GENOMIC DISORDERS The Genomic Basis of Disease doc

Genomic
Disorders
The Genomic
Basis of Disease
The Genomic
Basis of Disease
Edited by
James R. Lupski,
MD
,
P
h
D
Pawel Stankiewicz,
MD
,
P
h
D
Genomic
Disorders
Edited by
James R. Lupski,
MD
,
P
h
D
Pawel Stankiewicz,
MD
,

P
h
D
G
ENOMIC
D
ISORDERS

Edited by
JAMES R. LUPSKI, MD, PhD
PAWEL STANKIEWICZ, MD, PhD
Department of Molecular and Human Genetics
Baylor College of Medicine, Houston, TX
G
ENOMIC
D
ISORDERS
The Genomic Basis of Disease
/
© 2006 Humana Press Inc.
999 Riverview Drive, Suite 208
Totowa, New Jersey 07512
www.humanapress.com
All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or
by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission
from the Publisher.
The content and opinions expressed in this book are the sole work of the authors and editors, who have warranted due
diligence in the creation and issuance of their work. The publisher, editors, and authors are not responsible for errors
or omissions or for any consequences arising from the information or opinions presented in this book and make no
warranty, express or implied, with respect to its contents.

Due diligence has been taken by the publishers, editors, and authors of this book to assure the accuracy of the information
published and to describe generally accepted practices. The contributors herein have carefully checked to ensure that
the drug selections and dosages set forth in this text are accurate and in accord with the standards accepted at the time
of publication. Notwithstanding, as new research, changes in government regulations, and knowledge from clinical
experience relating to drug therapy and drug reactions constantly occurs, the reader is advised to check the product
information provided by the manufacturer of each drug for any change in dosages or for additional warnings and
contraindications. This is of utmost importance when the recommended drug herein is a new or infrequently used drug.
It is the responsibility of the treating physician to determine dosages and treatment strategies for individual patients.
Further it is the responsibility of the health care provider to ascertain the Food and Drug Administration status of each
drug or device used in their clinical practice. The publisher, editors, and authors are not responsible for errors or
omissions or for any consequences from the application of the information presented in this book and make no warranty,
express or implied, with respect to the contents in this publication.
Production Editor: Melissa Caravella
Cover design by Patricia F. Cleary
For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at
the above address or at any of the following numbers: Tel.: 973-256-1699; Fax: 973-256-8341, E-mail: orders@
humanapr.com; or visit our Website: www.humanapress.com
This publication is printed on acid-free paper. ∞
ANSI Z39.48-1984 (American National Standards Institute) Permanence of Paper for Printed Library Materials.
Photocopy Authorization Policy:
Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted
by Humana Press Inc., provided that the base fee of US $30.00 is paid directly to the Copyright Clearance Center at 222
Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the
CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users
of the Transactional Reporting Service is: [1-58829-559-1/06 $30.00].
Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1
eISBN 1-59745-039-1
Library of Congress Cataloging-in-Publication Data
Genomic disorders : the genomic basis of disease / edited by James R. Lupski, Pawe Stankiewicz.
p. ; cm.

Includes bibliographical references and index.
ISBN 1-58829-559-1 (alk. paper)
1. Genetic disorders Molecular aspects.
[DNLM: 1. Genetic Diseases, Inborn. 2. Chromosome Aberrations. 3. Genome Components. 4. Genome.
5. Genomics methods. QZ 50 G3354 2006] I. Lupski, James R., 1957- II. Stankiewicz, Pawe .
RB155.5.G465 2006
616'.042 dc22
2005020461
Dedication
To our many mentors who have nurtured our intellectual curiosity and to our
dedicated families for their love and support.
—J. R. L. and P. S.
v

In Memorium
In memory of Carlos A. Garcia (1935–2005) and his passion for medicine, science,
and the patients and families for whom he cared.
vii

Preface
ix
Uncovering Recurrent Submicroscopic Rearrangements As a Cause of Disease
For five decades since Fred Sanger's (1) seminal discovery that proteins have a specific
structure, since Linus Pauling's (2) discovery that hemoglobin from patients with sickle
cell anemia is molecularly distinct, and since Watson and Crick's (3) elucidation of the
chemical basis of heredity, the molecular basis of disease has been addressed in the
context of how mutations affect the structure, function, or regulation of a gene or its
protein product. Molecular medicine has functioned in the context of a genocentric world.
During the last decade it became apparent, however, that many disease traits are best
explained not by how the information content of a single gene is changed, but rather on

the basis of genomic alterations. Furthermore, it has become abundantly clear that architec-
tural features of the human genome can result in susceptibility to DNA rearrangements that
cause disease traits. Such conditions have been referred to as genomic disorders (4,5).
It remains to be determined to what extent genomic changes are responsible for disease
traits, common traits (including behavioral traits), or perhaps sometimes represent benign
polymorphic variation. The widespread structural variation of the human genome, alter-
natively referred to as large-copy number polymorphisms, large-scale copy number varia-
tions, or copy number variants has begun only recently to be appreciated (6–9).
High-resolution analysis of the human genome has enabled detection of genome changes
heretofore not observed because of technology limitations. Whereas agarose gel electro-
phoresis enables detection of changes of the genome up to 25–30 kb in size, and cytoge-
netic banding techniques can resolve deletion rearrangements only greater than 2–5 Mb
in size, alterations of the genome between more than 30 kb and less than 5 Mb defied
detection until pulsed-field gel electrophoresis and fluorescence in situ hybridization
became available to resolve changes in the human genome of such magnitude (10–12).
Those methods were limited to detection of specific genomic regions of interest and could
not evaluate genomic rearrangements in a global way.
The availability of a “finished” human genome sequence (13) and genomic microarrays
(14) have enabled approaches to resolve changes in the genome heretofore impossible to
assess on a global genome scale (i.e., simultaneously examining the entire genome rather
than discreet segments). Array comparative genome hybridization (aCGH) is one powerful
approach to high-resolution analysis of the human genome. The CGH determines differ-
ences by comparisons to a reference “normal genome,” whereas the array enables detec-
tion of such changes at essentially any resolution that is desired, limited only by
imagination and cost. Furthermore, the application of bioinformatic analyses to the
finished human genome sequence and comparative genomic analysis enable information
technology approaches to identify key architectural features throughout the entire
genome that are associated with known recurrent rearrangements causing genomic
disorders.
An increasing number of human diseases are recognized to result from recurrent DNA

rearrangements involving unstable genomic regions. A combination of high-resolution
genome analysis with informatics capabilities to examine individuals with well-
characterized phenotypic traits is a powerful approach to address the question: To what extent
are constitutional DNA rearrangements in the human genome responsible for human traits?
Such approaches may also yield insights into recurrent somatic rearrangements (15).
Genomic Disorders: The Genomic Basis of Disease attempts to survey the subject area of
genomic disorders in the beginning of the postgenomic era. After a short historical
presentation (Part I) describing the trials and tribulations involved in uncovering the recurrent
submicroscopic duplication associated with Charcot-Marie-Tooth disease type 1A, the book
is organized into parts on genome structure (II), genome evolution (III), genomic rearrange-
ments and disease traits (IV), functional aspects of genome structure (V), and modeling and
assays for genomic disorders (VI). Finally, Part VII includes appendices that delineate
disease traits and genomic features (listed in tabular form) for well-characterized genomic
disorders as well as clinical phenotypes for which chromosome microarray analysis may be
used to detect the responsible rearrangement mutation. We believe that the topics chosen for
individual chapters illustrate the genomic basis of disease.
James R. Lupski,
MD, PhD
Paw
el
Stankiewicz, MD, PhD
REFERENCES
1. Sanger F. The terminal peptides of insulin. Biochem J 1949;45:563–574.
2. Pauling L, Itamo HA, Singer SJ, Wells IC. Sickle cell anemia, a molecular disease. Science
1949;110:64–66.
3. Watson DA, Crick FHC. Molecular structure of nucleic acids. A structure for deoxyribose nucleic
acids. Nature 1953;171:737–738.
4. Lupski JR. Genomic disorders: structural features of the genome can lead to DNA rearrangements and
human disease traits. Trends Genet 1998;14:417–422.
5. Stankiewicz P, Lupski JR. Genome architecture, rearrangements and genomic disorders. Trends

Genet 2002;18:74–82.
6. Shaw-Smith C, Redon R, Rickman L, et al. Microarray based comparative genomic hybridisation
(array-CGH) detects submicroscopic chromosomal deletions and duplications in patients with
learning disability/mental retardation and dysmorphic features. J Med Genet 2004;41:241–248.
7. Iafrate AJ, Feuk L, Rivera MN, et al. Detection of large-scale variation in the human genome. Nat
Genet 2004;36:949–951.
8. Sebat J, Lakshmi B, Troge J, et al. Large-scale copy number polymorphism in the human genome.
Science 2004;305:525–528.
9. Tuzun E, Sharp AJ, Bailey JA, et al. Fine-scale structural variation of the human genome. Nat Genet
2005;37:727–732.
10. Schwartz DC, Cantor CR. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel
electrophoresis. Cell 1984;37:67–75.
11. Pinkel D, Straume T, Gray JW. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence
hybridization. Proc Natl Acad Sci USA 1986;83:2934–2938.
12. Lupski JR. 2002 Curt Stern Award Address. Genomic disorders: recombination-based disease resulting
from genomic architecture. Am J Hum Genet 2003;72:246–252.
13. International Human Genome Sequencing Consortium. Finishing the euchromatic sequence of the
human genome. Nature 2004;431:931–945.
14. Carter NP, Vetrie D. Applications of genomic microarrays to explore human chromosome structure
and function. Hum Mol Genet 2004;13:R297–R302.
15. Barbouti, A., Stankiewicz, P., Birren, B., et al. The breakpoint region of the most common isochro
mosome, i(17q), in human neoplasia is characterized by a complex genome architecture with large
palindromic low-copy repeats. Am J Hum Genet 2004;74:1–10.
x Preface
/
Contents
Dedication v
In Memorium vii
Preface ix
Contributors

xv
xi
PART IINTRODUCTION
1 The CMT1A Duplication: A Historical Perspective Viewed
From Two Sides of an Ocean 3
James R. Lupski and Vincent Timmerman
PART II GENOMIC STRUCTURE
2 Alu Elements 21
Prescott Deininger
3 The Impact of LINE-1 Retrotransposition on the Human
Genome
35
Amy E. Hulme, Deanna A. Kulpa, José Luis Garcia Perez,
and John V. Moran
4 Ancient Transposable Elements, Processed Pseudogenes,
and Endogenous Retroviruses
57
Adam Pavlicek and Jerzy Jurka
5 Segmental Duplications
73
Andrew J. Sharp and Evan E. Eichler
6 Non-B DNA and Chromosomal Rearrangements
89
Albino Bacolla and Robert D. Wells
7 Genetic Basis of Olfactory Deficits
101
Idan Menashe, Ester Feldmesser, and Doron Lancet
8 Genomic Organization and Function of Human
Centromeres
115

Huntington F. Willard and M. Katharine Rudd
PART III GENOME EVOLUTION
9 Primate Chromosome Evolution 133
Stefan Müller
10 Genome Plasticity in Evolution: The Centromere Repositioning 153
Mariano Rocchi and Nicoletta Archidiacono
xii Contents
PART IV GENOMIC REARRANGEMENTS AND DISEASE TRAITS
11 The CMT1A Duplication and HNPP Deletion 169
Vincent Timmerman and James R. Lupski
12 Smith-Magenis Syndrome Deletion, Reciprocal Duplication
dup(17)(p11.2p11.2), and Other Proximal
17p Rearrangements
179
Pawel Stankiewicz, Weimin Bi, and James R. Lupski
13 Chromosome 22q11.2 Rearrangement Disorders
193
Bernice E. Morrow
14 Neurofibromatosis 1
207
Karen Stephens
15 Williams-Beuren Syndrome
221
Stephen W. Scherer and Lucy R. Osborne
16 Sotos Syndrome 237
Naohiro Kurotaki and Naomichi Matsumoto
17 X Chromosome Rearrangements
247
Pauline H. Yen
18 Pelizaeus-Merzbacher Disease and Spastic Paraplegia Type 2

263
Ken Inoue
19 Y-Chromosomal Rearrangements and Azoospermia 273
Matthew E. Hurles and Chris Tyler-Smith
20 Inversion Chromosomes 289
Orsetta Zuffardi, Roberto Ciccone, Sabrina Giglio,
and Tiziano Pramparo
21 Monosomy 1p36 As a Model for the Molecular Basis
of Terminal Deletions
301
Blake C. Ballif and Lisa G. Shaffer
22 inv dup(15) and inv dup(22)
315
Heather E. McDermid and Rachel Wevrick
23 Mechanisms Underlying Neoplasia-Associated Genomic
Rearrangements
327
Thoas Fioretos
PART VFUNCTIONAL ASPECTS OF GENOME STRUCTURE
24 Recombination Hotspots in Nonallelic Homologous
Recombination 341
Matthew E. Hurles and James R. Lupski
25 Position Effects
357
Pawel Stankiewicz
/
/
PART VI GENOMIC DISORDERS: MODELING AND ASSAYS
26 Chromosome-Engineered Mouse Models 373
Pentao Liu

27 Array-CGH for the Analysis of Constitutional Genomic
Rearrangements
389
Nigel P. Carter, Heike Fiegler, Susan Gribble,
and Richard Redon
PART VII APPENDICES
Appendix A: Well-Characterized Rearrangement-Based
Diseases and Genome Structural Features at the Locus 403
Pawel Stankiewicz and James R. Lupski
Appendix B: Diagnostic Potential for Chromosome
Microarray Analysis
407
Pawel Stankiewicz, Sau W. Cheung, and Arthur L. Beaudet
Index
415
About the Editors
427
Contents xiii
/
/

xv
CONTRIBUTORS
NICOLETTA ARCHIDIACONO, PhD • Department of Genetics and Microbiology, University
of Bari, Bari, Italy
A
LBINO BACOLLA, PhD • Center for Genome Research, Texas A & M University System
Health Science Center, Texas Medical Center, Houston, TX
B
LAKE C. BALLIF, PhD • Signature Genomic Laboratories, LLC, Spokane, WA

A
RTHUR L. BEAUDET, MD • Department of Molecular and Human Genetics, Baylor
College of Medicine, Houston, TX
W
EIMIN BI, PhD • Department of Molecular and Human Genetics, Baylor College
of Medicine, Houston, TX
N
IGEL P. CARTER, DPhil • The Sanger Institute, Wellcome Trust Genome Campus,
Cambridge, UK
S
AU W. CHEUNG, PhD • Department of Molecular and Human Genetics, Baylor College
of Medicine, Houston, TX
R
OBERTO CICCONE, PhD • Biologia Generale e Genetica Medica, Universita di Pavia,
Pavia, Italy
P
RESCOTT DEININGER, PhD • Department of Epidemiology, Tulane Cancer Center,
Tulane University Health Sciences Center, New Orleans, LA
E
VAN E. EICHLER, PhD • Department of Genome Sciences, University of Washington,
Seattle, WA
E
STER FELDMESSER, MSc • Department of Molecular Genetics and the Crown Human
Genome Center Weizmann Institute of Science, Rehovot, Israel
H
EIKE FIEGLER, PhD • The Sanger Institute, Wellcome Trust Genome Campus,
Cambridge, UK
T
HOAS FIORETOS, MD, PhD • Department of Clinical Genetics, Lund University Hospital,
Lund, Sweden

S
ABRINA GIGLIO, MD, PhD • Ospedale San Raffaele, Milano, Italy
S
USAN GRIBBLE, PhD • The Sanger Institute, Wellcome Trust Genome Campus,
Cambridge, UK
A
MY E. HULME, BS, MS • Department of Human Genetics, The University of Michigan
Medical School, Ann Arbor, MI
M
ATTHEW E. HURLES, PhD • The Sanger Institute, Wellcome Trust Genome Campus,
Cambridge, UK
K
EN INOUE, MD, PhD • Department of Mental Retardation and Birth Defect Research,
National Institute of Neuroscience, National Center of Neurology and Psychiatry,
Kodaira, Tokyo, Japan
J
ERZY JURKA, PhD • Genetic Information Research Institute, Mountain View, CA
D
EANNA A. KULPA, BS, MS • Department of Human Genetics, The University of Michigan
Medical School, Ann Arbor, MI
N
AOHIRO KUROTAKI, MD, PhD • Department of Molecular and Human Genetics, Baylor
College of Medicine, Houston, TX
D
ORON LANCET, PhD • Department of Molecular Genetics and the Crown Human
Genome Center, Weizmann Institute of Science, Rehovot, Israel
xvi Contributors
P
ENTAO
L

IU
,
P
h
D
• The Sanger Institute, Wellcome Trust Genome Campus, Cambridge, UK
JAMES R. LUPSKI, MD, PhD • Department of Molecular and Human Genetics, Department
of Pediatrics, Baylor College of Medicine, Houston, TX
N
AOMICHI MATSUMOTO, MD, PhD • Department of Human Genetics, Yokohama City
University Graduate School of Medicine, Fukuura, Yokohama, Japan
H
EATHER E. MCDERMID, PhD • Department of Biological Sciences, University
of Alberta, Edmonton, Alberta, Canada
I
DAN MENASHE, MSc • Department of Molecular Genetics and the Crown Human
Genome Center, Weizmann Institute of Science, Rehovot, Israel
J
OHN V. MORAN, PhD • Department of Human Genetics and Internal Medicine,
The University of Michigan Medical School, Ann Arbor, MI
B
ERNICE E. MORROW, PhD • Department of Molecular Genetics, Albert Einstein College
of Medicine, Bronx, NY
S
TEFAN MÜLLER, PhD • Department of Biology II, Ludwig, Maximilians University,
Munich, Germany
L
UCY R. OSBORNE, PhD • Departments of Medicine and Molecular & Medical Genetics,
University of Toronto, Toronto, Canada
A

DAM PAVLICEK, PhD • Genetic Information Research Institute, Mountain View, CA
J
OSÉ LUIS GARCIA PEREZ, PhD • Department of Human Genetics, The University
of Michigan Medical School, Ann Arbor, MI
T
IZIANO PRAMPARO, PhD • Biologia Generale e Genetica Medica, Universita di Pavia,
Pavia, Italy
R
ICHARD REDON, PhD • The Sanger Institute, Wellcome Trust Genome Campus,
Cambridge, UK
M
ARIANO ROCCHI, PhD • Department of Genetics and Microbiology, University of Bari,
Bari, Italy
M. K
ATHARINE RUDD, PhD • Institute for Genome Sciences & Policy, Duke University,
Durham, NC
S
TEPHEN W. SCHERER, PhD • Program in Genetics & Genomic Biology, Sick Kids
Hospital, Toronto, Canada; Department of Molecular & Medical Genetics,
University of Toronto, Toronto, Canada
L
ISA G. SHAFFER, PhD • Signature Genomic Laboratories, LLC, Spokane, WA; Sacred
Heart Medical Center, Spokane, WA; Health Research and Education Center,
Washington State University, Spokane, WA
A
NDREW J. SHARP, PhD • Department of Genome Sciences, University of Washington,
Seattle, WA
P
AWEL STANKIEWICZ, MD, PhD • Department of Molecular and Human Genetics, Baylor
College of Medicine, Houston, TX

K
AREN STEPHENS, PhD • Departments of Medicine and Laboratory Medicine, University
of Washington, Seattle, WA
V
INCENT TIMMERMAN, PhD • Molecular Genetics Department, Flanders Interuniversity
Institute for Biotechnology, University of Antwerp, Antwerpen, Belgium
C
HRIS TYLER-SMITH, PhD • The Sanger Institute, Wellcome Trust Genome Campus,
Cambridge, UK
R
OBERT D. WELLS, PhD • Center for Genome Research, Texas A & M University Sys-
tem Health Science Center, Texas Medical Center, Houston, Texas
/
RACHEL WEVRICK, PhD • Department of Medical Genetics, University of Alberta,
Edmonton, Alberta, Canada
H
UNTINGTON F. WILLARD, PhD • Institute for Genome Sciences & Policy, Duke University,
Durham, NC
P
AULINE H. YEN, PhD • Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
O
RSETTA ZUFFARDI, PhD • Biologia Generale e Genetica Medica, Universita di Pavia,
Pavia, Italy
Contributors xvii

Chapter 1 / CMT1A Duplication 1
I
INTRODUCTION
2 Part I / Introduction
Chapter 1 / CMT1A Duplication 3

3
From: Genomic Disorders: The Genomic Basis of Disease
Edited by: J. R. Lupski and P. Stankiewicz © Humana Press, Totowa, NJ
1
The CMT1A Duplication
A Historical Perspective Viewed
From Two Sides of an Ocean
James R. Lupski, MD, PhD
and Vincent Timmerman, PhD
CONTENTS
FROM THE UNITED STATES
FROM EUROPE
REFERENCES
FROM THE UNITED STATES
I came to Houston, Texas in 1986 with one goal being to identify “the gene” for Charcot-
Marie-Tooth (CMT) disease. I was peripherally aware of the paper by Botstein and colleagues
(1) proposing the genetic mapping of human “disease genes” using linked restriction fragment
length polymorphisms (RFLPs) to position the gene within the human genome and indeed
became very excited as a graduate student when Gusella’s paper (2) appeared in Nature linking
the Huntington disease locus to markers on chromosome 4. It was a natural extension to think
this “positional cloning” approach might be applied to a host of other human traits. There was
a personal, one might say egocentric, reason to choose CMT because I have the disease (3) and,
in fact, the first blood samples collected for DNA linkage studies were from my own family
wherein CMT segregated as an apparent autosomal recessive trait.
The year 1986 was also somewhat historic for the opportunity to attend the Cold Spring
Harbor Symposium on Quantitative Biology, which that year was on “The Molecular Biology
of Homo sapiens” (4). It was there that Kary Mullis first announced publicly the polymerase
chain reaction (PCR) technique, and also some of the first “scientific public” debates surround-
ing the initiation of the Human Genome Project took place. I distinctly remember Kary Mullis
arguing during these discussions that if there was going to be a huge amount of DNA sequence

determined (like the three billion basepair human genome) then the “G” symbol for the base
guanine should be changed to “W” to distinguish it from “C,” which was difficult to do because
of the typewriters and printers available at the time. He argued that Crick already had one of
the symbols (“C” for cytocine) named after him and Watson should have one. I vaguely
remember Jim Watson smiling on the sidelines of the audience. I was married that week in
Huntington, New York.
4 Part I / Introduction
The move to Houston was also because of the decision to continue my clinical training and
begin internship and residency in pediatrics at Texas Children’s Hospital. This occupied my
time immensely and, thus, I was fortunate to be able to join Pragna Patel, a junior faculty
member in the Institute for Molecular Genetics, to bank CMT family samples and initiate our
genetic linkage studies.
Family collections began in earnest towards the end of my residency (1988–1989). Pragna
had known of a physician, Carlos Garcia (New Orleans), who followed a number of families
with CMT in Louisiana and we also contacted Jim Killian (Houston), at the time co-chairman
of Neurology at Baylor College of Medicine, who published a huge French Acadian pedigree
segregating CMT a decade earlier (5). He had also made the intriguing observation that appar-
ent homozygosity for the dominant CMT gene, a child of two affected parents, resulted in a
significantly more severe phenotype (5). Thus, like many other human traits, CMT is probably
better characterized as a semi-dominant disorder.
Carlos Garcia directed the Muscular Dystrophy Association clinics in New Orleans, Baton
Rouge, and Lafayette, LA. Once a month, Carlos’ wife Mona would always remark “It’s that
time of the month again,” I would fly to New Orleans and stay overnight Monday at the
Garcia’s. Carlos and I would awaken and drive a couple of hours to Baton Rouge and see
patients from morning until just after lunch, drive to Lafayette (where Carlos followed several
hundred CMT patients) and see patients until dinner time. We would have a wonderful Cajun
dinner, stay overnight in Lafayette, and the next day start seeing patients early in the morning
until late afternoon, then he would drive me back to the New Orleans airport with a suitcase
of blood samples in hand. Carlos would clinically examine and oversee nerve conduction
velocity (NCV) testing (NCVs are an objective laboratory test for type 1 CMT [CMT1]) while

I would draw pedigrees and obtain blood for DNA samples and to make permanent transformed
lymphoblastoid cell lines on my return to Houston.
One particular blood collection sticks out in my mind. It took place in a hospital clinic
adjacent to the emergency room of a local hospital in Lafayette. We first collected blood from
a teenage man distinguished by an unusual haircut and tattoos dressed in an outfit becoming
of a punk rocker. When we next began collecting blood from his younger sister, she passed out
and started to have myoclonic jerks. Her older brother started to shout “she is throwing a fit.”
He then proceeded to stand, look at both Dr. Garcia and I, and stated, “I am going to go get a
REAL doctor” and proceeded to the emergency room next door. Needless to say both he and
she were just fine and Dr. Garcia and I recovered from our ego bruising.
These monthly trips continued for a few years, but for the collection of very large families
we would sometimes arrange a family reunion. It was remarkable how there would be one
family member, often an unaffected individual, who could mobilize the entire family because
of their belief in the research efforts. Importantly, we had to perform the electrical studies
(NCVs) and collect blood samples from all family members. This included unaffected indi-
viduals, who were sometimes hesitant, or required further explanation of the need for their
samples. I often thought of the irony of the situation. At these reunion parties, Dr. Garcia would
oversee the administration of the electrical shocks accompanying nerve conduction studies, I
would draw blood from each family member, and they would feed us wonderful Cajun
barbeque. We similarly collected the large family reported by Dr. Killian using the family
reunion approach. In this case, Jim Killian rented the town hall of a small town in the French
Acadian countryside of Louisiana. I remember Dr. Killian asking other family members about
one particular family member, expressing some concern during the inquiry. Apparently, dur-
Chapter 1 / CMT1A Duplication 5
ing the examinations and home visits that led to the 1979 paper of Killian and Kloepfer (5), this
family member drew a gun on Dr. Killian thinking that he was either “the law,” or a tax
collector.
Meanwhile in Houston, Pragna had collected several polymorphic DNA markers from the
laboratory of Dr. Ray White in Utah and we analyzed systematically the family material that
was available. We began with the smaller chromosomes and essentially had ruled out several,

including initially chromosome 17, using sparse markers when Jeff Vance (Durham, NC) (6)
reported linkage of CMT1A to chromosome 17 using the same marker that revealed linkage
to NF1 on chromosome 17. We and others confirmed this chromosome 17 linkage (7).
Much effort was now focused on identifying, and/or making more informative, DNA mark-
ers for the pericentromeric region of chromosome 17. Yusuke Nakamura (Tokyo, Japan) had
provided some chromosome 17 cosmid clones, which were used to identify chromosome 17
polymorphic markers. Also, Pragna developed a novel method to obtain region specific chro-
mosome 17 markers using differential Alu-PCR (8). At the time Alu-PCR had been recently
developed in our Institute for Molecular Genetics by David Nelson in Tom Caskey’s lab (9).
To identify region specific markers, Alu-PCR was performed on somatic cell hybrids that
retain either intact human chromosome 17 or a deleted chromosome from a patient with Smith-
Magenis syndrome (SMS) [del(17)(p11.2p11.2)] (10). Amplification products were compared
and if a band was present in the amplification from the hybrid retaining intact chromosome 17,
but not from the amplification of the hybrid with the deletion chromosome, then this was
surmised to physically come from the specific deleted region. Of course, one also identifies Alu
polymorphisms this way. We found that the procedure could be remarkably simplified by first
reducing the genome complexities using restriction endonuclease digestion before the Alu-
PCR. This, in turn, lead to the development of “restricted-Alu PCR” (11). By 1989 we had
accumulated extensive mapping data to show that the CMT1A locus was on the short arm of
chromosome 17 and most tightly linked to markers that were physically located within the
common SMS deletion interval in 17p11.2.
Here I must digress to say that much of our daily business was centered around marker
genotyping using RFLPs. It was clear that some markers worked better than others, for some
the segregating alleles were easier to score than for others, and in general each DNA marker
had its own “personality.” There were clearly certain DNA markers that appeared to show an
artifact of different hybridization intensities for cross-hybridizing bands on genomic Southern
blots. However, individuals from the same families were not always run adjacent to each other
on the genomic Southerns. By no means did we initially recognize that the presumed artifact
of “dosage differences between cross-hybridizing bands” segregated in a Mendelian fashion.
Scoring of alleles was done independent of knowledge of affection status. Linkage analyses

were performed in collaboration with Aravinda Chakravarti (Baltimore, MD), and I worked
mostly with his student Susan Slaugenhaupt, who would input the data from the scoring sheets
for the analyses.
Although RFLP mapping was proceeding, much effort was also expended on screening the
proximal 17p linked probes for the presence of simple sequence repeats (SSR; e.g., [GT]
n
)
because these were just identified in the human genome (12,13), determined to be highly
polymorphic, and could be rapidly analyzed by PCR. Odila Saucedo-Cardenas cloned and
sequenced several different SSRs from CMT1A-linked markers and developed flanking primer
sets with Roberto Montes de Oca-Luna that could be used in the PCR to type CMT1A families.
Odila and Roberto were, at the time, both Research Technicians in the laboratory. Roberto
6 Part I / Introduction
made an interesting observation for one of the SSR markers, termed RM11-GT with SSR
(TA)
5
(GT)
17
(AT)
8
(see Fig. 1) (14). When primers were used to type the CMT1A families for
this marker, one often found three alleles rather than the usual two expected with one inherited
from each parent. Three alleles were observed in many individuals with CMT1A, but not in
all—the marker was not always fully informative. Three alleles were not observed in unaf-
fected family members with the exception of three individuals who were asymptomatic; how-
ever, these latter three seemingly unaffected individuals had not had nerve conduction studies.
Subsequent NCV studies revealed decreased motor NCVs consistent with CMT1 and con-
firmed our suspicion that these individuals had subclinical, not yet penetrant disease.
Roberto examined some of the Southern blots that utilized an RFLP marker from the same
locus and noted that often when there were three alleles revealed by RM11-GT (14), a dosage

difference could be observed between the two alleles if the affected individual was heterozy-
gous for that RFLP. These initial observations suggested that there may be three copies of the
genomic region that was being assayed, potentially reflecting genomic duplication at the
CMT1A locus. The entire laboratory now focused on the “duplication hypothesis” and, to keep
our hypothesis quiet, it was referred to as the “D” word within the laboratory because we all
focused on gathering data to support or refute the duplication hypothesis using multiple inde-
pendent molecular approaches. When now correcting for diagnosis (i.e., making sure that all
apparent unaffected individuals did not have subclinical disease by performing NCVs and
Fig. 1. Nucleotide sequence of the simple sequence repeat RM11-GT. Autoradiogram of a DNA sequenc-
ing gel showing the repeat, which lies at the basis of the polymorphic DNA marker RM11-GT. Initial
evidence for the Charcot-Marie-Tooth type 1A (CMT1A) duplication was revealed by this marker that
showed three alleles (i.e., triallelic) in fully informative CMT patients.