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Practical Preimplantation Genetic Diagnosis
Yury Verlinsky and Anver Kuliev
Practical
Preimplantation
Genetic Diagnosis
With 125 Figures
Yury Verlinsky, PhD
Anver Kuliev, MD, PhD
Reproductive Genetics Institute, Chicago, IL, USA
British Library Cataloguing in Publication Data
A catalogue record for this book is available from the British Library
Library of Congress Control Number: 2005923550
ISBN-10: 1-85233-920-9
Printed on acid-free paper
ISBN-13: 978-1-85233-920-3
C Springer-Verlag London Limited 2005
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Contents
Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
vii
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ix
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
xi
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
xiii
1. Primary Prevention of Genetic Disorders and Place of Preimplantation
Genetic Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
2. Approaches to Preimplantation Genetic Diagnosis . . . . . . . . . . . . . . . . . . . . .
2.1 Obtaining Biopsy Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.1 Polar Body Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.2 Preconception Testing for Paternally Derived Mutations
by Sperm Duplication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.3 Development of Artificial Human Gametes
in Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.4 Embryo Biopsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2 Single-Cell Genetic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1 DNA Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.2 FISH Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. preimplantation Genetic Diagnosis for Single Gene Disorders . . . . . . . . . .
3.1 Autosomal Recessive Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2 Autosomal Dominant Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1 Primary Torsion Dystonia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2 Charcot-Marie-Tooth Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4 Homozygous or Double Heterozygous
Recessive Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5 Conditions with No Available Direct Mutation Testing . . . . . . . . . . .
3.6 Late-Onset Disorders with Genetic Predisposition . . . . . . . . . . . . . . .
3.6.1 p53 Tumour-Suppressor Gene Mutations . . . . . . . . . . . . . . . . .
3.6.2 Neurofibromatosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6.3 Other Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6.4 Alzheimer Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.7 Blood Group Incompatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7
8
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CONTENTS
3.8 Congenital Malformations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
3.8.1 Sonic Hedgehog Mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
3.8.2 Currarino Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
3.8.3 Crouzon Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
3.9 Dynamic Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
4. Preimplantation HLA Typing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1 PGD for Fanconi Anemia with Preimplantation
HLA Matching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2 Current Experience of PGD with Preimplantation
HLA Matching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3 Preimplantation HLA Matching Without PGD . . . . . . . . . . . . . . . . . .
4.4 Worldwide Experience on Preimplantation HLA Typing . . . . . . . . .
111
5. Preimplantation Genetic Diagnosis for Chromosomal Disorders . . . . . . .
5.1 Aneuploidies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.1 Complex Errors and Aneuloidy Rescue
in Female Meiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.2 Mitotic Errors in Cleaving Embryos
in Relation to Meiosis Errors . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.3 PCR-Based Aneuploidy Testing
in Cleaving Embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.4 Practical Relevance of Autosomal
Monosomy Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.5 Uniparental Disomies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2 Chromosomal Rearrangements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
135
140
111
113
124
128
146
149
152
153
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157
6. Clinical Outcomes Following Preimplantation
Genetic Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
7. Preimplantation Genetic Diagnosis and Establishment of
Human Embryonic Stem Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1 Marfan Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2 Thalassemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3 Fragile-X Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.4 Myotonic Dystrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.5 Neufibromatosis Type 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.6 Becker Muscular Dystrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
175
179
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179
182
182
182
8. Ethical, Social, and Legal Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Foreword
The casual reader may not fully appreciate the significance of the well-chosen title for this
unique and welcome volume, Practical Preimplantation Genetic Diagnosis. The title is not
only apropos but provided by the team that truly made it possible.
Prenatal genetic diagnosis began in the 1950s, with detection of X-chromatin in amniotic
fluid cells. By the late 1960s amniocentesis allowed diagnosis of chromosomal abnormalities
and selected inborn errors of metabolism. Labs sprung up worldwide, and in the 1970s
antenatal testing became standard in certain patients. The 1980s brought two key advances.
First was noninvasive screening, with an invasive procedure like amniocentesis performed
only if the risk was high enough to be justified. Concurrently, there was movement toward
prenatal diagnosis earlier in pregnancy, as witnessed by chorionic villus sampling (CVS).
Still, diagnosis was not possible before 10 to 12 weeks, and, hence, clinical pregnancy
termination was still necessary.
Although proof of the principle for preimplantation genetic diagnosis (PGD) had been
shown in 1967 by Professor Robert Edwards through X-chromatin analysis in rabbit blastocysts, application in the human awaited advances in human assisted reproductive technologies (ARTs) and in molecular techniques. The first human PGD finally came only in
1990. During the next few years, there was great interest in PGD in the genetic community; however, relatively few cases were performed, mostly for mendelian disorders. With
introduction of fluorescent in situ hybridization (FISH), however, PGD was burgeoning.
By 2000, novel indications were added, including aneuploidy testing to identify chromosomally normal embryos. Indeed, this is now the most common indication for PGD.
Practical Preimplantation Genetic Diagnosis systematically covers indications and technology. One is taken through PGD from start (embryo) biopsy to end (consequences), and
we learn from the group that has performed half the world’s cases. The technique for obtaining embryonic DNA is first described (polar body biopsy or blastomere biopsy), followed
by single-cell genetic analysis (FISH or polymerase chain reaction–based approaches). The
spectrum of mendelian disorders and their detection follows. Once controversial topics like
late-onset disorders are covered, with especially lucid coverage on PGD to obtain human
leukocyte antigen (HLA)-compatible embryos. Preimplantation genetic diagnosis not only
avoids another affected offspring, but also provides umbilical cord–derived stem cells for
transplantation into an older moribund sib. Both numerical and structural chromosomal
abnormalities are discussed. We are given novel information on the unexpectedly high
sequential error rate in meiosis I followed by meiosis II. Clinical outcome and the safety
of PGD are discussed (and reassurance is given). Throughout, the discussion flows seamlessly between indication and counseling (reflecting the impeccable genetic credentials of
the authors) and laboratory performance (likewise). Illustrations are highly informative
and lucid. This is invaluable in understanding PGD for mendelian disorders, the diagnosis
of which requires linkage analysis. The illustrations make a potentially arcane topic quite
clear.
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FOREWORD
Overall, one comes away with a keen appreciation of how PGD has moved from “boutique” medicine, practiced only rarely by the cognoscenti, to a technique rapidly becoming
de rigueur in all in vitro fertilization (IVF) centers. Within 5 to 10 years, surely most, if not
all, ART will require genetic testing prior to embryo transfer.
Considerable information is new and not heretofore available even in peer review format.
The result is a pleasing combination of reading a textbook peppered with peer review
information. The sense of movement in PGD is thus palpable.
Practical Preimplantation Genetic Diagnosis is thus a gem. In this age of edited works,
a text written by only one or two authors is rare. That our authors practically invented
the field, and certainly have made it practical, makes this volume truly extraordinary.
This volume should thus be at the reach of every reproductive geneticist, ART lab, and
reproductive biologist. The avid student will come away fully informed and up to date, and
also ready for tomorrow’s advances.
Joe Leigh Simpson, M.D.
Ernst W. Bertner Chairman and Professor
Department of Obstetrics and Gynecology
Houston, Texas
June 2005
Preface
Although treatment is the major goal in the control of genetic disease, as in other fields
of medicine, this is not yet a reality for most inherited conditions. Even with dramatic
advancement in the field of gene therapy, there are not, unfortunately, many success stories
to allow predicting its sound impact in the near future. Therefore, in the absence of radical
treatment, prevention of genetic disorders is still one of the available options for genetically
disadvantaged people. Among the measures for prevention, the avoidance of the birth of
an affected child to couples at genetic risk has become a quite acceptable option in many
populations. This is based on population screening and prenatal diagnosis, and has been
quite successful, resulting in almost eradicating new cases of some genetic diseases from
several Mediterranean populations, such as those in Sardinia and Cyprus. However, this
has generated an increasing number of abortions following prenatal diagnosis, leading to
a growing concern and negative reaction in society to the preventive genetics programs.
For example, some ethnic groups cannot accept any control measures regarding congenital
diseases because pregnancy termination is not allowed due to social or religion reasons.
Even in those communities where abortion is allowed, some couples have to experience two
or more terminations of pregnancies before they can have a normal child. No doubt these
families might require an alternative option to achieve their desired family size without the
need for termination of pregnancy.
The present book is devoted to a principally new approach in prevention of genetic
disorders, which avoids the need for prenatal diagnosis and termination of pregnancy.
Accordingly, a novel concept of prepregnancy diagnosis is introduced, which is based on
the control of the processes of the oocyte maturation, fertilization, and implantation, so to
select and transfer back to patients only normal embryos, and achieve an unaffected pregnancy resulting in the birth of a healthy child. In this way, the couples at high risk of having
an offspring with genetic disease have an option to control the outcome of their pregnancy
from the onset. So the place of this approach in the context of other available approaches
for prevention of genetic disorders is discussed, together with other primary preventive
measures, which may allow presently avoiding up to a half of congenital malformations
presented at birth (Chapter 1). Although the option of prepregnancy diagnosis involves
ovarian stimulation and in vitro fertilization (IVF), the description of the available experience demonstrates that this has appeared to be an acceptable procedure in many ethnic
groups all over the world.
As described in the methodological section (Chapter 2), preimplantation genetic diagnosis (PGD) may be achieved by testing of the female, and perhaps in the future also
male, gametes or single cells from the preimplantation embryo. Both of these methods have
been used in clinical practice and have shown to be useful in prediction of the genotype
of the resulting pregnancy, although many problems still remain to be resolved, requiring careful consideration. The experience in the application of both of these approaches
and the progress in overcoming the existing problems are described, based on the original
practice of more than 3500 PGD cycles, presently comparable to the overall available world
x
PREFACE
experience, as well as the experiences from the other active PGD centres. Because basic
clinical methods for performing PGD are similar to those in IVF, which have been described previously in detail, the emphasis in this book is mainly on the laboratory aspects.
However, the clinical impact of PGD, and some of the ethical, social, and legal aspects are
also briefly explored (Chapters 6 and 8). The present progress of PGD makes clear that the
at-risk couples have to be aware of all available preventive options, including PGD, which
is a complement rather than an alternative to the available preventive genetics services,
allowing the couples to make their own choices.
Because PGD of single-gene disorders involves extensive preparatory work for each
particular condition, a special section is devoted to the description of the PGD strategy
for each group of these disorders. This includes the description of primer sequences, PCR
conditions, polymorphic markers, the experience of the application of conventional and
fluorescence PCR, allele drop out (ADO) rates, and other expected problems leading to
misdiagnosis, with the detailed presentation of relevant case reports (Chapter 3). A special
section is also devoted to preimplantation nongenetic testing, involving HLA typing, which
has recently become one of the most attractive PGD applications allowing improved access
to HLA-compatible stem cell transplantation (Chapter 4).
The application of PGD is of particular interest for assisted reproduction practices, so
this is described in detail in a special section, providing strong evidence of PGD impact
on pregnancy outcome (Chapters 5 and 6). This is not surprising, as the introduction of
methods for preselection of cytogenetically normal and avoiding the transfer of cytogenetically abnormal embryos should considerably improve the efficiency of IVF, based on
the fact that aneuploidies are major contributors to spontaneous abortions and may also
explain some of the implantation failures. Although more data are still needed to confirm positive impact on birth outcome, an extensive original experience of FISH analysis
of over 15,000 oocytes and embryos is presented (Chapter 5), together with the analysis
of previous reproductive histories of patients, demonstrating strong evidence of clinical
impact (Chapter 6). These chapters also include the follow-up studies of detected cases
with chromosomal abnormalities in the different stages of preimplantation development
to explore the problems of mosaicism and its biological significance at the cleavage stage.
It is presently being further confirmed that PGD is the most attractive option for couples carrying balanced translocations (Chapter 5). In light of these data, a pioneering work
on conversion of interphase nuclei of single biopsied blastomeres into metaphase chromosomes, based on the use of the modern nuclear transfer techniques, is described with
presentation of the original experience of the application of these methods for PGD of
translocations.
For the first time, the original experience of the latest applications of PGD to embryonic
stem cells is described, providing the possibility of obtaining preimplantation embryos
with known genotypes as a source for the establishment of custom-made embryonic stem
cells (Chapter 7).
Finally, the current status of PGD is summarized, with prospects of the application of
PGD to medical practice. This information may help not only in planning and organization of such services, but also in providing a working manual for the establishment and
performance of PGD in the framework of IVF and genetic practices.
Acknowledgements
We are indebted to our colleagues in DNA, FISH, and embryology laboratories, O. Verlinsky,
T. Sharapova, S. Ozen, K. Lazyuk, G. Wolf, Y. Illkevitch, N. Strelchenko, V. Galat,
V. Kuznyetsov, Z. Zlatoposlky, M. Chmura, V. Koukharenko, and I. Kirillova, and also
to our genetic counsellors, C. Lavine, R. Beck, R. Genoveze, and D. Pauling for their participation in acquisition of the data and technical assistance.
Contributors
Svetlana Rechitsky, PhD, Single-Gene Disorders
and HLA Typing
Jeanine Cieslak, MLT,
Micromanipulation and Chromosomal
Disorders