 187b
Leptotene, 16–17 in colorectal cancer, 80 MAO (monoamine oxidase) inhibitors, 155b
 Index 249

MAP kinase–mediated protein Metabolic disorder(s) (Continued) Mitosis, 14–15, 14b, 16f, 65–66, 66f
phosphorylation cascade, 70, 72f galactosemia as, 60–61, 60f–61f, 64t, nondisjunction in, 6, 21–22
MAPK pathway, 197–198 e8–e9, e9–e10 Mitotic spindle, 66b
MAPKKK (mitogen-activated protein kinase historical perspective on, 51–52 Mitral valve disease, in Marfan syndrome,
kinase kinase), 70 homocystinuria as, 58–60, 59f, e8 121
Maple syrup urine disease (MSUD), 61–62, hyperhomocystinuria as, 58–60 MLH1 gene, 76t, 81
62f, 63t–64t hyperphenylalaninemia as, 54–57, 55f, MLH2 gene, 76t
liver transplantation in, 64 56t–57t, e8 MM (mitochondrial myopathies), 41,
Marfan syndrome, 29b, 119–122, 120f–122f Lesch-Nyhan syndrome as, 29b, 135–136, 128–130, 129f–131f
Mastectomy, 82b 136f, e23 MMA (methylmalonic acidemia), 60, 63t
Maternal age maple syrup urine disease as, 61–62, 62f, MMR (mismatch repair), 10–11, 10f, 35
Down syndrome and, 19, 19f 63t–64t MnlI, 220t
Klinefelter syndrome and, 200 model of, 52–53, 52f MNS blood group, 94t
Mating newborn screening for, 53–54, 54t, e8 Möbius syndrome, e13
assortative, 214 organic acidemias as, 61–63, 62f Modified radical mastectomy, 82b
random, 209 treatment of, 63–64, 64t MODY (maturity-onset diabetes of young),
Maturity-onset diabetes of young (MODY), tyrosinemia as, 57–58, 64t 49, 49f, 49t, e6
49, 49f, 49t, e6 Metabolomics, 217 Molar pregnancy, e4–e5, e5
McArdle disease, e4 Metacentric chromosome, 12, 14f Molecular medicine, 217–238
Mean, 41 Metal ions, 187b allele-specific oligonucleotide testing in,
Mean corpuscular hemoglobin (MCH), 104b Metaphase, 3f, 12, 13f, 76f 225, 225f
Mean corpuscular hemoglobin of meiosis, 17 deletion detection in, 226
concentration (MCHC), 104b of mitosis, 15, 16f diagnostic paradigm for, 218–219, 218f
Mean corpuscular volume (MCV), 104b Metastasis, 69b DNA sequence analysis in, 222–226, 223f
Medical ethics, 228, 229t Methionine synthase, 58, 59f gene chip technique in, 236–237
Medullary thyroid carcinoma syndrome, Methionine synthase deficiency, 60 gene expression profiling in, 235–236
64t Methylation, DNA, 37–38, 38b, 145b gene replacement technique in, 232
Megacolon, aganglionic. See Hirschsprung 3-Methylcrotonyl-CoA carboxylase, 62 gene silencing technique in, 232, 233f
disease deficiency of, 62–63, 63t gene therapy in, 229–232, 230t
Meiosis, 16–17, 16f 5,10-Methylenetetrahydrofolate reductase, gene transfer procedures in, 230–232,
nondisjunction in, 6, 19–21, 20f 58, 59f 231t, 233t
recombination in, 7, 16–17, 16f deficiency of, 60 genetic counseling in, 228
MEK, 72f Methylmalonic acidemia (MMA), 60, 63t genetic testing in, 226–228, 226t
Melanocytes, 58b Methyltestosterone, 200b individualized, 232–234, 234f–236f
Melanoma, 9b, 76t Metopic suture, 120b microarray analysis in, 234–237, 235f
Melanosomes, 58b MHC (major histocompatibility complex), mutation detection in, 224–226, 225f,
MELAS (mitochondrial encephalomyopathy 46 236–237, 236f
with lactic acidosis and stroke-like Microarray analysis, 91f, 92 nucleic acid visualization in, 219
episodes), 29b, e18 Microdeletion syndromes, 7, 7b, 25, 26t pharmacogenomics in, 232–234
Membrane, erythrocyte, 97 Microfibrils, 115, 116f, 119–120 point mutation detection in, 224–225,
defects in, 98–100, 98f–99f, 99t–100t Microsatellite instability, 81 225f
lipids of, 97b Microtubules, 15b, 66b polymerase chain reaction in, 221–222
Membrane potential, 128 Middle repetitive DNA, 3–4 prenatal genetic testing in, 228, 228b
MEN (multiple endocrine neoplasia) type MIF (müllerian inhibitory factor), 17b, privacy rules in, 220b
II, 183 194–195, 194f, 204 recurrence risk assessment in, 228–229
Mendel, Gregor, 28 Migraine, bipolar disorder and, 157t restriction fragment length polymorphism
Mendelian inheritance, 28–35, 29b, 29t Miller-Dieker syndrome, 26t in, 219–221, 221f, 226t
autosomal dominant, 28–30, 29b, 30f Mismatch repair (MMR), 10–11, 10f, 35 Southern blot analysis in, 219–221, 220t,
autosomal recessive, 30–32, 31f, 31t Mismatch repair genes, e12 221f
codominant, 32 Missense mutation, 6 tools of, 217–226
consanguinity and, 32 Mitochondrial disease, 41, 128–130, 131f, trinucleotide repeat expansion detection
expressivity in, 35 e18 in, 226
late-acting genes in, 35 chronic progressive external Molecular mimicry, in diabetes mellitus, 47
penetrance in, 34–35, 34f ophthalmoplegia as, 131–132 Monoamine oxidase (MAO), 187b
X-linked dominant, 34, 34f Kearns-Sayre syndrome as, 131–132, Monoamine oxidase (MAO) inhibitors, 155b
X-linked recessive, 32–34, 33f e18 Monosomy, 6, 18–21, 20f
Menkes syndrome, 64t, 187–188, 189t, e28 myoclonic epilepsy with ragged red partial, 6–7
Merlin gene, 135 fiber syndrome as, 131, 131f Monozygotic (MZ) twins, 45b, 147–148
MERRF (myoclonic epilepsy with ragged ragged red fibers in, 130, 130f Morphine, 232b
red fibers), 29b, 131, 131f, e18, e21 treatment of, 132 Mosaicism, 21–22, 27, 39–40, 40f
Mesonephric ducts, 202b Mitochondrial DNA (mtDNA), 40–41, anaphase lag and, 21–22
Mesovarium, 17b, 85b 128–130, 129f, e21 chromosomal, 39–40, 200
Messenger RNA (mRNA), 5, 5f Mitochondrial encephalomyopathies, 130 in Down syndrome, 21–22
MET oncogene, 71t Mitochondrial encephalomyopathy with germ-line, 40, 40f
Meta-analysis, 156 lactic acidosis and stroke-like episodes nondisjunction and, 21–22
Metabolic acidosis, 62b (MELAS), 29b, e18 MPS (mucopolysaccharidoses), 64t,
Metabolic blockade, model of, 52–53, 52f Mitochondrial inheritance, 40–41, 40f, e4 142–144, 143t–144t, e23
Metabolic disorder(s), 51–64 Mitochondrial myopathies (MM), 128–130, MRD (multicystic renal dysplasia), 178–180,
aminoacidopathies as, 54–60 129f–131f 179f, 180t, 181b
biotinidase deficiency as, 62–63, e10 ocular, 130b, 131–132 mRNA (messenger RNA), 5, 5f
 250 Index

MSH2 gene, 76t, 81 Musculoskeletal disorders (Continued) Neurofibromatosis type 1 (NF1), 29b,
MSH6 gene, 76t, 81 Kearns-Sayre syndrome as, 130b, 79–80, 79b, 133–135
MSR1 gene, 76t, 78t, 87–88 131–132 café-au-lait spots in, 35
MSUD (maple syrup urine disease), 61–62, mitochondrial myopathies as, 128–130, genetics of, 135
62f, 63t–64t 129f–130f, 130b incidence of, 133
liver transplantation in, 64 muscular dystrophies as, 125, Lisch nodules in, 135, 135f
mtDNA (mitochondrial DNA), 40–41, 126f–127f neurofibromas in, 79–80, 79f, 133–135,
128–130, 129f, e21 myoclonic epilepsy with ragged red 135f
MTHFR (5,10-methylenetetrahydrofolate fiber syndrome as, 131, 131f vs. neurofibromatosis type 2, 134t
reductase) deficiency, 60 Mutagens, 7–8, 8b Neurofibromatosis type 2 (NF2), 29b, 135,
Mucopolysaccharidoses (MPS), 29b, 64t, Mutation(s), 4, 6, 28 e21
142–144, e23 in cancer, 68, 70f café-au-lait spots in, 35, 133, 134f
dysostosis multiplex with, 144b de novo, 7, 40 vs. neurofibromatosis type 1, 134t
type I, 142–143, 143f, 143t–144t detection of, 224–226, 225f vestibular schwannoma in, 135, 136f
type II, 143–144, 143f, 143t–144t frameshift, 6–7, e1 Neurofibromin gene, 79–80, 135
type III, 143f–144f, 143t, 144, 158 gain-of-function, 29t, 35, 70. See also Neurologic disease(s), 133–158
Müllerian inhibitory factor (MIF), 17b, Oncogenes complex, 147–157
194–195, 194f, 204 gene chip detection of, 236–237 Alzheimer disease as, 148–150, 158
Multicystic renal dysplasia (MRD), inversion, 7, 7f, 26, 26f, e1 behavioral genetics in, 147
178–180, 179f, 181b loss-of-function, 29t, 35. See also Tumor bipolar disorders as, 155–157,
bilateral, 179–180 suppressor genes 155t–156t
familial, 180 in metabolic disorders, 52–53, 52f Parkinson disease as, 150–151, 150f,
malformations with, 180t missense, 6 151t, e20
unilateral, 179–180 in mitochondrial disease, 41 schizophrenia as, 151–155, 151t,
Multifactorial inheritance, 28, 41, 41b nonsense, 6 152f–153f
characteristics of, 44 point, 6, 224–225, 225f single-gene, 133–147
in diabetes mellitus, 44, 46t, e5. See also spontaneous, 7, 40 Fabry disease as, 64t, 137f, 138–139,
Diabetes mellitus (DM) transition, 6, e1 139f, e23
environmental factors in, 43–44 transversion, 6 fragile X syndrome as, 145–146, 146f,
epigenetic factors in, 43–44 triplet repeat, 35–37, 36t 146t, e24
gender and, 42–43 MYB oncogene, 71t Gaucher disease as, 137f, 139–141,
liability in, 42, 42t, 43f MYC gene, 66f, 74b, e27 139b, 140f, 140t, e22–e23
phenotypic distribution in, 41, 42f translocation of, 70–71, 72f Huntington disease as, 146–147, 147f,
relatives’ risk in, 42, 43f n-MYC oncogene, 71t e20, e21–e22
Multifactorial trait, 42 amplification of, 73–74 Krabbe disease as, 137f, 142
Multiple endocrine neoplasia (MEN) type MYC protein, 74b Lesch-Nyhan syndrome as, 135–136,
II, 183 Myelodysplastic syndrome, e13 136f, e23
Multiple polyposis of colon, 64t Myocardial infarction (MI), 160b lysosomal storage diseases as, 136–144
Multiplex fluorescence in situ hybridization in heterozygous familial mucopolysaccharidoses as, 142–144,
(M-FISH), 15f hypercholesterolemia, 161 143t–144t, e23
Muscle cell disease(s), 125–132 Myoclonic epilepsy with ragged red fibers neurofibromatosis as, 133–135,
chronic progressive external (MERRF), 29b, 131, 131f, e18, e21 134f–136f, 134t, e21
ophthalmoplegia as, 130b, 131–132 Myosin binding protein C, 175 Niemann-Pick disease as, 137f,
Kearns-Sayre syndrome as, 130b, Myotonic dystrophy, 29b, 35, 36t 141–142
131–132 MZ (monozygotic) twins, 45–46, 45b Sandhoff disease as, 137–138, 137f,
mitochondrial myopathies as, 128–130, e22
129f–130f, 130b N sphingolipidoses as, 136–142, 137f
muscular dystrophies as, 125, 126f–127f Na+-dependent norepinephrine transporter Tay-Sachs disease as, 137–138, 137f,
myoclonic epilepsy with ragged red fiber (NET), 58b 158
syndrome as, 131, 131f NAD (nicotinamide adenine dinucleotide), triplet repeats and, 144–147
Muscular dystrophy, 125 163b Neurturin (NRTN), 182–183, 183t
Becker, 125 NADP (nicotinamide adenine dinucleotide Neutrophil elastase, 172–174, e25
Duchenne, 125, 127f, 230t, e17, e36 phosphate), 163b Newborn
Musculoskeletal disorders, 114–132 NADPH (nicotinamide adenine of diabetic mother, 50
connective tissue and bone, 114–122 dinucleotide phosphate), 100, G6PD deficiency in, 102
anatomic basis for, 114, 115t, 116f 100b–101b, 101f, e14 hemolytic disease of, 96–97, 113
differential considerations in, 122, Naked DNA vaccination, 233t kernicterus in, 102b
123t Negative assortative mating, 214 Newborn screening, 53–54, 54t, e8
Ehlers-Danlos syndrome as, 119, 119f, Negative predictive value, 226–227, 226t NF. See Neurofibromatosis type 1 (NF1);
120t Nephrolithiasis, 180 Neurofibromatosis type 2 (NF2)
Marfan syndrome as, 119–122, NES (nuclear export signal), 145b NF1 gene, 76t, 79–80
121f–122f NET (Na+-dependent norepinephrine NF2 gene, 76t
osteogenesis imperfecta as, 117–118, transporter), 58b NHL (non-Hodgkin lymphoma), 71b
117t, 118f Neu (ERB-B2) oncogene, 71t Niacin, 163b
growth factor receptor defect, 122–125 Neural tube defects, 43 in familial hypercholesterolemia, 163
achondroplasia as, 122–125, 123f–125f Neuregulin 1 (NRG1) gene, 154 Nicotinamide adenine dinucleotide
muscle cell, 125–132 Neuroblastoma, 73–74, 76f phosphate (NADPH), 100, 100b–101b,
chronic progressive external Neurocristopathy, 181 101f, e14
ophthalmoplegia as, 130b, NEUROD1 gene, 49t NIDDM. See Non–insulin independent
131–132 Neurofibrillary tangles, 148–149 diabetes mellitus (NIDDM)
 Index 251

Niemann-Pick disease, 137f, 141–142 Oncogenes (Continued) p21, 66f, 67–68, 68b, 85–86, 87f
Nitrogenous base, 1 gene amplification and, 73–74, 75f p27, 66f, 67–68, 68b
NLS (nuclear localization signal), 145b translocation activation of, 70–73, p53, 66f, 68b, 85–86, 87f. See also TP53
Nociceptors, 139b 72f–73f gene
Non-Hodgkin lymphoma, 71b One gene–one enzyme thesis, 52 p57, 67–68
Nondisjunction, 6 One gene–one polypeptide thesis, 52 p arm, 12, 14f
in Down syndrome, 19–21, 20f, 25 Online Mendelian Inheritance of Man, 4 P blood group, 94t
meiotic, 6, 19–21, 20f Oocytes, 17, 201b Pachytene, 16–17
mitotic, 21–22 Oogenesis, 17 Packaging signal (psi) sequence, 231b
in penta-X syndrome, 27 Ophthalmoplegia, chronic progressive PAH (phenylalanine hydroxylase)
Nonessential amino acids, 54b external, 130b, 131–132 deficiency. See Phenylketonuria
Non–insulin independent diabetes mellitus Opioids, 232b PAH gene, 56, 236
(NIDDM), 44–45 Opitz G/BBB syndrome, 178b Pain, 139b
familial, 45 Optic neuropathy, hereditary, Leber Palindromes, 4b
obesity and, 47 (LHON), 29b, 41, 130 Pancreas, 45b
risk assessment for, 48–49 Organic acidemia(s), 61–63, 62f, 63t Pancreatic islets of Langerhans, 44b, 46–47
screening for, e38 biotinidase deficiency as, 62–63 destruction of, 47, 48f
susceptibility genes in, 47–48 maple syrup urine disease as, 61–62, 62f, Panmixia, 209
twin studies of, 45–47 63t–64t PAR (pseudoautosomal region), 193, 193f,
Nonmalfeasance, 229t Ornithine transcarbamylase (OTC) 197
Nonmendelian inheritance, 29b, 35–41 deficiency, 212b Paracentric inversion, 7, 7f, 26, 26f
genomic imprinting in, 37–39, 38f–39f Orofaciodigital syndrome, 29b Paramesonephric ducts, 202b
mitochondrial, 40–41, 40f Orotic aciduria, 64t PARK genes, 150, 151t
mosaicism in, 39–40, 40f Ossification, 123b Parkin, 151t
triplet repeats in, 35–37, 36t, 37f Osteogenesis, 115t Parkinson disease, 150–151, 150f, 151t, e20
Nonpenetrance, 34–35, 34f Osteogenesis imperfecta (OI), 117–118, Partial monosomy, 6–7
Nonpolyposis colorectal cancer. See 117t, e17, e19 Partial thromboplastin time (PTT), 111b
Hereditary nonpolyposis colorectal vs. child abuse, 118 Partial trisomy, 25
cancer (HNPCC) dentinogenesis imperfecta in, 117, 118b, Patau syndrome, 21, 21f, 22t
Nonsense mutation, 6 118f PAX3 gene, 29t, 182
Nonviral vectors, 172, 172b, 231–232, 233t hearing loss with, 117b PCR (polymerase chain reaction), 221–222,
Noonan syndrome, 29b, 197–198, e31 type I, 117, 117t, 118f 222f, 222t, e38
NOR (nucleolar-organizing region), 25b type II, 117t, 118 in sickle cell anemia, 107, 107f
NPC1 gene, 141 type III, 117t, 118 Pedigree, 28–29
NPC2 gene, 141 type IV, 117t, 118 in autosomal dominant inheritance, 29,
NRG1 (neuregulin 1) gene, 154 type V, 117t 30f
NRTN (neurturin), 182–183, 183t type VI, 117t in autosomal recessive inheritance,
NRTN (neurturin) gene, 183t type VII, 117t 30–31, 31f, e6–e7, e6f, e7
Nuclear export signal (NES), 145b wormian bones in, 118, 119f in germline mutation, 40, 40f
Nuclear localization signal (NLS), 145b Ovarian cancer, 76t, 83–85, 85f in hemophilia A, 33, 33f
Nuclear ribonucleoproteins, small (snRNPs), Ovarian follicles, 17b in Hunter syndrome, e22f
5b Ovarian ligament, 85b in Huntington disease, e20, e20f
Nucleic acid visualization, 219 Ovary (ovaries), 17b, 85b in Li-Fraumeni syndrome, 86, 88f
Nucleolar-organizing region (NOR), 25b follicle of, 201b in mitochondrial inheritance, 40, 40f
Nucleosome, 1, 2f streak, 196 in prostate cancer, 88f
Nucleotide excision repair, 8–9, 9f Ovotesticular disorder of sexual in retinoblastoma, 78f
Nucleotides, 1, 6b development, 201–202, 202b, 202f, symbols for, 30f
Null allele, 95–96, 173 e32, e32–e33 in X-linked dominant disorder, 50
Nullisomy, 20–21 Ovulation, 17 in X-linked dominant inheritance, 34, 34f
Nutrigenomics, 217 Oxidative phosphorylation (OXPHOS), in X-linked recessive inheritance, 33, 33f
128–130 Penetrance, 34–35, 34f, e1, e4
O Oxidative stress, 150b Penicillin, in sickle cell anemia, 105b
Obesity, 43 glucose-6-phosphate dehydrogenase and, Penta-X syndrome, 27
type 2 diabetes mellitus and, 47 101b, 101f Pentose phosphate pathway, 100, 100b
Occipital lobe, in schizophrenia, 153f OXPHOS (oxidative phosphorylation), Pericentric inversion, 7, 7f
Ochronosis, 52 128–130 Persephin (PSP), 182–183
OCT (ornithine transcarbamylase) Oxycephaly, 120 Peutz-Jeghers syndrome, e11
deficiency, 212b Oxygen PGD (preimplantation genetic diagnosis),
Ocular mitochondrial myopathy, 130b, hemoglobin binding of, 105b 214, 228
131–132 toxicity of, 150b Ph (Philadelphia) chromosome, 25, 72–73,
Oculocutaneous albinism, 57, e9 Oxyhemoglobin dissociation curve, 105b 72f–73f, e3, e13
Oculocutaneous tyrosinemia, 58 Oxymetholone, 200b Pharmacogenomics, 217, 232–234
Odontoblasts, 118b Phenotype, 4, 28
OI (osteogenesis imperfecta), 117–118, P in autosomal recessive inheritance, 31t
117t, 119f, e17, e19 p14ARF, 66f in codominant inheritance, 32
Oligohydramnios, 178, e27 p15, 67–68, 68b in multifactorial inheritance, 41, 41f
Oligonucleotides, allele-specific, 225, 225f p16, 67–68, 68b penetrance and, 34–35
Oncogenes, 69–74, 71t, 72f p16INK4, 66f, 68f, 76t Phenylalanine
in Burkitt lymphoma, 70–71, 72f p18, 67–68 accumulation of. See Phenylketonuria
in chronic myeloid leukemia, 72–73, 72f p19, 67–68, 68b (PKU)
 252 Index

Phenylalanine (Continued) Prenatal diagnosis, 214, 228 QT interval, 166b

metabolism of, 54–57, 55f of achondroplasia, 125, 125f long. See Long QT syndrome
testing for, 56–57 of Down syndrome, 19b, 27 Quinacrine staining, 12–14
Phenylalanine hydroxylase deficiency. See Presbycusis, 117b
Phenylketonuria (PKU) Presenilin 1 (PSEN1) gene, 149 R
Phenylketonuria (PKU), 29b, 31, 54–57, Presenilin 2 (PSEN2) gene, 149 Radiation, ionizing, 7–8, 8b
e8–e9 Primaquine sensitivity, 101–102 Radical mastectomy, 82b
carrier of, 211t, 213–214, 213f Privacy rules, 220b, e39 RAF, 70
classic, 54–57 PRL3 gene, 80f Ragged red fibers (RRFs), 130, 130f
clinical manifestations of, 57, 57t Procollagen, 115 myoclonic epilepsy with, 131, 131f, e18,
consanguinity and, 213, 213t PRODH gene, 154 e21
incidence of, 162t Prometaphase, 15 Random mating, 209
maternal, 57, 57t Promoter, 5, 5b, 5f RAS oncogene, 66f, 69, 71t
pathogenesis of, 54–56, 55f, 56t Proofreading repair, 11 in colorectal cancer, 80, 80f
treatment of, 56, 63, 64t Prophase, 15, 16f in neurofibromatosis type 1, 135
types of, 56, 56t Propionic acidemia, 63t K-RAS oncogene, 71t
Phenylpyruvic acid, 54–56, 55f Propionyl-CoA carboxylase, 62 RAS protein, 69–70, 72f
Philadelphia (Ph) chromosome, 25, 72–73, deficiency of, 62–63, 63t in neurofibromatosis, 79–80
72f–73f, e3, e13 Propionyl-CoA carboxylase deficiency, Rb. See Retinoblastoma (Rb)
Phosphodiester bond, 3b 62–63 RB1 gene, 67, 74–79, 76t, 78t
PHOX2B gene, 183t Prostate, 86b RBC. See Red blood cell (RBC)
Phytohemagglutinin, 12 Prostate cancer, 76t, 86–88, 88f Reactive oxygen species (ROS), 60b
Pima Indians, 47–48 risk factors for, 87b Reading frame, 6–7, 128
PKU. See Phenylketonuria (PKU) Prostate-specific antigen (PSA), 86b, 237 Rearrangement, balanced, 24–25
Plagiocephaly, 120 Protein(s) Receptor(s), 124b
Pleiotropic consequences, 12 mutant, 29t androgen, 204, 204b
Pleiotropy, e18 posttranslational modification of, 53b catecholamine, 58b
PMP22 gene, 29t Protein 4.2, 98, 98f, 99t dihydropyridine, 175
PMS2 gene, 81 Protein C, 112 dopamine D4, 156, e20
Point mutation, 6 Proteoglycans, 142, 142b low-density lipoprotein, 160–161,
detection of, 224–225, 225f Proteomics, 217 160f–161f, 175–176
pol gene, 69b Prothrombin time (PT), 111b opioid, 232b
Polar body, 17 Proto-oncogenes, 69 ryanodine, 175
POLH gene, 9t–10t PSA (prostate-specific antigen), 86b, 237 transferrin, 185, 186f
Polyadenosine (polyA), 5b PSEN1 gene, 149 Receptor serine/threonine kinases, 195b
Polycation conjugates, 233t PSEN2 gene, 149 Receptor tyrosine kinases, 124b, 182–183,
Polymerase chain reaction (PCR), 221–222, Pseudoautosomal region (PAR), 193, 193f, 183f
222f, 222t, e38 197 Recessive allele, 28, 29t
in sickle cell anemia, 107, 107f Pseudogenes, 103–104 Recessive inheritance
Polymorphisms, 4 PSP (persephin), 182–183 autosomal, 29t, 30–32, 31f, 31t
Polyploidy, 18 PstI, 220t characteristics of, 31–32
Polyposis, adenomatous, familial (FAP), 76t, Psychosine, 142b genotype in, 30, 31t
80–81, 80f PT (prothrombin time), 111b consanguinity and, 32, 212–214, 212t,
Population genetics, 209–216 PTEN-induced putative kinase 1, 151t 213f
assortative mating and, 214 PTEN/MMAC1 gene, 82 X-linked, 29b, 32–34, 33f
consanguinity and, 212–214, 212t–213t, PTPN11 gene, 197–198 characteristics of, 33–34
213f PTPN11 protein, 197–198 Recessive mutation, dominant effects of, 29t
DNA technology and, 214–215, PTT (partial thromboplastin time), 111b Reciprocal translocation, 23–24, 23f
227–228 Pulmonary disorders Recombination, 7, 16–17, 16f
Hardy-Weinberg equilibrium in, α1-antitrypsin deficiency as, 173–174, Recurrence risk assessment, 228–229
209–212 173f–174f Red blood cell (RBC) antigens, 93–97, 94t.
historical perspective on, 209 cystic fibrosis as, 167, 167t, 168b, See also ABO blood group
Positive assortative mating, 214, 214b, e36 168f Red blood cell (RBC) hemoglobin defect(s),
Positive predictive value, 226–227, 226t Pulmonary stenosis, 198b 102–110, 103f–105f
Posttranslational modification, 53b Punnett, R. C., 209 hemoglobinopathies as, 102–104, 103f
Potter facies, 178, 179f, e27 Punnett square, 209–210, 210f sickle cell anemia as, 104–107, 104f–
Prader-Willi syndrome, 7b, 26t, e7 Purines, 1–2, 2b 105f, 105t–106t, 106b, 107f. See
clinical manifestations of, 39 Pycnodysostosis, 32, 212–213, e6–e7 also Sickle cell anemia
epigenetics of, 43 Pyloric stenosis, 42–43 thalassemias as, 107–110, 108f, 109t
genetics of, 29b, 38, 39f Pyridoxine, 59, 59b Red blood cell (RBC) indices, 104b
uniparental disomy in, 38–39 deficiency of, e9 Red blood cell (RBC) membrane, 97
pRB protein, 67, 68f, 74, 85–86 Pyridoxine challenge test, 60b defects of, 98–100, 98f–99f, 99t–100t
Predictive value, 226–227, 226t Pyrimidines, 1–2, 2b lipids of, 97b
Pregnancy Pyropoikilocytosis, hereditary, 98f–99f, Red blood cell (RBC) metabolic defects,
diabetes with, 49–50 99–100, 100t 100–102, 100b, 102t
phenylketonuria in, 57, 57t Pyruvate carboxylase, 62 5α-Reductase, 196b, 203f
Preimplantation genetic diagnosis (PGD), 5α-Reductase deficiency, 203–204, 203b,
214, 228 Q 203f
Premessenger RNA, 5 q arm, 12, 14f Reelin (RELN) gene, 154
Premutation, 36, 145 Q-banding, 12–14 RELN gene, 154
 Index 253

Renal agenesis, 178, 178b, 179f RNA interference (RNAi), 232 Sensitivity, test, 226–227, 226t
Renal development, 178, 178t RNA polymerase II, 5b, 5f Sensorineural hearing disorder, 117b
Renal disorder(s), 177–181 RNA processing, 5b Serine proteases, 111b
agenesis as, 178, 178b, 179f Robertsonian translocation, 7, 24, 24f Sertoli cells, 17b
anatomic, 177b–178b Romano-Ward (RW) syndrome, 164, 165t, Severe achondroplasia with developmental
cystinuria as, 180–181, 181b, 181t 166, 216 delay and acanthosis nigricans
multicystic dysplasia as, 178–180, 179f, ROS (reactive oxygen species), 60b (SADDAN), 124
180t, 181b RRFs (ragged red fibers), myoclonic epilepsy Sex chromatin, 22–23
Renal dysplasia, multicystic, 178–180, 179f, with, 131, 131f, e18, e21 Sex chromosomes, 4, 12, 13f. See also X
180t, 181b rRNA (ribosomal RNA), 4, 25b chromosome(s); Y chromosome
Renal stones, 180 Rubella, congenital, 46 numerical abnormalities of, 19–20, 22–23
Renin, 206b RW (Romano-Ward) syndrome, 164, 165t, Sex-determining region of the Y
Restriction endonucleases, 4b, 219, 220t 166, 216 chromosome (SRY) gene, 193–194,
Restriction fragment length polymorphism Ryanodine receptor, 175 193f, 202, 208, e33–e34
(RFLP), 219–221, 221f, 226t Sex differentiation, 192–201, 194f
mutation-specific, 224–225, 225f S Sex-linked inheritance. See X-linked
RET gene, 71t, 182–183, 183f, 183t, e27 S (synthesis) phase, 14, 14b, 65–66, 66f inheritance
Retina, 75b G1 transition to, 67–68, 67f Sex reversal, dosage-sensitive (DSS), 195
Retinal detachment, 75b SADDAN (severe achondroplasia with Sex steroidogenesis, 201, 203f, 205f
Retinal dystrophin, e17 developmental delay and acanthosis disorders of, 201–205
Retinal pigment epithelium, congenital nigricans), 124 adrenocortical disorders and, 205–208,
hypertrophy of, e2, e11, e29 Saggital suture, 120b 205f, 207f–208f, 207t
Retinoblastoma (Rb), 29t, 68f, 74–79, 76t, Salt wasting, 206b ovotesticular disorder of sexual
e11–e12, e12 SAM (S-adenosylmethionine), 58, 58b, development as, 201–202, 202b,
calcification in, 77f, 79b 154–155 202f
computed tomography of, 77f Sandhoff disease, 137–138, 137f, e22 46,XX disorder of sexual development
familial, 77, 78f, 78t Sanfilippo syndromes, 143t, 144, 144f, 158 as, 204–205
leukocoria in, 74, 77f Sanger dideoxynucleotide chain-terminating 46,XY disorder of sexual development
loss of heterozygosity in, 77–78 technique, 222, 223f, 224b as, 202–204, 203f
magnetic resonance imaging of, 77f Sarcoma, 69b Sexual development, 192–201
pathology of, 79b Scaphocephaly, 120 disorders of, 192
RB1 gene in, 67, 74–79, 76t, 78t Scheie syndrome, 142–143, 144t adrenocortical disorders and, 205–208,
spontaneous, 78–79, 78f, 78t Schizophrenia, 151–155, 153f 205f, 207f–208f, 207t
Retinoblastoma gene product (pRB), 67, brain areas affected in, 152, 152f–153f chromosomal mosaicism in, 200
68f, 74, 85–86 chromosomal abnormalities in, 154–155 Klinefelter syndrome as, 198–200,
Retrolental fibroplasia, 150b DAO gene in, 154 198b, 199f–200f, e32
Retroviral vectors, 230, 231t DAOA gene in, 154 nomenclature for, 192, 193t
Retroviruses, 69, 69b, 231b dopamine metabolism in, 154–155, 155f ovotesticular, 201–202, 202b, 202f
Reverse genetics, 133 DTNBP1 gene in, 154 triple-X female as, 201
Reverse transcriptase, 69b environmental factors in, 151–152, 153f Turner syndrome as, 196–198, 197b,
RFLP (restriction fragment length family risks of, 151, 153f e31–e32, e33
polymorphism), 219–221, 221f, 226t GAD1 gene in, 154 46,XX disorder of sexual development
Rh blood group, 94, 94t, 95b, 95f genetics of, 154–155 as, 204–205
incompatibility in, 97, 97f NRG1 gene in, 154 46,XY disorder of sexual development
Rh immune globulin, 97 prevalence of, 151, 151t as, 202–204, 203f
RHCE gene, 94, 95f RELN gene in, 154 XYY syndrome as, 200
RHD gene, 94, 95f twin studies in, 151 SF-1 (steroidogenesis factor 1) gene,
Rhinosinusitis, chronic, 171, e26 Schwannoma, vestibular, 135, 136f 194–195, 194f
Ribonucleic acid. See RNA (ribonucleic acid) SCN5A gene, 165t, 166 Short-limb dwarfism. See Achondroplasia
Ribonucleoproteins, small nuclear (snRNPs), SCN4B gene, 165t Short-segment disease, in Hirschsprung
5b Screening disease, 182
Ribonucleotides, 224b for cancer, 91f, 92, e38 SHOX gene, 197–198
Ribosomal RNA (rRNA), 4, 25b carrier, 227–228 SHOXa protein, 197
Ribozyme therapy, 232 in cystic fibrosis, 170–171, 171t, 215, Sick sinus syndrome with bradycardia, 165t
Rickets, hypophosphatemic, 29b 215t, e36–e37 Sickle cell anemia, 104–107, 104f, e15
Risk for Down syndrome, 19b acute chest syndrome in, 106, 106b
empirical, 229 genetic, 227–228 carrier for, 106, 211t, 216, e37
liability and, 42, 43f carrier, 227–228 codominant expression in, 32
recurrence, 42 newborn, 227 diagnosis of, 215b
Risk assessment, 228–229 vs. testing, 227 DNA analysis in, 107, 107f, 224–225,
RNA (ribonucleic acid) for inborn errors of metabolism, 53–54, 225f
hairpin structure of, 35b 54t, e8 genetic aspects of, 105–106, 105f
heterogenous nuclear (hnRNA), 5 for type 2 diabetes mellitus, e38 incidence of, 162t
messenger (mRNA), 5, 5f SD (standard deviation), 41 infection in, 105b
nontranslated, 4 Secondary sexual characteristics, 196b pathophysiology of, 32b, 106
premessenger, 5 Security of diagnosis, 123t, e19 penicillin prophylaxis in, 105b
ribosomal (rRNA), 4, 25b Selective advantage, 210 physiologic alterations of, 106, e15
silencing (siRNA), e40 Selective disadvantage, 210 survival with, 105t
transfer (tRNA), 4 Seminiferous tubules, 17b Sickle cell hemoglobin S (Hb S), 32, 32b,
uracil in, 2b in Klinefelter syndrome, 198, 199f 103, 105–106, 105f, 106t
 254 Index

Sickle cell trait, 101b, 106, 106t, 211b Steroid response elements (SREs), 164b TFR2 gene, 184–185, 184t
Sideroblastic anemia, 59b Steroid sulfatase (STS) gene, 198–199 TGFBR2 (transforming growth factor-β
SIDS (sudden infant death syndrome), 164 Steroidogenesis, adrenal, 205, 205f receptor 2) gene, 81
Simvastatin, 163b Steroidogenesis factor 1 (SF-1) gene, Thalassemia, 107–110
Single nucleotide polymorphism (SNP), 194–195, 194f α-, 107–108, 108f, 109t, e15
234, 234f–235f, 236–237, e38–e39 Stratum corneum, 115b β-, 108–110, 108f, e15
SIP1 gene, 183t Stratum germinativum, 115b classification of, 109, 109t
SIS oncogene, 71t Streak ovary, 196 Thalassemia intermediate, 109–110, 109t
Sister chromatid, 12, 17 Stroke, 112, 160b Thalassemia major, 109–110, 109t
Skewed inactivation, 33–34 STS (steroid sulfatase) gene, 198–199 Thalassemia minor, 109–110, 109t
Skin, 115b. See also Connective tissue (CT) Submetacentric chromosome, 12, 14f α-Thalassemia trait, 108, 109t
angiokeratoma of, 138b, 139f Substance abuse, bipolar disorder and, 157t Theca cells, 201b
hyperelasticity of, 119, 119f Succinylcholine, 232b Threshold level, 41, 42f
tumors of, 9b Sudden infant death syndrome (SIDS), 164 Thrombophilia, 112
SKY (spectral karyotyping), 14, 15f Suicide, bipolar disorder and, 157t Thromboplastin time, partial (PTT), 111b
SLC3A gene, 180–181 Superoxide dismutase (SOD), 187b, 189f Thumb sign, 121f, e17
SLC7A9 gene, 180–181, 182f Supranuclear gaze palsy, 141b Thymine (T), 1–2, 2b, 3f
SLC18A2 gene, 154–155 Susceptibility genes, in diabetes mellitus, Tibia vara, 195b
SLC40A1 gene, 184, 184t 47–48 Timothy syndrome, 165t
Small nuclear ribonucleoproteins (snRNPs), Suspensory ligament, 85b Tissue-specific genes, 52, 53t
5b Synapsis, 17b Tobacco smoke, α1-antitrypsin deficiency
Smith-Magenis syndrome, 7b, 26t Synaptonemal complex, 17b and, 173–174, 173f–174f
SNP (single nucleotide polymorphism), 234, Syncope, 164 Torsades de pointes (TdP), 164, 165f
234f–235f, 236–237, e38–e39 Syncytiotrophoblast, 195b Toulouse-Lautrec, Henri de, 32, 212–213
snRNPs (small nuclear ribonucleoproteins), Synpolydactyly, 29b TP53 gene, 85–86, 92
5b Synthesis (S) phase, 14, 14b, 65–66, 66f in colorectal cancer, 78t, 80f
SNRPN gene, 38 G1 transition to, 67–68, 67f in Li-Fraumeni syndrome, 76t, 86
SNTA1 gene, 165t Syntrophin, 127f Transcription, 1, 4–5, 5b, 5f
SOD (superoxide dismutase), 187b, 189f α-Synuclein, 151t, e22 Transcriptomics, 217
Solenoid structure, 1, 2f Transferrin, 185, 185b, 186f
SOS repair, 11 T Transferrin receptor, 185, 186f
Southern blot analysis, 219–221, 220t, 221f T (thymine), 1–2, 2b, 3f Transferrin receptor 2 (TFR2) gene,
SOX9 gene, 194, 194f, e27 T lymphocytes, 47b 184–185, 184t
SOX10 gene, 183t Tandem mass spectrometry, 56–57 Transforming growth factor-β (TGF-β), 195b
Specificity, test, 226–227, 226t TART (testicular adrenal rest tumor), 176, Transforming growth factor-β receptor 2
Spectral karyotyping (SKY), 14, 15f 207, e34 (TGFBR2) gene, 81
Spectrin, 97–100, 98f, 99t TATA box, 5 Transition mutation, 6, e1
Spermatocytes, 17 TATAAAA sequence, 5 Translation, 1, 4–5
Spermatogenesis, 17 Tay-Sachs disease, 137–138, 137f, 158 Translocation, 7, 23–25, 23f–24f
Spherocytosis, hereditary, 64t, 98, 98f–99f, carrier for, 211t, 227–228 oncogene activation by, 70–73, 72f–73f
100t, e14 incidence of, 162t reciprocal, 23–24, 23f
mutations in, 98, 99t neuropathy in, 215b robertsonian, 7, 24, 24f
Sphingolipid(s), 136–137, 137f TDF (testis determining factor), 193 Translocation Down syndrome, 18f, 24, 24f,
Sphingolipidosis(es), 136–142, 137f TdP (torsades de pointes), 164, 165f 27, e2
Fabry disease as, 137f, 138–139, 139f, Telomerase, 88–89, 89f Transversion mutation, 6
e23 Telomeres, 88–89, 89f Trichothiodystrophy, 10t
Gaucher disease as, 137f, 139–141, 139b, Telophase Triglycerides, 159t
140f, 140t, e22–e23 of meiosis, 17 Trigonocephaly, 120
Krabbe disease as, 137f, 142 of mitosis, 15, 16f Trinucleotide repeat. See Triplet repeats
Niemann-Pick disease as, 137f, 141–142 Template strand, 4–5, 5f Trinucleotide repeat expansion detection,
Sandhoff disease as, 137–138, 137f, e22 Test 226
Tay-Sachs disease as, 137–138, 137f, 158 efficiency of, 226–227, 226t Triphalangeal thumb, e17
Sphingomyelin, 136–137 sensitivity of, 226–227, 226t Triple helix, 115, 116f
accumulation of, 141–142 specificity of, 226–227, 226t Triple screen, 19b, 27, 228b
Sphingomyelinase deficiency, 137f, 141–142 validity of, 226–227, 226t Triple-X syndrome, 201, e31
Spina bifida, 42 Testes, in Klinefelter syndrome, 198, Triplet, of DNA code, 4, 6b
Spinocerebellar ataxia, 29b, 36t 199f–200f Triplet repeats, 29b, 35–37, 36t, 37f,
Spliceosome, 5b Testicular adrenal rest tumor (TART), 176, 144–147
Spontaneous mutation, 7, 40 207, e34 detection of, 226
SPRY genes, 178, 178t Testis determining factor (TDF), 193 in fragile X syndrome, 145–146, 146f,
Spur cell anemia, 97b Testosterone, 194–195, 194f, 196b, 201b 146t, 226
Squamous cell carcinoma, 9b in Klinefelter syndrome, 199–200 in Huntington disease, 146–147, 147f
SREs (steroid response elements), 164b replacement of, 200b Trisomy, 6, 18, 20–21
SRY (sex-determining region of the Y synthesis of, 202, 203f partial, 25
chromosome) gene, 193–194, 193f, Tetrad chromosome, 16–17 Trisomy 13, 21, 21f, 22t, e6
202, 208, e33–e34 Tetrahydrobiopterin, e9 Trisomy 15, 39
Standard deviation (SD), 41 Tetrahydrobiopterin deficiency, 56 Trisomy 16, 21
Statins, 163b Tetrahydrobiopterin synthase (BH4), 55f Trisomy 18, 21, 21f, 22t, 181b, e2
Steinberg sign, 121f, e17 Tetraploidy, 27 Trisomy 21. See Down syndrome
Stem cells, 230 Tetrasomy, 18 Trisomy 22, 21
 Index 255

Tropocollagen, 115, 116f Uniparental disomy, 38–39, e7 X

Tryosinemia, 55f Unique DNA, 3–4 X chromosome(s), 12, 13f
TTD-A gene, 10t Uracil (U), 2b, 5b inactivation of, 23, 33–34, 198, e15
Tubulin, 65–66 Ureter, ectopic, 177b loss of, in Turner syndrome, 196–198,
Tumor necrosis factor (TNF) receptor, 86b UV (ultraviolet) radiation, 8, 8b 197b, 197f
Tumor suppressor genes, 74–80, 76t multiple, e31
apoptotic, 76t, 85–88, 87f V
X inactivation center (XIC), 34
in breast cancer, 83 Vacor, 47 X-linked inheritance, 28, e4
cell division–controlling, 74–79, 76t VACTERL association, 181b, 191 of androgen insensitivity syndrome, 204
DNA repair, 76t, 80–85, 82f Validity, test, 226–227, 226t dominant, 29b, 34, 34f
in familial adenomatous polyposis, 81 Valine, 61, 62f of Fabry disease, 138
in hereditary nonpolyposis colon cancer, Valproate, 157, 157b of fragile X syndrome, 145–146
81 Vas deferens, congenital bilateral absence of of glucose-6-phosphate dehydrogenase
in Li-Fraumeni syndrome, 85–86 (CBAVD), 167, 167t, 170–171, 171b, deficiency, 102
in neurofibromatosis type 1, 76t, 79–80 176 of hemophilia A, 33, 33f, 110t, 111
in ovarian cancer, 83–85, 85f Velocardiofacial syndrome (VCFS), 26t, of hemophilia B, 110t, 111
in prostate cancer, 86–88 154, 178b of Hunter syndrome, 143–144, e22
in retinoblastoma, 74–79, 76t Venous thrombosis, 112 of Lesch-Nyhan syndrome, 135–136
Tunica albuginea, 17b activated protein C resistance and, 112 of muscular dystrophy, 125, 127
Turkheimer, Eric, 147 anatomy of, 112b of ornithine transcarbamylase deficiency,
Turner syndrome, 19–20, 196–198, epidemiology of, 112 212b
e31–e32, e33 Factor V Leiden and, 112, 112t recessive, 29b, 32–34, 33f
chromosomal mosaicism in, 200, e33 pathology of, 112b X-linked loci, 212, 212f
clinical features of, 196–197, 197b Ventricular arrhythmias, 164, 165f, 167 Xanthelasma, 161, 163f
epidemiology of, 197 Vertical supranuclear gaze palsy, 141b Xanthine oxidase inhibitor, 136b
genetics of, 197, 197b Very-low-density lipoprotein (VLD), 159t Xanthomas, 161, 162f, e25
genotypes in, 197b, 200 Vesicular monoamine transporter (VMAT2) Xeroderma pigmentosum, 9, 9t, 11, e1
history of, 23 protein, 154–155 XIC (X inactivation center), 34
pathogenesis of, 197 Vestibular schwannoma, 135, 136f XIST gene, 34, 208
streak ovaries in, 196 VHL gene, 76t, 78t XPA gene, 9t–10t
treatment of, 197 Viral infection XPC gene, 9t–10t
Turricephaly, 120 with human-derived products, 175b 45,X disorder of sexual development,
Twin studies, 147–148 in type 1 diabetes mellitus, 46–47 196–198, 197b, 197f
in bipolar disorder, 156 Viral vectors, 171b, 230–231, 231t, 233t, 46,XX disorder of sexual development,
in diabetes mellitus, 45–46 e40 201–202, 202b, 202f, e34
in schizophrenia, 151–152, 152f–153f Vitamin B6, 59, 59b 47,XXX disorder of sexual development,
Twins, 45b VLDL (very-low-density lipoprotein), 159t 201
Two-hit hypothesis, 74–75, 78f Von Hippel–Lindau kidney cancer, 76t 47,XXY disorder of sexual development,
Tyrosinase, 187b Von Willebrand disease, 110–112, 110t 198–200, 198b, 199f–200f
Tyrosine Von Willebrand factor, 110 46,XY disorder of sexual development,
elevated levels of, 56–57 202–204, 203f, 207f, e33–e34
metabolism of, 57–58 W
46,XY disorder of sexual development with
Tyrosine kinase, 25 Waardenburg syndrome, 29t, 181b maternal virilization, 204–205
Tyrosine receptor signaling, 69, 72f Walker sign, 121f, e17 XYY syndrome, 200
Tyrosinemia Watson, James, 1
neonatal, 57–58 Weight, distribution of, 41 Y
treatment of, 64t Weinberg, Wilhelm, 209 Y chromosome, 12, 13f
type I, 58 Wernicke’s area, in schizophrenia, 153f anatomy of, 193, 193f
type II, 58 Williams syndrome, 26t pseudoautosomal regions of, 193, 193f
Wilson disease, 64t, 188–190, 189t, e28, sex-determining region of, 193–194, 193f,
U e28–e29, e29 202, 208, e33–e34
U (uracil), 2b, 5b epidemiology of, 189 testis determining factor gene of, 193
UBE3A gene, 38 Kayser-Fleischer ring in, 190, 190f
Ubiquitin, 38b treatment of, 190 Z
Ubiquitin carboxyl-terminal esterase L1, WNT genes, 178t Z-form DNA, 1b
151t WNT proteins, 81b, e27 ZDHHC8 gene, 154
Ubiquitin ligase, 38 Wobble effect, 6b, e1 Zinc finger motif, 193b
UCSNP-43, 47–48 Wolf-Hirschhorn syndrome, 7b, 147 Zona fasciculata, 205b
Ulcer, duodenal, 96 Worm, Olaus, 118 Zona glomerulosa, 205b
Ultraviolet (UV) radiation, 8, 8b Wormian bones, 118, 119f Zona reticularis, 205b
Unequal crossing-over, 36, 37f, e15 Wrist sign, 121f Zygotene, 16–17
Unifactorial inheritance, 28 WT-1 gene, 178t, 194f Zymogens, 111b
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