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Preimplantation
Genetic Testing
 Preimplantation
Genetic Testing
Recent Advances in Reproductive Medicine

Edited by
Darren K. Griffin, BSc, PhD, PGCertHE,
CBiol, FRSA, FRSB, FRCPath, DSc
Professor of Genetics, School of Biosciences
Director, Centre for Interdisciplinary Studies of Reproduction
University of Kent
Canterbury, United Kingdom

Gary L. Harton, PhD

Global Director, Market Development PGT
PerkinElmer, Inc.
Waltham, Massachusetts, USA
Honorary Senior Lecturer, University of Kent
Canterbury, United Kingdom
Teacher, Eastern Virginia Medical School
Norfolk, Virginia, USA
Teacher, Bryant University
Smithfield, Rhode Island, USA
First edition published 2020
by CRC Press
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and by CRC Press

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© 2020 by Taylor & Francis Group, LLC

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Contents

Foreword................................................................................................................................................... vii
Editors........................................................................................................................................................ ix
Contributors............................................................................................................................................... xi

1. Preimplantation Genetic Testing and Reproductive Genetics from a Physician’s Perspective.......1

Kateřina Veselá

2. Genetic Counseling for Preimplantation Genetic Testing...........................................................11

Lauri D. Black and Jill M. Fischer

3. Preimplantation Genetic Testing for Aneuploidies: Where We Are and Where We’re
Going................................................................................................................................................ 25
Andrea Victor, Cagri Ogur, Alan Thornhill, and Darren K. Griffin

4. Preimplantation Genetic Testing for Structural Rearrangements............................................ 49

Cagri Ogur and Darren K. Griffin

5. Preimplantation Genetic Testing for Monogenic Disorders....................................................... 77

Martine De Rycke and Pieter Verdyck

6. Novel Methods in Preimplantation Genetic Testing: Comprehensive Preimplantation

Genetic Testing................................................................................................................................ 87
Olga Tsuiko and Joris Robert Vermeesch

7. Time-Lapse Imaging and Preimplantation Genetic Testing...................................................... 97

Alison Campbell

8. Mosaicism Mechanisms in Preimplantation Embryos...............................................................111

Maurizio Poli and Antonio Capalbo

9. Transfer of Mosaic Embryos........................................................................................................ 127

Francesco Fiorentino, Ermanno Greco, Maria Giulia Minasi, and Francesca Spinella

10. Noninvasive Methods of Preimplantation Genetic Testing for Aneuploidies......................... 139

Luis Navarro-Sánchez, Carmen María García-Pascual,
Lucía Martínez-Merino, Carlos Simón, and Carmen Rubio

11. Preimplantation Genetic Testing in the Future..........................................................................151

Joe Leigh Simpson, Svetlana Rechitsky, and Anver Kuliev
Index....................................................................................................................................................... 157

v
Foreword

Thirty years on from the first pregnancies and live births following IVF, embryo biopsy, and single-cell
genetic analysis in couples at risk of X-linked diseases [1], preimplantation genetic diagnosis (PGD), or
preimplantation genetic testing for monogenic disease (PGT-M), as it has recently been renamed, is now
well established as a valuable alternative to prenatal diagnosis for a wide range of inherited conditions
[2,3].
In the early days, embryos could only be cultured for 2 or 3 days post insemination (Day 2 or 3)
before they were transferred to the uterus, and even delaying transfer to Day 4 reduced implantation and
pregnancy rates. The slow freezing protocols used for cryopreservation of embryos at these early stages
were rudimentary, and often embryos were destroyed or partially damaged in the thawing process. The
challenge, therefore, was to remove a single cell from each embryo early on Day 3 at the 6- to 10-cell
stage to minimize the damage to the embryo, perform the genetic analysis for the mutation on single
copies of the parents’ chromosomes within a few hours, and then select unaffected embryos for transfer
later the same day. This could only be contemplated because of the emergence in the mid-1980s of the
polymerase chain reaction (PCR), which allowed the million-fold amplification of short fragments of
DNA. In addition, human genetics was still in its infancy and the mutations causing only a handful of
common inherited diseases had been identified.
Today, there have been major advances in all aspects of preimplantation genetics that have revolutionized
our ability to interrogate the genetics of human gametes and embryos. Embryo culture to the blastocyst
stage on Days 5 to 7 and biopsy of multiple trophectoderm cells using laser-assisted hatching and excision
is now routine. Cryopreservation of biopsied blastocysts, using ultra-rapid freezing, or vitrification, is
highly efficient and minimally damaging, removing any time constraints on the genetic analysis. Most
importantly, the ability to amplify the whole genome of single or small numbers of cells efficiently and
generate micrograms of DNA has revolutionized our ability to perform genetic tests enabling the use of
genome-wide methods including microarrays and next-generation sequencing (NGS). Alongside these
developments, the sequencing of the human genome in the early 2000s and progress in identifying
the genetic basis of thousands of monogenic diseases and other conditions has expanded the range of
applications for PGT. These include rare severe childhood lethal diseases, late-onset diseases, genetic
predisposition to cancers, and tissue typing to enable cord blood stem cells to be used for transplantation
to existing affected children in the family.
Chromosome aneuploidy is a major cause of pregnancy failure and loss following natural conception.
The use of various molecular cytogenetic methods for PGT of aneuploidy (PGT-A) has confirmed the
high incidence of aneuploidy in human gametes and embryos, particularly in oocytes in which the
incidence increases exponentially in women above the age of 35 years. PGT-A following blastocyst biopsy
is therefore widely used for euploid embryo selection for infertile patients and there is accumulating
evidence for significantly improved implantation rates and live birth outcomes, particularly following
single vitrified-warmed embryo transfer in later managed cycles. However, the use of quantitative, mainly
NGS-based methods, for 24-chromosome copy number analysis has revealed a relatively high incidence
of intermediate copy number changes interpreted as indicating mosaicism for aneuploidies among the
sampled trophectoderm cells. The clinical significance of low-level mosaicism is not fully understood
since there are now reports of apparently healthy live births following transfer of embryos with only
this type of copy number change. One explanation is that the affected trophectoderm cells may not be
representative of the inner cell mass from which the fetus is derived. Alternatively, these cells may simply
not be viable, and die out during early development.
Inherited diseases typically affect all the cells and tissues of the body, and the clinical manifestations
are often multisystemic. This makes them challenging to treat. However, there has been some progress.
For example, there is now a drug that can effectively arrest the neurological degeneration in children

vii
viii Foreword

affected by Batten disease [4] and there has been some success with gene therapy for beta thalassemia [5].
However, these therapies remain highly expensive and limited in their availability. For couples who know
that they are at risk of having affected children either because of a clinical diagnosis in an existing child
or family member or increasingly because of the availability of preconception screening for extensive
panels of serious recessive monogenic diseases, PGT or prenatal diagnosis following natural conception
remain the only options for them to have their own healthy children.
Thirty years ago, it was anticipated that early noninvasive methods of diagnosing inherited disease from
trophoblast cells recovered from the lower pole of the uterus or from cell-free fetal DNA in maternal blood
would be developed and would, within a few years, replace PGT because of the expense and difficulty of
the IVF process, especially for fertile couples. In the event, it would be over two decades later following
the development of NGS that noninvasive prenatal testing (NIPT) for common aneuploidies became
possible and only now that this approach is being extended to genome-wide aneuploidies, microdeletion
syndromes, and some monogenic diseases [6].
Live birth rates following IVF have increased steadily over the last 30 years with improvements to
culture media and laboratory conditions, and with PGT-A, implantation rates per embryo transfer can
now average 60%–80% irrespective of maternal age. In the early years, many women in their 40s having
PGT-M had repeated cycles without success, presumably because the majority or all of the unaffected
embryos that were transferred had one or more aneuploidies and were not viable. Today, using combined
PGT-M and aneuploidy testing (PGT-M/A), clinical outcomes are similarly improved by transferring
only euploid-unaffected embryos, and patients can make an informed choice about continuing treatment.
Preimplantation genetic testing remains technically challenging and demanding and requires a
multidisciplinary team, including expert genetic counseling to ensure the couple are fully aware of their
options and the possible outcomes. Nevertheless, the birth of thousands of unaffected children worldwide
is a testament to the value couples place on starting a pregnancy knowing that it is unaffected, and a
reward for all the dedicated work of those involved in helping them to have healthy children.

Alan H. Handyside
School of Biosciences
University of Kent
Canterbury, United Kingdom

REFERENCES
1. Handyside AH, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human
preimplantation embryos sexed by Y-specific DNA amplification. Nature. 1990;344.
2. Handyside AH. ‘Designer babies’ almost thirty years on. Reproduction. 2018;156:F75–9.
3. Niederberger C et al. Forty years of IVF. Fertil Steril. 2018;110:185–324.e5.
4. Kohlschütter A, Schulz A, Bartsch U, Storch S. Current and emerging treatment strategies for neuronal
ceroid lipofuscinoses. CNS Drugs. 2019;33:315–25.
5. Ikawa Y, Miccio A, Magrin E, Kwiatkowski JL, Rivella S, Cavazzana M. Gene therapy of
hemoglobinopathies: Progress and future challenges. Hum Mol Gen. 2019;28(R1):R24−30.
6. Hayward J, Chitty LS. Beyond screening for chromosomal abnormalities: Advances in non-invasive
diagnosis of single gene disorders and fetal exome sequencing. Semin Fetal Neonatal Med. 2018;23:94–101.
Editors

Darren K. Griffin earned his Bachelor of Science and Doctor of Science degrees from the University
of Manchester and his PhD from University College London. After postdoctoral stints at Case Western
Reserve University (Cleveland, Ohio) and the University of Cambridge, he had his first academic post
at Brunel University (London), before settling at the University of Kent (Canterbury, United Kingdom),
where he has worked for over 15 years. He has worked under the mentorship of Professors Joy Delhanty,
Christine Harrison, Terry Hassold, Alan Handyside, and Malcolm Ferguson-Smith.
He is president of the International Chromosome and Genome Society and a Fellow of the Royal
College of Pathologists, the Royal Society of Biology, and the Royal Society for the Encouragement of
Arts, Manufactures and Commerce. He sits on the faculty of CoGEN (Controversies in Genetics) and
has previously sat on the board of the Preimplantation Genetic Diagnosis International Society (PGDIS),
organizing its annual meeting in 2014.
Professor Griffin is a world leader in cytogenetics. He performed the first successful cytogenetic PGD
(using X and Y FISH probes for sexing) and, more recently, played a significant role in the development
of karyomapping, an approach he now applies to cattle. Throughout a scientific research career of over
30 years he has co-authored over 200 scientific publications, mainly on the cytogenetics of reproduction
and evolution, most recently providing insight into the karyotypes of dinosaurs.
He is a prolific science communicator, making every effort to make scientific research publicly
accessible (both his own and others’) and is an enthusiastic proponent for the benefits of interdisciplinary
research endeavours. He has supervised over 35 PhD students to completion, and his work appears
consistently in the national and international news. He currently runs a vibrant research lab of about 20
people (including a program of externally supervised students) and maintains commercial interests in the
outcomes of research findings, liaising with companies in the field.

Gary L. Harton earned his Bachelor of Science degree from James Madison University (Harrisonburg,
Virginia) and his PhD at the University of Kent (Canterbury, United Kingdom). His thesis title was
“Facilitating the widespread use of preimplantation genetic diagnosis and screening through best practice
and novel technology development.”
After more than 20 years serving in various roles, including both clinical laboratory and management
positions, he began his commercial career performing business development and market development
roles with two prominent genetics companies, a fertility startup and a large reference genetics laboratory.
Dr. Harton has been involved in a number of exciting breakthroughs in the field of reproductive genetics,
including performing the first clinical preimplantation genetic diagnosis (PGD) for an autosomal dominant
disease (Marfan syndrome) and performing the first clinical PGD for spinal muscular atrophy (SMA).
In addition, Dr. Harton was involved in the team that pioneered non-disclosing Huntington disease
(HD) PGD, where a patient at risk for HD could ensure that their children were free from the mutation
without discovering their own HD status. He was also involved in the team that discovered and patented
karyomapping, a revolutionary new technology allowing practically any genetic defect to be diagnosed
in embryos using one single array-based test.
Dr. Harton is certified by the American Board of Bioanalysis (ABB) as a technical supervisor in
molecular diagnostics and is a member of the American Society for Reproductive Medicine (ASRM) as
well as the European Society of Human Reproduction and Embryology (ESHRE), and the Preimplantation
Genetic Diagnosis International Society (PGDIS). Dr. Harton is currently a member of the board of
directors for PGDIS and a member of the Special Advisory Group for United Kingdom-NEQAS, which
performs external quality assessment schemes for preimplantation genetics. He has also served as chair
of the Steering Committee for the ESHRE PGD and has been an author on numerous peer-reviewed
scientific articles, abstracts, and book chapters.

ix
Contributors

Lauri D. Black Darren K. Griffin

Pacific Reproductive Genetic Counseling Centre for Interdisciplinary Studies of
Pacifica, California Reproduction (CISoR)
School of Biosciences
Alison Campbell University of Kent
CARE Fertility Canterbury, United Kingdom
Nottingham, United Kingdom
Alan H. Handyside
Antonio Capalbo
Centre for Interdisciplinary Studies of
IGENOMIX
Reproduction (CISoR)
Marostica (VI), Italy
School of Biosciences
and University of Kent
Canterbury, United Kingdom
Dipartimento di Scienze Anatomiche, Istologiche
Medico-Legali e dell’Apparato Locomotore Anver Kuliev
Sezione Istologia ed Embriologia Medica Department of Human and Medical
Sapienza University of Rome Genetics
Rome, Italy Herbert Wertheim College of Medicine
Martine De Rycke Florida International University
Centre for Medical Genetics Miami, Florida
Universitair Ziekenhuis Brussel and
and Reproductive Genetic Innovations (RGI)
Reproduction and Genetics Northbrook, Illinois
Vrije Universiteit Brussel (VUB)
Brussels, Belgium Lucía Martínez-Merino
R&D Department
Francesco Fiorentino IGENOMIX
GENOMA Group Valencia, Spain
Molecular Genetics Laboratories
Rome and Milan, Italy Maria Giulia Minasi
Reproductive Medicine
Jill M. Fischer
European Hospital
New Jersey Center for Science, Technology &
Rome, Italy
Mathematics (NJCSTM)
Kean University
Luis Navarro-Sánchez
Union, New Jersey
R&D Department
Carmen María García-Pascual IGENOMIX
R&D Department Valencia, Spain
IGENOMIX & IGENOMIX Foundation
INCLIVA Cagri Ogur
Valencia, Spain Bahҫeci Genetic Diagnosis Center
Altunizade
Ermanno Greco and
Reproductive Medicine Department of Bioengineering
European Hospital Yildiz Technical University
Rome, Italy İstanbul, Turkey

xi
xii Contributors

Maurizio Poli Francesca Spinella

IGENOMIX GENOMA Group
Marostica (VI), Italy Molecular Genetics Laboratories
and Rome and Milan, Italy
REPROOMICS Alan Thornhill
Amsterdam, Netherlands Centre for Interdisciplinary Studies of
Svetlana Rechitsky Reproduction
Department of Human and Medical School of Biosciences
Genetics University of Kent
Herbert Wertheim College of Medicine Canterbury, United Kingdom
Florida International University and
Miami, Florida
Igenomix United Kingdom, Ltd
and Surrey, United Kingdom
Reproductive Genetic Innovations (RGI)
Northbrook, Illinois Olga Tsuiko
Laboratory of Cytogenetics and Genome Research
Carmen Rubio Department of Human Genetics
R&D Department KU Leuven
IGENOMIX & IGENOMIX Foundation Leuven, Belgium
INCLIVA
Valencia, Spain Pieter Verdyck
Centre for Medical Genetics
Carlos Simón Universitair Ziekenhuis Brussel
R&D Department Reproduction and Genetics
IGENOMIX and
and Vrije Universiteit Brussel (VUB)
Department of Obstetrics and Gynaecology Brussels, Belgium
Valencia University
and Joris Robert Vermeesch
IGENOMIX Foundation Laboratory of Cytogenetics and Genome Research
INCLIVA Department of Human Genetics
Valencia, Spain KU Leuven
Leuven, Belgium
and
Department of Obstetrics and Gynecology Kateřina Veselá
School of Medicine REPROMEDA Biology Park
Stanford University Brno, Czech Republic
Stanford, California
Andrea Victor
Joe Leigh Simpson Centre for Interdisciplinary Studies of
Department of Human and Medical Genetics Reproduction
Herbert Wertheim College of Medicine School of Biosciences
Florida International University University of Kent
Miami, Florida Canterbury, United Kingdom
and and
Reproductive Genetic Innovations (RGI) Zouves Fertility Center
Northbrook, Illinois Foster City, California
1
Preimplantation Genetic Testing and Reproductive
Genetics from a Physician’s Perspective

Kateřina Veselá

CONTENTS
Introduction................................................................................................................................................. 1
Cytogenetic Testing..................................................................................................................................... 2
Multiple-Gene NGS Panels......................................................................................................................... 2
Description of Molecular Genetic Methods Used in the “PANDA” Panel Clinically at Repromeda,
Brno, Czech Republic................................................................................................................................. 2
Cystic Fibrosis....................................................................................................................................... 3
Spinal Muscular Atrophy....................................................................................................................... 3
Deafness (Mutation in Connexin 26)..................................................................................................... 3
Fragile X Syndrome............................................................................................................................... 3
Diagnosis of Other Potential Fertility Disorders................................................................................... 4
Genetic Testing of Gamete Donors............................................................................................................. 5
Therapeutic Procedures for Fertility Disorders and Genetic Defect Risks Using Genetic Methods.......... 5
PGT-A.................................................................................................................................................... 6
Mosaicism.............................................................................................................................................. 6
Genetic Methods to Detect the Window of Implantation........................................................................... 7
Genetic Testing in the Surrogacy Program................................................................................................. 7
Genetic Methods in the Control of Therapeutic Results............................................................................. 8
Summary..................................................................................................................................................... 8
References................................................................................................................................................... 9

Introduction
Reproductive genetics is now inseparably linked to reproductive medicine procedures and is becoming
an integral part thereof. It has been applied in the diagnosis of factors of infertility and genetic disorder
carrier status. This is specifically used for screening assessments before admitting a couple to an
in vitro fertilization (IVF) program for targeted diagnosis of hereditary diseases or defects, or as part
of preimplantation genetic testing (PGT) procedures. In many cases it is becoming an integral part of
controlling therapeutic results in the form of noninvasive or invasive prenatal diagnosis.
Reproductive-genetic counseling in infertile couples is a comprehensive communication process
purposed to evaluate a risk for genetic disorder in an offspring and to discuss measures to minimize that
risk and improve the results of the treatment. Options for optimal testing and screening, interpretation
and implications of test results, further education about a potential genetic diagnosis and prognosis, and
emotional support are highly important aspects of a reproductive-genetic counseling visit. The core
ethical principles of genetic counseling are the autonomy of the individual or couple, their right to full
information, and the highest standards of confidentiality.
Although crude monetary arguments are not generally used to justify the provision of genetic counseling
services, financial analysis might show a net cost savings to society/family by the reduction of expenditure
on caring for those with serious genetic disease or disability.
1
2 Preimplantation Genetic Testing

Our contemporary professional goals in reproductive genetics are quite different from former ideas
of eugenic medicine (“the health of the people,” i.e., of the race). Respect for individual autonomy takes
precedence over measures of the impact of genetic services on the population. We have always to comply
with the wish for having a healthy baby, which is universal in all couples. Reproductive genetics is a part
of medicine whose general purpose is to prevent or at least minimize suffering and distress.

Cytogenetic Testing
Basic cytogenetic tests used in reproductive medicine include karyotyping, performed for many years
using G-banding [1] in cultured peripheral blood cells (lymphocytes). One of the best-established means
of genetic diagnosis, adaptations of the original approach are used to this day, supplemented by enhanced
banding techniques and digitized analysis. Given the dynamic development of molecular biology, this
method has, in part, been successfully converted to a molecular genetic platform for routine use in a short
time horizon. Currently, flatbed microarrays and the single nucleotide polymorphism (SNP) array method
[2] are used for closer specifications of the findings of classical karyotyping if there are any unclear
aspects, and in special indications. Microarrays provide considerably higher resolving power compared
to G-banding, and can be used to detect microdeletions or microduplications as low as 0.5 Mb.
Expanded carrier screening (ECS) of patients as well as gamete donation is performed ever more
commonly as part of testing before inclusion in the IVF program. Similarly, as in the case of new
preimplantation and prenatal testing methods, the development of ECS has been made possible by the
arrival of newer developments in molecular genetics [3].

Multiple-Gene NGS Panels

The introduction of next-generation sequencing (NGS) (including massive parallel sequencing [MPS])
has allowed a substantially wider and more detailed analysis of the genetic causes and relationships of
pathological conditions [4]. Multiple-gene panels allow testing of many genes in a very short time. Such
panels are typically a set of genes tested in parallel; physically, it is a set of DNA segments used to prepare
a library, subsequently used as the basis for sequencing [5,6].
Tests using multiple-gene panels are most commonly used in oncology to detect inherited mutations
in oncogenes [7], and in cardiological diagnosis [8]. Currently, their use is rapidly increasing for use in
reproductive medicine [4–6]. Some reproductive clinics use standardized commercial panels, while others
design an individual structure and assign the production of panels created based on their own specific
requirements.
The purpose of using panels in modern reproductive medicine is to determine any potential genetic
causes of fertility disorders in the man and woman, to uncover any gene mutations that might lead to
the development of serious inherited diseases in the offspring, and finally to detect any factors posing
potential risks to infertility therapy which could have potentially adverse impacts on the result.
Regularly tested gene mutations that cause the most common serious inherited diseases include mutations
in the CFTR gene causing cystic fibrosis, the SMN1 gene causing spinal muscular atrophy, the GJB2 gene
causing nonsyndromic deafness, and the FMR1 gene causing fragile X syndrome. Sites in different countries
may include isolated tests of various mutations or implement these in multiple-gene panels with respect to
habitual practice, patient requirements, legal regulations, or guidelines of a local professional association.

Description of Molecular Genetic Methods Used in the “PANDA”

Panel Clinically at Repromeda, Brno, Czech Republic
The scientists and clinicians at Repromeda have developed a custom NGS panel that assesses a number
of specific mutations as noted previously. Details of this test, called PANDA, are included next to
highlight one specific use of custom panels used during an IVF cycle.
Preimplantation Genetic Testing and Reproductive Genetics from a Physician’s Perspective 3

In brief, the test includes steps for performing preparation of the library: Custom Amplicon Library
NEXTFlex NOVA-4301-03 Bioo Scientific Corp. (Perkin Elmer), sequencing using the MiSeq system
(Illumina), evaluation using SeqPilot software (JSI Medical Systems), Repromeda bioinformatics
software—SOP00417.
Additional detection test (CGG)n—Fragile X, Repromeda CGG RP PCR detection and AmplideX
FMR1 PCR Kit (Asuragen), fragmentation analysis—SOP 00415. This is a diagnostic genetic test
designed for the testing of mutations that cause the most common genetic diseases in the Central European
population; furthermore, thrombophilic mutations and genes/variants clinically related to infertility,
hormonal stimulation, embryonal development, and pregnancy losses are tested.

Cystic Fibrosis
The 5’UTR region of the CFTR gene, the entire coding region of the gene CFTR±10 bp exon/intron
overlap, poly-T/TG allelic variant in intron 8, mutations of CFTRdele2,3 kb, 1811+1,6 kbA>G,
3272−26A>G and 3849+10 kbC>T mutation are tested. The testing cannot detect changes in
copy number variants (CNV) of individual exons except the deletion of exons 2 and 3 (mutation
CFTRdele2,3,kb), or intra-gene rearrangements in the CFTR gene. If the test result is negative (i.e.,
no evidence of carrier status), the residual risk for the presence of a pathogenic variant in the CFTR
gene in the subject is ≈ 1:900.
Besides its application for excluding the risk of cystic fibrosis being passed to the next generation,
CFTR gene testing is also important in the diagnosis of male infertility. Mutations can cause congenital
absence of the vas deferens (bi- or unilateral), bilateral obstruction of the ductus ejaculatorius, or bilateral
obstruction of the ductus epididymis. All men with idiopathic obstructive azoospermia should thus have
their CFTR gene examined.

Spinal Muscular Atrophy

Deletion of exons 7 and 8 of the SMN1 gene is tested, as this deletion is present in most carriers of spinal
muscular atrophy. The testing is associated with 5% false negative results. Carriers exist who have two
copies of exons 7 and 8 of the SMN1 gene on one chromosome and no copy on the other chromosome,
which cannot be differentiated using any available method. Approximately 6% of patients with SMA may
have a de novo deletion or another congenital intra-gene mutation in the SMN1 gene. If the test result is
negative (no evidence of carrier status), the residual risk for the presence of a pathogenic variant in the
SMN1 gene in the subject is ≈ 1:1500.

Deafness (Mutation in Connexin 26)

We test the entire coding region of the GJB2 gene, which has only one exon. The test cannot detect
changes in the CNVs of the GJB2 gene and/or any intra-gene rearrangements. If the test result is negative
(no evidence of carrier status), the residual risk for the presence of a pathogenic variant in the GJB2 gene
in the subject is ≈ 1:740.

Fragile X Syndrome
We test CGG repeats in the FMR1 gene. According to the European Society of Human Genetics, (CGG)
n < 45 is a normal finding, the range of 45–54 is a normal finding in the “grey zone,” 55–200 means
premutation, and n > 200 is full mutation. FMR1 gene mutation testing falls both in the group of gene
mutation tests and of infertility cause tests given that premature ovarian failure is more common in female
carriers of the premutation, and therefore decreased ovarian reserve in young women should also be an
indication for the testing of this gene.
Additional genes tested in the previously named panel include thrombophilic mutations—coagulation
factor V Leiden mutation (G169A) and R2 (H1299R) mutation; factor II prothrombin (G2021A mutation);
MTHFR (methylene tetrahydrofolate reductase—C667T and A1298C mutations); plasminogen activator
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