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List of Contributors ix
Preface to the Seventh Edition of Emery and Rimoin’s Principles and Practice of Medical Genetics and Genomics xi
Preface to Perinatal and Reproductive Genetics xiii
1 Introduction to Perinatal Disorders and Reproductive Genetics 1
10.14 Conclusion 245 References 245 Further Reading 248
11 Preimplantation Genetic Testing 249
Svetlana A. Yatsenko and Aleksandar Rajkovic
11.1 Introduction 249
11.2 Milestones in PGT 250
11.3 Indications for Preimplantation Genetic Testing 251
11.4 Technical Approaches 253
11.5 Testing and Analysis of Embryonic Nuclear DNA 254
11.6 Embryo Testing for Monogenic Conditions (PGT-M) 254
11.7 PGT-M for Mitochondrial Conditions 256
11.8 Preimplantation Genetic Testing for Structural Chromosome Rearrangements 256
11.9 Preimplantation Genetic Testing for Aneuploidy 258
11.10 Interpretation of PGT Results and Clinical Dilemmas 259
11.11 PGT-A: Mosaicism 262
11.12 Advantages and Limitations of PGT 262
11.13 Prenatal Follow-Up and Confirmatory Testing 263
11.14 Genetic Counseling 264
11.15 Future Technological Advances in ART and PGT 265
11.16 Regulatory Policies, Ethical Considerations, and Challenges in PGT 267 References 268
12 Expanded Carrier Screening 281
Ronald J. Wapner, Katherine Johansen Taber, Gabriel Lazarin and James D. Goldberg
12.1 Introduction 281
12.2 History of Reproductive Carrier Screening 281
12.3 Expanding Carrier Screening: One Gene at a Time 283
12.4 Introduction of Expanded Carrier Screening Panels 284
12.5 Changes in Technology from Genotyping to Sequencing Drive Carrier Screening Performance Improvements 285
12.6 Introduction of Expanded Carrier Screening into Clinical Practice 285
12.7 Process of Carrier Screening 286
12.8 Pretest Counseling 286
12.9 Interpretation of Molecular Findings 287
12.10 Reproductive Options for Carrier Couples Identified During Pregnancy 290
12.11 Reproductive Options for Carrier Couples Identified Before Pregnancy 290
12.12 Posttest Counseling of Pregnant Carrier Couples 290
12.13 Preimplantation Genetic Testing for Carrier Couples 291
12.14 Use of PGT-M for Identifying Potential HLA Donor Embryos for Affected Siblings 292
12.15 Conclusions 292 References 292
Index 295
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LIST OF CONTRIBUTORS
Robert G. Best
Department of Biomedical Sciences, University of South Carolina School of Medicine Greenville, Greenville, SC, United States
Vineet Bhandari
Department of Pediatrics, Cooper Medical School of Rowan University, The Children’s Regional Hospital at Cooper, Camden, NJ, United States
Jeffrey S. Dungan
Department of Obstetrics and Gynecology, Division of Clinical Genetics, Feinberg School of Medicine of Northwestern University, Chicago, IL, United States
Richard W. Erbe
Departments of Pediatrics and Medicine, University at Buffalo, Division of Genetics, Oishei Children’s Hospital, Buffalo, NY, United States
James D. Goldberg
Department of Obstetrics and Gynecology, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, NY, United States; Myriad Women’s Health, South San Francisco, CA, United States
John Paul Govindavari
Division of Molecular Pathology, Medical Genetics & Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, United States
Anthony R. Gregg
Department of Obstetrics and Gynecology, PRISMA Health, Columbia, SC, United States
Susan J. Gross
Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, United States; Cradle Genomics, San Diego, CA, United States
Jeffrey R. Gruen
Departments of Pediatrics, Genetics and Investigative Medicine, Yale University School of Medicine, Yale Child Health Research Center, New Haven, CT, United States
Csilla Krausz
Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence, Italy
Gabriel Lazarin
Department of Obstetrics and Gynecology, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, NY, United States; Myriad Women’s Health, South San Francisco, CA, United States
Aaron R. Prosnitz
Sanger Heart and Vascular Institute, Levine Children’s Hospital at Atrium Health, Charlotte, NC, United States
Aleksandar Rajkovic
Department of Pathology, University of California San Francisco, San Francisco, CA, United States; Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco, San Francisco, CA, United States; Institute of Human Genetics, University of California San Francisco, San Francisco, CA, United States
Viktoria Rosta
Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence, Italy
Inderneel Sahai
Department of Pediatrics, University of Massachusetts Medical School, New England Newborn Screening Program, Worcester, MA, United States
Rhona Schreck
Division of Molecular Pathology, Medical Genetics & Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, United States
Lee P. Shulman
Department of Obstetrics and Gynecology, Division of Clinical Genetics, Feinberg School of Medicine of Northwestern University, Chicago, IL, United States
Joe Leigh Simpson
Departments of Human and Molecular Genetics and of Obstetrics and Gynecology, Herbert Wertheim College of Medicine, Florida International University Miami, FL, United States
Charles M. Strom
UCLA School of Dentistry, Center for Head and Neck Oncology, Los Angeles, CA, United States
Ronald S. Swerdloff
Division of Endocrinology, Department of Medicine, Harbor-UCLA Medical Center and The Lundquist Institute, Torrance, CA, United States
Katherine Johansen Taber
Department of Obstetrics and Gynecology, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, NY, United States; Myriad Women’s Health, South San Francisco, CA, United States
Andrew F. Wagner
Department of Obstetrics and Gynecology, Division of Clinical Genetics, Feinberg School of Medicine of Northwestern University, Chicago, IL, United States
Christina Wang
Division of Endocrinology, Department of Medicine, Harbor-UCLA Medical Center and The Lundquist Institute, Torrance, CA, United States
Ronald J. Wapner
Department of Obstetrics and Gynecology, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, NY, United States
John Williams III
Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, Los Angeles, CA, United States
Svetlana A. Yatsenko
Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States; Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States; Magee-Womens Research Institute, Pittsburgh, PA, United States; Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, United States
PREFACE TO THE SEVENTH EDITION OF EMERY AND RIMOIN’S PRINCIPLES AND PRACTICE OF MEDICAL GENETICS AND GENOMICS
The first edition of Emery and Rimoin’s Principles and Practice of Medical Genetics appeared in 1983. This was several years prior to the start of the Human Genome Project in the early days of molecular genetic testing, a time when linkage analysis was often performed for diagnostic purposes. Medical genetics was not yet a recognized medical specialty in the United States, or anywhere else in the world. Therapy was mostly limited to a number of biochemical genetic conditions, and the underlying pathophysiology of most genetic disorders was unknown. The first edition was nevertheless published in two volumes, reflecting the fact that genetics was relevant to all areas of medical practice.
Thirty-five years later we are publishing the seventh edition of Principles and Practice of Medical Genetics and Genomics. Adding “genomics” to the title recognizes the pivotal role of genomic approaches in medicine, with the human genome sequence now in hand and exome/ genome-level diagnostic sequencing becoming increasingly commonplace. Thousands of genetic disorders have been matched with the underlying genes, often illuminating pathophysiological mechanisms and in some cases enabling targeted therapies. Genetic testing is becoming increasingly incorporated into specialty medical care, though applications of adequate family history, genetic risk assessment, and pharmacogenetic testing are only gradually being integrated into routine medical practice. Sadly, this is the first edition of the book to be produced without the guidance of one of the founding coeditors, Dr. David Rimoin, who passed away just as the previous edition went to press.
The seventh edition incorporates two major changes from previous editions. The first is publication of the text in 11 separate volumes. Over the years, the book had grown from two to three massive volumes, until the electronic version was introduced in the previous
edition. The decision to split the book into multiple smaller volumes represents an attempt to divide the content into smaller, more accessible units. Most of these are organized around a unifying theme, for the most part based on specific body systems. This may make the book more useful to specialists who are interested in the application of medical genetics to their area but do not wish to invest in a larger volume that covers all areas of medicine. It also reflects our recognition that genetic concepts and determinants now underpin all medical specialties and subspecialties. The second change might seem on the surface to be a regressive one in today’s high-tech world—the publication of the 11 volumes in print rather than strictly electronic form. However, feedback from our readers, as well as the experience of the editors, indicated that access to the web version via a password-protected site was cumbersome, and printing a smaller volume with two-page summaries was not useful. We have therefore returned to a full print version, although an eBook is available for those who prefer an electronic version.
One might ask whether there is a need for a comprehensive text in an era of instantaneous Internet searches for virtually any information, including authoritative open sources such as Online Mendelian Inheritance in Man and GeneReviews. We recognize the value of these and other online resources, but believe that there is still a place for the long-form prose approach of a textbook. Here the authors have the opportunity to tell the story of their area of medical genetics and genomics, including in-depth background about pathophysiology, as well as giving practical advice for medical practice. The willingness of our authors to embrace this approach indicates that there is still enthusiasm for a textbook on medical genetics; we will appreciate feedback from our readers as well.
The realities of editing an 11-volume set have become obvious to the three of us as editors. We are grateful to our authors, many of whom have contributed to multiple past volumes, including some who have updated their contributions from the first or second editions. We are also indebted to staff from Elsevier, particularly Peter Linsley and Pat Gonzalez, who have worked patiently with us in the conception and production of
this large project. Finally, we thank our families, who have indulged our occasional disappearances into writing and editing. As always, we look forward to feedback from our readers, as this has played a critical role in shaping the evolution of Principles and Practice of Medical Genetics and Genomics in the face of the exponential changes that have occurred in the landscape of our discipline.
PREFACE TO PERINATAL AND REPRODUCTIVE GENETICS
Mention the term “genetics” to most laypeople and they will think first of “inheritance,” the transmission of inborn traits from one generation to the next. In the case of Homo sapiens, this process involves sexual reproduction via gametogenesis, fertilization, embryonic and fetal development during gestation, followed by labor, delivery, and the immediate newborn period. These processes in aggregate comprise the perinatal period, and the myriad ways in which any of these steps can go wrong constitute the content of this volume. In that sense, this volume represents the quintessential aspect of genetics for many people.
This volume boasts state-of-the-art updates of key chapters in previous editions dealing with prenatal diagnosis, infertility, newborn screening, fetal loss, and other critical topics. In addition, several new chapters not present in the previous editions have been introduced, reflecting the latest advances in molecular and bioinformatic technology to enable such impressive applications as noninvasive prenatal screening, preimplantation genetic testing, and highly expanded carrier screening by next-generation DNA sequencing.
As with all such technological advances, ethical and legal dilemmas often come to light, and the authors in this volume do not shy away from discussion of those, either. Some of the ethical/legal challenges are specific to the particular techniques and their respective intellectual property, while others are overarching across the entire field of maternal–fetal medicine and genetics. Included in that latter category are restrictions on access to needed reproductive services, due either to inequities in health insurance coverage for expensive procedures or to politically motivated intrusions into reproductive decision-making, such as legislative obstacles to pregnancy termination after specific (sometimes very early) gestational ages or even for specific fetal diagnoses (such as Down syndrome).
It is hoped that this volume will address the most current needs of medical geneticists, genetic counselors, obstetricians, and all other healthcare professionals interested in this most fundamental area of clinical genetics and patient care.
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Introduction to Perinatal Disorders and Reproductive Genetics
Susan J. Gross1,2
1Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, United States, 2Cradle Genomics, San Diego, CA, United States
1.1 INTRODUCTION
There are multiple time points that one could point to as signifying the “start” of the field of perinatal genetics. Certainly, the concept of heritable disease appears early in human history. The Talmud, an ancient legal text compiled around 500 CE, rules against circumcising an infant if his older brothers had died from uncontrolled bleeding following their circumcisions—likely the first description and management of an X-linked disorder, specifically hemophilia (BT Yevamot 64b).
While clinical genetics as a focus area dedicated to the management and understanding of heritable disorders advanced considerably during the middle of the last century, reproductive genetics remained hindered by the very limited ability to evaluate the fetus in any meaningful way. Fetal dysmorphology and even the simplest metabolic or genetic tests were simply not possible. Thus, the real breakthrough in the field of reproductive genetics would have to wait for technological advances that would overcome these limitations. This chapter will highlight key fetal imaging and diagnostic and screening milestones that have brought us to where we can now “see” what had been hidden for millennia. Having achieved the ability to assess the fetus, the future holds much promise but there remain some important concerns that still need to be addressed.
1.2 IMAGING DURING PREGNANCY—A FIRST LOOK
1.2.1 Radiography
While ultrasound is often the first technology that usually comes to mind when thinking of obstetrical imaging, radiography in pregnancy was first reported in 1923 [1]. Abdominal X-rays to assess adequacy of a maternal pelvis for delivery was first described in 1944 and, despite concerns regarding teratogenic effects, remained in use or under study until relatively recently [2]. This modality can assess fetal osseous structures and therefore identify important birth defects, including single gene disorders (e.g., skeletal dysplasias) as well as multifactorial anomalies (e.g., anencephaly). However, radiography specifically for obstetrical reasons is usually avoided due to concerns regarding potential teratogenicity and limited clinical utility.
1.2.2 Ultrasound Imaging
There is no dispute among reproductive geneticists that ultrasound was one of the breakout technologies that changed the field forever. Based on the work of Christian Doppler and other physicists in the 19th and early 20th century, the first known medical application was reported to the public in 1949 to detect gallstones, based on different echo signals from reflected sound waves that were recorded on an oscilloscope
screen [3]. The next major technological step, and what many consider the actual beginning of medical ultrasound, begins with the report of 2D ultrasound in the 1950s for breast anatomy and neck, although the patient was immersed in water to overcome the artificial echoes that would otherwise be generated. Direct skin “contact” 2D ultrasound arrived a few years later, thanks to the Scottish Obstetrician Ian Donald and his engineering colleague Tom Brown [4]. Reproductive sonographers and geneticists will usually point to the seminal paper by Donald, Brown, and gynecologist John MacVicar [5] that described their findings related to abdominal masses, which included not only ovarian cancer but also the first ultrasound image of a fetal head. While a sonographer with experience would be able to make sense of these images, the resolution was less than optimal. Furthermore, these images were generated over time and were “static” and were not actually captured in “real time.” Nevertheless, as described by Dr. Stuart Campbell, a pioneer in the field in his own right, the publication of this paper signaled that “the starting gun had been fired and the ultrasound race had begun” [3].
One cannot overstate the role of ultrasound in the field of perinatal genetics. Below is a brief overview as the technology has continued to advance from the “snowstorm” images in the 1950s to current 3D (images that add depth) and 4D (incorporates time, allowing for assessment of movement) technologies.
Pregnancy Dating: Ultrasound dating, rather than date of first menstrual period, has become the standard of care for dating pregnancies. Precise dating is important for many reasons but is critical when trying to determine if a fetus is small for gestational age when working up a potential genetic issue. Likewise, an erroneous gestational age can result in false-positive or false-negative fetal aneuploidy or neural tube defect screening test results.
Fetal Dysmorphology: It is standard of care to offer women routine fetal anatomy scanning during the first half of pregnancy [6]. In many centers, this detailed scan occurs between 18 and 20 weeks. However, as the technology continues to improve, a detailed sonogram, including fetal echocardiogram, can be obtained in the first trimester [7]. Abnormal fetal anatomy on ultrasound exam remains one of the major reasons for referral to centers with expertise in fetal medicine and prenatal genetics.
Fetal Aneuploidy Screening: Standard screening for fetal aneuploidy is still used as a frontline method in many parts of the world and incorporates ultrasound along with protein markers to determine risk for trisomy 21 and other chromosomal anomalies. Nuchal translucency refers to a fluid-filled space normally seen behind the fetal neck on ultrasound performed in the first trimester of pregnancy. A measurement that is enlarged relative to gestational age is associated with Down syndrome, as well as other genetic disorders such as Noonan syndrome [8] and skeletal dysplasias [9]. Some centers look for “soft markers” that are not considered structural anomalies, but do confer increased risk for Down syndrome, such as increased renal pyelectasis found on second trimester sonogram [10].
Ultrasound-Guided Diagnostic Procedures: Ultrasound was not initially used routinely to direct fetal diagnostic procedures. Amniocentesis was available in the 1960s, but the quality and availability of ultrasound technology was still quite limited. Fetal injury from the needle was a significant risk that was discussed with a patient deciding whether to undergo a procedure. However, with the incorporation of ultrasound into prenatal care, use of this technology for needle guidance has become standard of care. Perfumo and Jauniaux [11] in their review point out the pivotal role of this technology as “it is only the use of ultrasound-guided amniocentesis in the 1980s that made it a very safe procedure during the first half of pregnancy.” While amniocentesis in the early days was possible without ultrasound guidance, procedures such as chorionic villus sampling (CVS) of the placenta or cordocentesis (also known as percutaneous umbilical blood sampling) would not have been possible.
Counseling and Surgical Aid: 3D ultrasound that provides depth to the fetal image has provided geneticists with a helpful tool when counseling patients for certain fetal anomalies. For example, the surface rendering provides a clearer image of certain anomalies, especially cleft lip and palate. In addition to defining the extent of the finding for diagnostic purposes, having this more recognizable image can help with patient counseling as well as help the cleft lip and palate interdisciplinary care team prepare [12].
1.2.3 MR Imaging
While CT imaging is sometimes used for maternal reasons, due to increased risk for fetal radiation exposure,
its use is limited prenatally. However, MRI can be an important adjunct to prenatal ultrasound and has demonstrated good sensitivity for fetal CNS malformations [13]. While neither ultrasound nor MRI is associated with fetal risk, it is still recommended that this technology be used judiciously, when the results could provide medical benefit [14].
Currently, amniocentesis and CVS remain the mainstays when it comes to confirming genetic disease during the prenatal period. In the past, cordocentesis was used more frequently for genetic diagnoses. For example, TAR syndrome that was suspected on prenatal ultrasound would be confirmed based on thrombocytopenia and anemia observed in fetal cord blood analysis [15]. However, molecular diagnosis can now be made using cells derived from an amniocentesis or CVS sample, which is considered a safer alternative [16].
1.3.1 Amniocentesis
Amniocentesis was first described in the 1800s when fluid was removed to treat polyhydramnios [17]. However, although used for other reasons over the intervening years, it was really not until the 1950s that the procedure became a part of obstetric care when it was demonstrated that spectrophotometric analysis of bilirubin in the third trimester could be used to diagnose and manage Rh disease [18]. This was quickly followed by genetic diagnosis of sex using Barr body analysis [19]. However, the big hurdle that needed to be overcome was the ability to culture the cells from the amniotic fluid. With that achievement, prenatal diagnostics could begin in earnest in the 1960s with a seminal publication that described fetal chromosomal analysis [20]. 1968 saw the first reports of fetal Down syndrome as well as galactosemia diagnoses [21,22]. Multiple case series quickly followed, and amniocentesis has remained the cornerstone for genetic screening confirmation and diagnosis to the present day.
1.3.2 CVS
A limitation of traditional amniocentesis has remained its timing during pregnancy. It is a second trimester test, generally offered after 15 weeks gestation. Early amniocentesis was proposed as a solution. However, a
randomized controlled trial demonstrated an increased risk for talipes equinovarus in the early amniocentesis group [23]. CVS, which entails sampling the placenta either through an abdominal or transvaginal approach, proved to be a first trimester diagnostic alternative. First performed in 1983 [24], the technique has become well established. While there is a small risk of false results due to placental mosaicism, the chromosomal complement of the placental cells used for this procedure closely mirrors that of the fetal cells obtained through amniocentesis.
1.3.3 Preimplantation Genetic Testing
Preimplantation genetic testing has become more widely available within IVF programs and is performed prior to embryo transfer, following conception. Usually, a biopsy is performed at the blastocyst stage, allowing 5 to 10 cells to be removed for further genetic testing. The goal is to identify unaffected embryos for transfer [25] and consequently avoid issues related to potential termination of pregnancy.
1.3.4 Cytogenetic and Molecular Techniques Used for Prenatal Diagnosis
Once fetal or placental cells could be retrieved, the evolution of prenatal diagnosis tracked with available cytogenetic and molecular technologies that were concurrently available. The first paper documenting 46 chromosomes in humans was not published until 1956 [26]. In the early days of amniocentesis, G-banding was not available and would not become part of cytogenetic practice until the 1970s. The next major milestone was the addition of molecular approaches to chromosomal analysis. FISH probes allowed for the identification of microdeletions that could not be seen using standard karyotyping alone. Currently, microarrays are considered standard of care for prenatal diagnosis in the United States, especially in the setting of fetal anomalies or stillbirth. If no structural anomalies are seen, conventional karyotype and microarray should be discussed with the patient [27]. Nor is prenatal exome sequencing still considered experimental. In the presence of fetal anomalies or a single major anomaly suggestive of a genetic disorder where microarray is negative or unavailable, exome sequencing becomes an option, similar to the postnatal setting [28].
Noninvasive prenatal diagnosis is the next technological phase that is garnering a lot of activity and attention. It holds out the promise of removing the risk for fetal loss
that is associated with amniocentesis or CVS. While the risk is low, 0.1%–0.3% in expert hands [29] and may not even confer excess risk especially if the fetus is not anomalous [30], many women prefer to avoid invasive testing if possible. The initial avenues explored were the isolation of trophoblasts from the endocervical canal [31] and fetal cells from the maternal circulation [32]. The focus is on the separation and extraction of these cells, as once isolated, current molecular sequencing techniques and various analytic approaches become possible. Isolation of intact fetal cells has now largely been superceded by direct sequencing of cell-free fetal DNA, as discussed below.
1.4 PRENATAL SCREENING FOR GENETIC DISORDERS—ANEUPLOIDY AND SINGLE GENE
1.4.1 Fetal Aneuploidy Screening
It is notable that even during the 1960s and 1970s, when amniocentesis was the only genetic testing option, women were still involved in a screening program. Amniocentesis was not universally available and therefore age alone was the clinical feature, absent any personal or family risk, used to determine who would be offered a diagnostic procedure. The age cut-off at 35 was used based on a few factors including resource allocation and the “balance” of 1/200 risk of fetal loss versus 1/200 risk of any fetal chromosomal anomaly at that maternal age. However, the medical community always appreciated that despite the increased risk in this older maternal age group, most children with Down syndrome are born to women less than 35. Even in patients with affected offspring, the risk is still only a few percentage points at most. Therefore, conceptually, whether we are looking at the first “AFP only” single marker aneuploidy screening test, standard first trimester screening or the latest cell-free DNA noninvasive prenatal screening (NIPS) approach, they all came about to help refine the initial “age alone” risk algorithm [33]. NIPS has dramatically changed the landscape with positive predictive values (PPVs) that are several times better than standard first trimester screening that combines first trimester ultrasound NT and biomarkers (45.5% vs. 4.2% for trisomy 21% and 40.0% vs. 8.3% for trisomy 18) [34]. However, despite this major leap forward in test performance, NIPS has not been without controversy. Additional disorders have been added with poor PPVs and varied clinical utility, such as rare aneuploidies and
microdeletion syndromes [35]. Most problematic is an ongoing confusion regarding the difference between a screening test that can only provide a risk assessment versus a true diagnostic test. In response, leading professional organizations have created open access calculator tools to help healthcare professionals provide accurate information to patients regarding PPVs and negative predictive values (NSGC PQF NIPT Calculator https://www.perinatalquality.org/Vendors/NSGC/NIPT/). It is also worth noting that the entire fetal genome has already been sequenced [36] using shotgun sequencing of maternal plasma DNA. The approach is not practical for broad clinical testing at this time, but it demonstrates that noninvasive fetal sequencing can already be performed with currently available technologies.
1.4.2 Carrier Screening for Genetic Disorders
Even prior to molecular diagnostics, fetal risk assessment for Mendelian disorders was possible. A good pedigree analysis could provide valuable information in the case of a woman with a family history of Duchenne Muscular Dystrophy or a previous child with cystic fibrosis. The population-based Tay Sachs screening program was successfully executed using maternal enzyme analysis and was the first multi-disease panel as some of the programs also screened for familial hypercholesterolemia using cholesterol levels. Hemoglobin electrophoresis and a simple MCV are considered the first-line screening tests for hemoglobinopathies [37].
However, there is no doubt that molecular technologies, in particular next-generation sequencing (NGS), have altered the carrier screening landscape. The current approach is to sequence the mother and if a pathogenic or likely pathogenic variant is identified, then the father of the baby also undergoes genetic testing in the case of an autosomal recessive disorder. While carrier screening is on one hand a diagnostic for the mother (if a pathogenic cystic fibrosis variant is found, she is indeed a carrier), the term “screening” is used because the purpose of the test is to assess the risk to fetus. The benefits of NGS technology are manifold, including the ability to test for more disorders in a highly precise and efficient way. However, the larger the panels, the more likely a patient will receive a “positive” screen result. As more variants will be found in genes associated with increasingly rare disorders, the odds that the other parent will likewise have a pathogenic variant in that same gene become more unlikely. Thus, there is significant
concern that larger panels will result in downstream anxiety and costs but will not necessarily provide useful information specific to the current pregnancy. Similar to aneuploidy screening, single gene variant detection has already been reported using cell-free DNA in maternal plasma [38] using droplet PCR. Other approaches have also been reported [39,40]. A clinical test is already on the market for select de novo and paternally inherited variants, although it is not considered to be sufficiently validated to be incorporated into standard of care [41].
1.5 THE END OF THE BEGINNING AND WHAT LIES AHEAD
From a broad perspective, the above survey of prenatal genetics tells us that we have attained what would have seemed like a far-off achievement only a few decades ago. We already have the technology to interrogate the fetal genome during pregnancy and the preimplantation period. Treatments will become available and newer diagnostic methodologies seem poised to fulfill the promise of noninvasive testing. There remains much to be done with respect to scalability and cost reduction; however, technological advances will continue and one can expect within a few years to see prenatal diagnostics move forward on all fronts as well, opening the door to true precision medicine prior to delivery. While there is much to celebrate, the same questions that have concerned the specialty in the past have not diminished and perhaps take on more urgency as our ability to finally access the fetal genome has arrived.
1.5.1 We Can Do It, but Should We Do It?
There has always been the push and pull between our ability to “do more” to benefit patients versus primum non nocere—first do no harm. Thoughtful clinicians and leaders in the field addressed this problem even when screening panels were still just a few disorders in size [42]. Andermann et al. [43], in a WHO bulletin, applied the well-known Wilson and Jungner principles of screening criteria to the genomic era. Many of the key concepts still hold, including the “North Star” of clinical utility. Even if a screening test works consistently well in the laboratory and can even detect disorders of interest in the clinical setting, should it be offered if there is no demonstrable positive impact on outcomes? Certainly, there are challenges as often specific genetic disorders tend to be rare, and large broad-based research studies
comparable to diabetes or coronary heart disease may not be feasible. Some screening and even diagnostic tests may require more “shared decision-making” approaches in the future. However, there is still the need for rigorous analytic, clinical validity and ultimately clinical utility studies if testing is to be provided to millions of women worldwide who are or seek to become pregnant. Professional bodies have tried to address the question with an approach that does not necessarily provide a defined panel of diseases, but rather seeks to specify characteristics of disorders that may warrant screening, for example, whether the condition could result in significant disability or knowledge of the condition could enhance delivery planning. Conversely, guidance also can address what disorders should be excluded, such as adult-onset disorders or high allele frequency variants but low penetrance such as MTHFR [44]. Others have looked closely at allele frequency and the identification of carrier couples rather than just one parent. Assessing only 40 genes with carrier rates >1.0% would identify a substantial number of panethnic carrier couples, while the addition of genes with lower carrier rates followed the principle of “diminishing returns” [45]. Genome sequencing will ensure that this conversation regarding prenatal test expansion will become more, not less, important in the future.
1.5.2 Women’s Autonomy
Related to the above discussion of what prenatal tests should be offered is the question of who gets to decide. For example, Canadian guidelines recommend invasive prenatal diagnosis be offered to women at high risk [46], while in the United States, all women have the option of screening versus diagnostic testing [47]. Some authors have approached the issue of women’s autonomy via the lens of informed consent and the “routinization” of prenatal testing, such that women are making decisions but based on limited knowledge. In addition, “[s]upport for access to prenatal genetic tests and abortion services and advocacy for robust informed consent processes grow out of the same ethical commitment to respect for autonomy” [48]. Other authors have noted that historically, the focus has been on the risk for fetal loss following invasive testing. Rather, an autonomy-based approach would help women identify what risk most concerns them personally. For some it may indeed be the risk of fetal loss but for other women, it may be the risk of having a child with a significant genetic abnormality [49].
1.6 CONCLUSION
Advances in reproductive genetics have been inextricably tied to the technologic achievements that have greatly accelerated over the past decade. Not only can we image the fetus, but we can sequence its genome using both invasive and noninvasive approaches which can conceivably lead to treatment and cures. However, the same concerns are still present, no different than decades ago when the field of reproductive genetics was just beginning. In his landmark paper on the first prenatal diagnosis of an inborn error of metabolism (galactosemia) using amniocentesis, the author ends his abstract with the following “… until considerably more experience is gained with these techniques, these procedures should be considered experimental in nature” [21]. Despite ethical concerns, science and medicine will continue to move forward with the goal of enhancing the well-being of individuals and communities. Further detail regarding the past milestones and future promise of reproductive genetics will be found in the pages of this volume. Nevertheless, while celebrating the many achievements that have improved the health of mothers and families, it is worth reflecting on a key moment in Dr. Charles Epstein’s Presidential Address to the American Society of Human Genetics in 1996 [50]. The address was meaningful for many reasons, not the least of which was the fact that Dr. Epstein, a recognized expert in the genetics of Down syndrome, had recently survived injuries caused by an explosion set by the “Unabomber,” an individual who was on a violent campaign to stop technology. Toward the end of his presentation, and in very compassionate terms, Dr. Epstein made the case that medical geneticists have a responsibility to educate and inform the public about what medical geneticists actually do. However, most important and especially relevant to those in the field of prenatal genetics, he recommended that the medical genetic community “continue to be –and, if anything, become more – involved in the social and ethical debate that increasingly surrounds everything that we do.”
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FURTHER READING
Brock DJ, Bolton AE, Monaghan JM. Prenatal diagnosis of anencephaly through maternal serum-alphafetoprotein measurement. Lancet 1973;2(7835):923–4. https://doi. org/10.1016/s0140-6736(73)92592-0
Committee on Obstetric Practice, the American Institute of Ultrasound in Medicine, and the Society for Maternal-Fetal Medicine. Committee opinion no 700: methods for estimating the due date. Obstet Gynecol 2017;129(5):e150–4. https://doi.org/10.1097/AOG.0000000000002046.
Cuckle H, Maymon R. Development of prenatal screening–A historical overview. Semin Perinatol 2016;40(1):12–22. https://doi.org/10.1053/j.semperi.2015.11.003.
Hobbins JC. Overview of imaging in pregnancy: history to the present, including economic impact. Semin Perinatol 2013;37(5):290–1. https://doi.org/10.1053/j.semperi.2013.06.002.
National Society of Genetic Counselors and Perinatal Quality Foundation: NIPT/cell free DNA screening predictive value calculator. [Accessed 17 August 2020].
Scott F, Chan FY. Assessment of the clinical usefulness of the “Queenan” chart versus the “Liley” chart in predicting severity of rhesus iso-immunization. Prenat Diagn 1998;18(11):1143–8.
Prenatal Screening for Neural Tube Defects and Aneuploidy*
Robert G. Best
Department of Biomedical Sciences, University of South Carolina School of Medicine Greenville, Greenville, SC, United States
2.1 INTRODUCTION
Population-based prenatal screening using biomarkers began in the 1980s following successful pilot studies that demonstrated that open neural tube defects (ONTDs) including spina bifida and anencephaly could be effectively identified by studying the presence of alpha-fetoprotein (AFP) in the maternal circulation in the second trimester of pregnancy [1]. Soon thereafter, it was discovered that AFP levels were statistically lower in pregnancies affected with Down syndrome (DS), and this observation was exploited to provide the first maternal serum biomarker screening for aneuploidy [2]. Clinical screening for these two conditions is based on successful characterization of the different distributions of these biomarkers between the population as a whole and the subset of pregnancies affected by the condition for which screening is seen as valuable. This approach has led to the discovery of dozens of other biomarkers for which affected pregnancies exhibit differences from the underlying population that can be exploited for screening purposes alone, or in combination with other biomarkers. This is not only limited to protein markers such as AFP but also smaller peptides [3,4], steroid hormones [5], nucleic acids [6], fetal morphometric measurements [7–10], and other attributes of the maternal–fetal environment [11,12]. Although neural tube defects (NTDs) and DS remain at the center
of prenatal screening, the methodology extends well beyond these two conditions.
Biomarker concentrations during pregnancy tend to be dynamic, so it is generally necessary to standardize measurements relative to gestational age by comparing a patient’s measured value against the median value in the overall pregnant population for that specific point in pregnancy. A risk-based approach that is shared among most of the conditions for which clinical prenatal screening is offered uses Bayesian probability modifications constructed from the statistical distributions of the biomarkers to modify a priori risks from the population to calculate a patient-specific risk for each condition of interest. This construct has led to dramatic improvements in the ability to detect DS prenatally and has opened an approach to identify other birth defects and potentially serious adverse pregnancy conditions (e.g., preeclampsia, low birth weight, etc.).
This chapter is intended to highlight the general approach to prenatal screening currently in use in clinical settings with a central focus on ONTD and DS and secondary focus on other aneuploidies and the great variety of other conditions that can be identified in the context of prenatal screening. This chapter begins with an historical perspective and ends with a reminder of the clinical public health context of prenatal screening. The introduction of cell-free fetal DNA (cfDNA) as a mode for screening has dramatically influenced prenatal screening for aneuploidies since its introduction in practice in 2012 (see Chapter 10); however, multiple marker testing remains as a vital component of prenatal screening in the United States and around the world.
2.2 PRENATAL SCREENING FOR BIRTH DEFECTS
Screening tests are designed to identify the potential for health disorders from among an otherwise healthy population. Screening differs from diagnostic testing in that false positives and negatives are expected and are incorporated into the schema. Prenatal screening focuses primarily on the risk of adverse health conditions of the fetus that are both serious and common. Current standards for healthcare screening advanced by the World Health Organization based on iterative improvements of earlier criteria proposed by Wilson and Junger [13,14] require that the screening test responds to a recognized need for a defined target population, reflects scientific evidence that the screening program is effective, is designed to be equitable across the entire target population, that the benefits outweigh any harms, and that the program integrates education, testing, clinical services, and program management [13].
Research around the Health Belief Model exploring the motivation of patients to accept available testing identifies the patient’s own perceptions of susceptibility, severity, benefits, and barriers as critically important, conditioned on beliefs of self-efficacy (i.e., an ability to take effective action) [15,16]. Decisions to participate are also affected by a variety of modifying factors (e.g., race/ ethnicity, age, education, etc.) and internal or external cues that trigger action (e.g., receiving information from trusted sources). Thus, the mere availability of a test or demonstration that screening is possible is not sufficient in terms of public policy nor patient demand. Two prenatal conditions that seem to fully meet all criteria for screening are ONTD and DS. In addition to these two conditions, there are several other conditions for which information arises while testing for ONTD or DS that bear sufficient clinical utility to merit inclusion in the overall screening program.
2.2.1 Neural Tube Defects
NTDs are among the most common of the serious birth defects in the population. These are major structural developmental defects affecting the central nervous system that arise from an error in the maturation of the neural tube early in pregnancy, between 14- and 28-days postfertilization (4–6 weeks by menstrual dating). During this 2-week period, the embryonic tissues that give rise to the spine and brain begin with a relatively
flat geometry, develop a transverse fold that deepens into a groove along the axis of development, that ultimately circularizes to give rise to a tubular structure as the leading edges of the neural fold begin to touch and connect with each other [17,18], providing the foundational structures for the brain and spine. Failure of the neural tube to close completely results in a disruption of these central structures of the nervous system. Although failure to close can result in a complete failure of the formation of the neural tube and all resulting structures downstream (complete dysraphism), most commonly the errors are confined to incomplete closure at one end or the other. When the failure involves the caudal end, the developmental failure results in an opening along the spine (spina bifida), whereas failure at the cephalic end results in a dramatic disruption of the primary structures of the brain and cranial vault (encephalocele, anencephaly). These two anomalies are almost equally common and account for approximately 90% of all NTDs [19].
Morbidity and mortality are variable depending upon the size, location, and fine structure of the defect. Spina bifida is typically associated with paralysis or weakness of the lower structures of the body but the extent is highly variable and ranges from a lack of clinical impairment to fetal or neonatal death [18]. Anencephaly is considered to be uniformly fatal with death early in the postnatal period for babies that survive to term [20].
When NTDs are covered by skin or other membranes, they are considered to be closed defects. Most often, NTD lesions are not covered with skin, and are therefore considered to be open defects. This is an important distinction because the mechanism that leads to differences in AFP concentrations between the affected and unaffected populations is limited to open defects. Only 15%–20% of spina bifida cases are closed defects but, in general, the prognosis is more favorable [21,22], whereas most cases of anencephaly are open [23]. Biochemical screening is therefore restricted to open NTD because the open lesion is directly related to the increased release of fetal protein into maternal circulation. It is not the intent of this chapter to fully characterize the range of NTDs and their various clinical presentations.
Most commonly, NTDs occur without other structural anomalies unrelated to the development of the neural tube and are considered to be isolated or nonsyndromic. Their occurrence is estimated to be 7/10,000 live births in the general population of the United
States [24] with notable differences in birth prevalence around the world [25] and variability related to race, geographical location, and the availability of folic acid in the diet [26]. Isolated NTDs are genetically complex traits with a heritability of approximately 60% [27,28] with many genes associated and few genes having been identified that clearly demonstrate major effects [29]. Like other complex traits, recurrence is increased when there are affected first-degree relatives [30] at a rate of approximately the square root of the population birth prevalence and less so for more distant affected relatives [28]. A number of environmental factors have been identified that influence the development of the neural tube including folic acid, folate antimetabolites, and type I diabetes [31–33]. Since the great majority of NTDs occur in the absence of a positive family history, prenatal identification is largely dependent upon general population screening through AFP or ultrasound examination.
NTDs can also appear in syndromic forms associated with structural defects unrelated to the neural tube. Recurrence risks for syndromic NTDs are highly variable and are dependent on the etiologic mechanisms. For example, Meckel–Gruber syndrome is a rare disorder with a birth prevalence of 2.6 per 100,000, inherited in a single-gene autosomal recessive pattern and is associated primarily with encephalocele [34]. In contrast, complete or partial aneuploidy may also involve disruption of the neural tube during development, with recurrence risks dependent on the mechanism through which the chromosomal imbalance arose. Most syndromic forms of NTDs are relatively rare and are therefore challenging to study for recurrence and the degree to which environmental factors might be involved. While it is not the intent of this chapter to address the fine points of the occurrence and distribution of all forms of NTDs, it is important to recognize differences in recurrence risks as a limiting factor in the estimation of patient-specific risk calculations in screening. Biomarker screening is effective for any ONTD independent of its causal mechanism.
2.2.2 Down Syndrome and Aneuploidies in Pregnancy
Studies in the 1970s showed that chromosomal abnormalities affect approximately 1 in 160 live births [35]. A more recent European study of second trimester amniocenteses demonstrated that this incidence may
be higher when minor alterations such as mosaicism are included [36]. The majority of chromosome abnormalities are sex chromosome alterations involving extra copies of the X or Y chromosomes, monosomy X or autosomal trisomies involving chromosomes 21, 18, or 13. Approximately 1 in 700–800 children are born with trisomy 21 (DS), 1 in 6000 are born with trisomy 18 (Edwards syndrome), and 1 in 10,000 with trisomy 13 (Patau syndrome) [37,38]. Most autosomal trisomies are caused by nondisjunction during maternal meiosis, a process that is more frequent with advancing maternal age [39].
2.2.2.1
Down Syndrome
DS is a complex clinical phenotype that results from trisomy of part or all of chromosome 21. DS is the most common autosomal aneuploidy occurring in humans, with a current birth prevalence of approximately 1:700 live births [38] and higher birth frequency among older mothers. People with DS typically have an IQ in the mildly to moderately low range with characteristic facial features that may include epicanthal folds, upward slanting palpebral fissures, flattened facial profile, short neck and small ears, hypotonia, hyperflexibility, single transverse palmar creases, and a variety of other benign or mild features [40]. Individuals with DS are susceptible to duodenal atresia, Hirschsprung disease, patent ductus arteriosus, early-onset Alzheimer disease, and acute leukemia [41,42]. Their personalities are frequently described as affectionate and pleasant albeit somewhat complex [43]. The combination of a relatively high birth prevalence, complexity of the clinical phenotype, older maternal age at birth, and relatively long life expectancy no doubt contribute to the high level of interest in prenatal screening and diagnosis.
2.2.2.2
Trisomy 18
Another autosomal aneuploidy, trisomy 18 (Edwards syndrome), demonstrates an altered biomarker profile compared with unaffected pregnancies and is therefore also detectable in multiple marker screening [44]. Edwards syndrome has a significantly higher morbidity and mortality compared with DS, is subject to higher rates of spontaneous abortion, and is far less common [45–47]. Because of its rarity and differences in severity and life expectancy, the public health rationale for trisomy 18 screening is considerably weaker
