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To protect the rights of the author(s) and publisher we inform you that this PDF is an uncorrected proof for internal business use only by the author(s), editor(s), reviewer(s), Elsevier and typesetter Toppan Best-set. It is not allowed to publish this proof online or in print. This proof copy is the copyright property of the publisher and is confidential until formal publication. These proofs may contain color(colour) figures. Those figures may print black and white in the final printed book if a color(colour) print product has not been planned. The color(colour) figures will appear in color(colour) in all electronic versions of this book.

Abstract abs0010 The collection of single nucleotide variations and copy number vari- ants within an individual forms a genetic backdrop that influences how a person looks, grows, and physiologically responds to stressors such as a disease or medication. High-frequency sequence variants in the population are part of the normal backdrop of the human genome, and for the most part are not thought to have functional consequences. Overt genetic pathology, in contrast, usually arises from genetic vari- ants that disrupt the normal expression or function of one or more genes. The old term has been replaced by variant, which can be benign, likely benign, of unknown significance, likely pathogenic, or pathogenic. This chapter reviews the molecular (DNA-based) tech- niques available to detect, identify, or diagnose disease-associated as well as inconsequential genetic variants, specifically pertaining to screening and diagnostic needs during pregnancy. The extent and specificity of the resulting information depends on the source of genetic material, as well as the testing platform employed. General benefits of genetic testing include identification of disorders for which in utero treatment may provide benefit, optimization of neonatal out- comes by planning appropriate delivery staffing and location, provid- ing family preparation for caring for a child with a , the option of pregnancy termination, and providing reassurance when results are normal. With rapidly developing technologies at our dis- posal, reproductive and obstetric professionals have a responsibility to connect patients with resources to ensure full understanding of the benefits and limitations of various screening and diagnostic options. Exciting new technologies are in development, and should continue to be held to high standards of scientific rigor and clinical validation before entering mainstream practice.

KEYWORDS molecular genetic karyotype mosaicism cell-free fetal DNA amniocentesis chorionic villus sampling carrier screening sequencing PCR next-generation sequencing noninvasive prenatal screening variant single nucleotide polymorphism copy number variant microarray comparative genomic hybridization oligonucleotide preimplantation genetic screening preimplantation genetic diagnosis embryo

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2 c00002 Molecular Genetic Technology

JENNIFER GILNER, MD, PhD | ALEKSANDAR RAJKOVIC, MD, PhD | JEFFREY A. KULLER, MD

challenge, such as an invading pathogen or ultraviolet ray expo- s0010 Detecting the Genetic sure from the sun. Understanding in the form Basis of Disease of SNPs and CNVs and their biological influence can reveal predisposition toward disease, variable susceptibility to infec- s0015 GENETIC VARIATION tions, and diverse responses to pharmacotherapy. p0010 Each human being’s complete genetic sequence reveals a sig- nificant amount of genetic variation. The collection of single GENETIC PATHOLOGY s0020 nucleotide variations and copy number variations within an indi- vidual forms a genetic backdrop that influences how a person High-frequency sequence variants in the population are part of p0025 looks, grows, and physiologically responds to stressors such as the normal backdrop of the human genome, and for the most a disease or medication. The allelic options at any given genetic part are not thought to have functional consequences. Overt locus derive from single nucleotide substitutions within the DNA genetic pathology, in contrast, usually arises from genetic vari- sequence. Population sampling has demonstrated that, among ants that disrupt the normal expression or function of one or healthy individuals, the genetic sequence differs at around 10 more genes (Table 2.1). Gene expression and function may be million sites (out of 3.2 billion total DNA base pairs [bp]). disrupted by less frequent gene variants in the coding portion These naturally occurring differences are called single nucleo- of a gene, increases or decreases in the relative dose of a DNA tide polymorphisms (SNPs). To be classified as an SNP, two or segment (CNV), changes in the normal amount of a gene more versions of the nucleotide sequence must be present in at product, or sequence changes in regulatory regions that prevent least 1% of the general population. Disease-causing nucleotide the cell from normally expressing the intended gene product. changes are relatively rare, thus the term single nucleotide poly- The old term mutation has been replaced by variant, which can morphism describes genetic variation of healthy individuals. Spe- be benign, likely benign, of unknown significance, likely patho- cific SNP-associated phenotypes are relatively inconsequential, genic, or pathogenic.7 Pathogenic and likely pathogenic variant such as SNPs in the trichohyalin gene (TCHH) that cause straight designations are reserved for changes in the genetic code that versus curly hair.1 Conversely, SNPs within the cytochrome P-450 lead to altered function and clinical consequences, based on isozyme 2D6 gene (CYP2D6) produce phenotypes with drastic several pieces of supporting information, including consistent modifications in an individual’s rate of opioid or antidepressant human case reports, identification of the type of variant, and metabolism, greatly altering drug side effect profiles.2,3 Identifica- verification by functional studies, animal models, or both. A tion of SNPs, their location, and their impact on gene function variant may involve changing a single nucleotide base or a larger has the potential to predict the future health of the individual, segment, in which bases are removed, duplicated, or inserted. even though an SNP does not directly cause disease. For example, in sickle cell anemia there is one identified disease- p0015 In addition to individual sequence variation through SNPs, associated variant in the hemoglobin gene; alternatively, in comparative genome studies between individual sequences have (CF) more than 1000 disease-associated variants revealed a far more pervasive form of genetic variation, termed or alleles have been described to date in the CF transmembrane copy number variations (CNVs).4,5 These are structural variants conductance regulator gene (CFTR). Both sickle cell anemia made up of relatively large DNA segments (ranging in size and CF are examples of single-gene disorders (also called men- from 1000 to 500,000 bp or more) that can be duplicated or delian disorders) in which the complete disease phenotype can deleted at a given genetic locus, and cumulatively affect 360 be accounted for by the abnormal function of a single gene. million nucleotides, or about 12% of the human genome.6 A Other human diseases or syndromes, such as autism, have a CNV can be benign (no known effect on the phenotype) or genetic basis, but the genotype-phenotype correlation is far pathogenic (well-documented effect on the phenotype), and a more complex and may involve multiple genes in addition to significant proportion of identified CNVs have as-yet unknown environmental influences.8 significance (may be changed to benign or pathogenic based on Larger genetic alterations, which risk disruption or loss of p0030 future data). multiple genes and their function, most commonly occur during p0020 Thus, whereas SNPs introduce genetic variation at the level cell division by mitosis or . A variety of chromosomal of individual base substitutions, CNVs represent variation in abnormalities may occur during chromosome alignment and the “dose” of a relatively large DNA segment. The collection of segregation (see Chapter 1), leading to chromosome breaks or SNPs and CNVs influences how an individual responds to a rearrangements or uneven distribution of into N 15

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16 PART 1 Scientific Basis of Perinatal Biology

t0010 TABLE daughter cells. Chromosome breaks and rearrangements may be 2.1 Genetic Pathology genetically balanced (normal phenotype) or genetically imbal- Prevalence of anced (producing abnormalities resulting from gain or loss of one Disorder Risk Age Disorder or more genes), or may completely disrupt a critical gene at the breakpoint site on the chromosome, leading to loss of function- 21, 13, 18 > 35 years 1/500 (0.2%) Sex chromosome Any 1/1000 (0.1%) ally relevant gene products. The molecular resolution of a regular karyotype (a technique for counting the chromosomal content Balanced Any 1/500 (0.2%) of a cell under light microscopy) is above 5 megabases (Mb), chromosomal which allows for detection of chromosome number changes and abnormalities relatively large structural chromosome rearrangements. In the Pathogenic Any 1/10,000 (0.01%) (unbalanced) past decade, advances in molecular cytogenetic technology, such chromosomal as array comparative hybridization (also termed chromosomal abnormalities microarrays), have improved resolution for assessing cellular Mendelian Any 1/280 (0.4%) DNA content below 5 Mb, down to as low as 1 kilobase (kb), disorders De novo variants Any ? and revealed a new class of chromosomal syndromes caused by Pathogenic Any 1/90 (1.2%) microdeletions and microduplications. These syndromes involve microdeletions/ two or more contiguous genes (genes in adjacent locations on duplications the chromosome). Table 2.2 lists some of the more commonly PHENOTYPIC INCIDENCE recognized syndromes caused by microdeletions and microdu- Structural/functional birth defects in 1/33 (3%) plications. The phenotypes of these conditions are the result of newborns the absence (or duplication) of multiple contiguous (adjacent) Autism 1/88 (1.2%) genes within the involved region. Total prevalence of genetic pathology 1/10 (10%) (30 million people in United States) PRECONCEPTIONAL AND PRENATAL s0025 Genetic pathology may arise from variation in a single gene, multiple DIAGNOSIS OF GENETIC PATHOLOGY genes, segments of chromosomes, or total number of chromosomes in a cell. The focus of this chapter is to review the molecular (DNA- p0035 based) techniques available to detect, identify, or diagnose

t0015 TABLE 2.2 Microdeletion or Microduplication Syndromes Genomic Disorder Associated With Location Genomic Disorder Associated With Deletions Duplications Size (Mb) 1q21.1 Thrombocytopenia–absent radius syndrome (TAR) Not known 0.347–0.357 region (OMIM 274000) 1q21.1 1q21.1 microdeletion (OMIM 612474) 1q21.1 microduplication (OMIM 612475) 1.19 2q13 Carrier juvenile nephronophthisis Benign copy number variation 0.150 3q29 3q29 (OMIM 609425) 3q29 microduplication syndrome (OMIM 1.6 611936) 7q11.23 Williams-Beuren syndrome (WBS) (OMIM 194050) WBS duplication syndrome (OMIM 609757) 1.5–1.8 15q11-q13 (OMIM 105830)/Prader-Willi 15q11-q13 duplication syndrome (OMIM 0.50 to ~6.0 syndrome (OMIM 176270) 608636) 15q13 15q11-q13 syndrome (CHRNA7) (OMIM Unclear (CHRNA7) (OMIM 612001) 0.400 612001) 1.5–1.8 16p11.2 16p11.2 deletion syndrome (OMIM 611913) 16p11.2 duplication syndrome (OMIM 0.593–0.706 611913) 16p11.2-p12 16p11.2 deletion syndrome (OMIM 613604) Pathogenic—Genoglyphix .7 Aberration Database (GCAD) (OMIM 23196) 16p13.1 16p13.1 microdeletion predisposing to autism 16p13.1 microduplication 1.3 and/or mental retardation 17p11.2 Smith-Magenis syndrome (OMIM 182290) Potocki-Lupski syndrome (OMIM 610883) 3.7 17p12 Neuropathy, hereditary, with liability to pressure Charcot-Marie-Tooth disease CMT1A (OMIM 1.4 palsies (HNPP) (OMIM 162500) 118220) 17q11.2 Neurofibromatosis type I (OMIM613675 ) NF1 critical region microduplication 1.2–1.4 syndrome 17q12 17q12 deletion syndrome (OMIM 614527) 17q12 duplication syndrome (OMIM 614526) 1.5 17q23 17q23.1-q23.2 deletion syndrome (OMIM 613355) 17q23.1-q23.2 duplication syndrome (OMIM 2.1 613618) 22q11.21 DiGeorge syndrome/velocardiofacial syndrome 22q11.2 microduplication (OMIM 608363) 1.5–3.0 (OMIM 188400/192430) 22q11.23 22q11.2 distal microdeletion (OMIM 611867) Unclear ~2.5 Microdeletion or microduplication syndromes are caused by an aberrant copy number (gain or loss) of a specific subchromosomal region. The common use of array comparative genomic hybridization has resulted in rapid increase in identification of benign as well as disease-associated N copy number variations.

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2 Molecular Genetic Technology 17

disease-associated as well as inconsequential genetic variants, approach to maximize identification of couples at increased risk specifically pertaining to screening and diagnostic needs when for having a pregnancy affected by a mendelian disorder. First, planning pregnancy or during pregnancy. The extent and speci- patients may not have accurate awareness of their ethnic ances- ficity of the resulting information depends on the source of try. Second, although the carrier frequency for disorders on genetic material, as well as the testing platform employed. Fig. ethnicity-based panels is individually high, these disorders 2.1 summarizes the potential sources of genetic material (DNA) account for only a small percentage of known mendelian dis- for evaluation (Fig. 2.1A) and the currently available genetic orders. For example, ACOG recommends at a minimum pan- assessments for preconceptional or prenatal screening or diag- ethnic screening for SMA and CF, while currently over 3000 nosis (Fig. 2.1B). genes are known to cause mendelian disorders. Third, so-called p0040 Genetic risk assessment begins with the evaluation of the next-generation sequencing technologies have enabled assaying biological parents. Family history and prior obstetric history hundreds or thousands of genes simultaneously in a cost- may reveal indications for targeted testing, such as DNA effective manner. These technological breakthroughs have led sequencing or biochemical analysis for single-gene disorders, or to widespread availability of expanded carrier screening, in parental karyotyping in the setting of recurrent pregnancy loss. which a large number of both common and rare disorders are Alternatively, indications to pursue screening or diagnostic screened for simultaneously in the general population. testing may include increased risk of related to Expanded carrier screening uses a highly customized, mul- p0060 maternal age; racial or ethnic background, with increased tiple molecular inversion probe assay13–15 to convert the infor- carrier frequency of certain diseases; or simply population- mation content of a genetic variant into fluorescently labeled based screening risk. In addition, it is acceptable for any patient, tag sequences.13 This approach identifies both the disease- regardless of risk, to choose diagnostic prenatal testing after associated and the wild-type alleles of each variant. At least informed consent. Prenatal diagnosis may allow reproductive 100 conditions have available screening, and that number is options, or interventions before disease is clinically detected in rapidly increasing.16 With such extensive availability of screen- a child. In some rare cases, diagnosis in utero may allow the ing options, ACOG has provided consensus recommendations opportunity to prevent irreversible changes in early for the design of expanded carrier screening panels in order development. to maintain focus on the ultimate goal of providing patients with meaningful information to guide pregnancy planning. s0030 Parental Carrier Screening Expanded carrier panels should only include disorders that 11 p0045 Certain populations have an increased frequency of specific, meet at least several of the following criteria : identifiable, disease-associated genetic variants. This may occur • Have a carrier frequency of 1 in 100 or greater u0010 because the population has remained relatively isolated, because • Have a well-defined phenotype u0015 many individuals in the population are descended from a few • Have a detrimental effect on quality of life u0020 common relatives having a specific variant (founder effect), or • Cause cognitive or physical impairment u0025 because the carrier state has a beneficial effect on survival in a • Require surgical or medical intervention u0030 particular environment (sickle cell carrier state granting protec- • Have onset early in life u0035 tion from malaria). Carrier screening refers to genetic testing of • Have the ability to be diagnosed prenatally u0040 asymptomatic individuals to determine if they carry one or As with all screening tests, expanded carrier screening is risk p0100 more such genetic variants. Traditionally, carrier screening has reducing rather than risk eliminating, because not all variants been targeted to specific ethnic populations known to be at for any disorder can be identified. A common example of this increased risk for particular disorders (ethnic-based screening). is CF, in which a negative pan-ethnic variant panel significantly However, as the population becomes more racially and ethni- reduces the risk of a patient being a carrier but does not elimi- cally diverse, pan-ethnic screening for a panel of disorders nate it given the large total number of variants in the gene. offered to all individuals regardless of ethnicity has also been Furthermore, positive finding rates are high in expanded carrier made available. Patients can undergo genetic carrier screening screening panels, and some positive findings are inconclusive prior to conception or during pregnancy.9 because many rare variants remain uncharacterized. Srinivasan p0050 The goal of carrier screening is to provide individuals with and colleagues have recently reported an expanded carrier panel information that will permit them to make informed reproduc- for more than 100 mendelian disorders, with multiple variants tive decisions.10 Table 2.3 lists the currently recommended tested per allele, in which 35% of individuals were found to be ethnic-based or pan-ethnic carrier screening tests that should a carrier for at least one variant, and the rate of carrier couples be discussed with patients considering pregnancy or who are was approximately 0.6% to 0.8%.16 Although this carrier rate currently pregnant, according to the American College of appears high, it should be considered that every individual is Obstetricians and Gynecologists (ACOG).10,11 Screening for suspected to carry a dozen or more deleterious variants. spinal muscular atrophy (SMA) and CF carrier status is recom- The appropriate extent of screening must be individualized p0105 mended for all patients. Additional screening may be tailored for each patient, and take into account both identified genetic by family history or ethnicity. Such testing is of maximal benefit risks and personal values after counseling by a qualified profes- when it is part of a comprehensive screening program including sional. Informed patient consent is required prior to offering patient education, genetic counseling, timely disclosure of test expanded carrier screening, and should meet the following results to patients, and availability of invasive diagnostic testing ACOG guidelines11,17: 12 when needed. 1. Carrier screening of any nature is voluntary, and it is o0045 reasonable to accept or decline. s0035 Expanded Carrier Screening 2. Results of genetic testing are confidential and protected o0050 p0055 Although ethnic-based carrier screening has been accepted in health insurance and employment by the Genetic practice for years, there are limitations to the ability of this Information Non-Discrimination Act of 2008. N

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18 PART 1 Scientific Basis of Perinatal Biology

Prenatal/ Sources Embryonic NONINVASIVE: Cell-free DNA Sources: Sources: ͺͺͺͺͺͺͺͺͺͺͺͺͺͺͺͺͺͺͺͺͺͺͺͺͺͺͺͺ Gametes Embryo biopsy INVASIVE: Chorionic villi Amniocytes Fetal biopsy

Antenatal

Preconception

Sources: Any nucleated cells Postnatal Adulthood (cheek swab, blood sample, etc.) Pregnancy Sources: Genomic DNA failure from nucleated cells Sources: (blood, skin, hair, cheek Products of swab, saliva, etc.) conception f0010 A

SCREENING OPTIONSDIAGNOSTIC OPTIONS Resolution

MACRO (chromosome) Karyotype Carrier screening Microarray Parental DNA MICRO CGH (subchromosome) SNP array

FISH PGS

Embryo biopsy

Southern Sequencing Next- SEQUENCE blot generation NIPS VARIANTS PGD PCR sequencing Cell-free fetal DNA

B Increasing genomic “coverage” Figure 2.1 Schematic of preconceptional and prenatal molecular genetic screening and testing options. (A) Genomic DNA is the essential substrate needed for risk assessment or diagnosis of diseases that have a genetic basis. The range of options available for genetic testing throughout the reproductive process are shown. (B) Summary of the various molecular genetic technologies in use for reproductive genetic assessment. The techniques on the left side of the panel represent screening options, which will identify a level of risk that a pregnancy may be affected by a genetic disorder. The techniques on the right side of the panel are designed for diagnosis of genetic variants that may be associated with clinical pheno- types. Diagnostic tests require genomic DNA substrate from the embryo or fetus, and can be used to validate positive findings on a screening test. Each of the listed techniques is described in detail in this chapter. CGH, Comparative genomic hybridization; FISH, fluorescence in situ hybridization; NIPS, noninvasive prenatal screening; PCR, polymerase chain reaction; PGD, preimplantation genetic diagnosis; PGS, preimplantation genetic screening; SNP, single nucleotide polymorphism. N

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2 Molecular Genetic Technology 19

t0020 TABLE 2.3 Disorders Recommended for Ethnic-Based or Pan-ethnic Carrier Screening by ACOG Screening Test Group Genetic Disorder Carrier Frequency Available Detection Rate (%) ALL WOMEN Spinal muscular atrophy Caucasian, 1 : 35 DNA variants Caucasian: 95% considering Hispanic, 1 : 117 (carrier screening Hispanic: 91% pregnancy or Ashkenazi Jew, 1 : 41 requires quantitative Ashkenazi Jew: 90% currently Asian, 1 : 53 PCR) Asian: 93% pregnant African-American, African-American: 1 : 66 71% Cystic fibrosis Caucasian, 1 : 25 DNA variants (>1300 Caucasian: 88% Hispanic, 1 : 58 disease-associated Hispanic: 72% Ashkenazi Jew, 1 : 24 alleles identified) Ashkenazi Jew: 94% Asian, 1 : 94 Most common screen is Asian: 49% African-American, panel of 23 pan- African-American: 1 : 61 ethnic variants 65% Hemoglobinopathy Hgb S CBC with RBC indices (includes sickle cell disease [Hgb S], African-American, for all women α-thalassemia, and β-thalassemia) 1 : 10 Hemoglobin Also in high frequency: electrophoresis if Mediterranean, ethnicity-based risk Middle Eastern, or abnormal RBC Southeast Asian, or indices West Indian descent α-Thalassemia African, 1 : 3 Mediterranean, 1 : 30 Southeast Asian, Middle Eastern, 1 : 20 β-Thalassemia African-American, <1 : 8 Asian, 1 : 20 Mediterranean, 1 : 7 Family history of 1 : 259 in general DNA-based molecular fragile X–related (related disorders include premature population analysis (Southern disorders or ovarian insufficiency and fragile X– blot and PCR) for intellectual associated tremor/ataxia syndrome) triplet repeat disability suggestive of fragile X syndrome Ashkenazi Jewish Tay-Sachs disease Ashkenazi Jew, 1 : 30 Biochemical: 98% (Eastern and (Note: screening also recommended if French Canadian, hexosaminidase A Central European patient is French Canadian or Cajun Cajun, 1 : 30 to 1 : 50 level descent; should descent) Non-Jewish groups, include Jews of 1 : 300 unknown Canavan disease 1 : 41 DNA variants 97% descent) Familial dysautonomia 1 : 31 DNA variants 99% Additional Fanconi anemia 1 : 89 DNA variants 99% autosomal group C recessive Bloom syndrome 1 : 100 DNA variants 95%–97% conditions for Niemann-Pick 1 : 90 DNA variants 95% which screening disease type A should be Mucolipidosis 1 : 127 DNA variants 95% considered in type IV patients of Gaucher disease 1 : 15 DNA variants 95% Ashkenazi Jewish Familial 1 : 52 DNA variants descent hyperinsulinism Glycogen storage 1 : 71 DNA variants disease type IA Joubert syndrome 1 : 92 DNA variants Maple syrup urine 1 : 81 DNA variants disease type 1B Usher syndrome 1 : 95 (type III) DNA variants ACOG, American College of Obstetricians and Gynecologists; CBC, complete blood count; PCR, polymerase chain reaction; RBC, red blood cell count.

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20 PART 1 Scientific Basis of Perinatal Biology

o0055 3. Conditions included on expanded carrier screening which are laboratory-based techniques for evaluating DNA panels vary in severity. Many are associated with signifi- sequence variation in an embryo or fetus. The benefits and cant adverse outcomes such as cognitive impairment, limitations of the various types of genetic assessment are dis- decreased life expectancy, and need for medical or surgi- cussed for each technique. General benefits of genetic testing cal intervention. include identification of disorders for which in utero treatment o0060 4. Pregnancy risk assessment depends on accurate knowl- may provide benefit, optimization of neonatal outcomes by edge of paternity. If the biological father is not available planning appropriate delivery staffing and location, providing for carrier screening, accurate risk assessment for reces- family preparation for caring for a child with a genetic disorder, sive conditions is not possible. the option of pregnancy termination, and providing reassur- o0065 5. A negative screen reduces but does not eliminate risk to ance when results are normal. offspring. o0070 6. Because expanded carrier screening includes a large Sources of Parental Genetic Material s0045 number of disorders, it is common to identify carriers for one or more conditions. In most cases, being a carrier of Genomic DNA is relatively stable; therefore it can be obtained p0165 an autosomal recessive condition has no clinical conse- from any cell with a nucleus, even if the cells are no longer quences for the individual carrier. If each partner is iden- viable. Samples for molecular testing can include blood lym- tified as a carrier of a different autosomal recessive phocytes, skin scrapings, hair, cheek cells or saliva, semen, urine, condition, offspring are not likely to be affected. and paraffin tissue blocks. At the individual gene level, disease- o0075 7. In some instances, an individual may learn that he/she has specific testing for families that carry known genetic variants two pathogenic variants for a condition (homozygous or may be performed using standard polymerase chain reaction compound heterozygous) and thus learn through carrier (PCR) amplification and Sanger DNA sequencing methods screening that he/she has an autosomal recessive condi- (see Hybridization Techniques: Southern Blot, Polymerase tion that could affect his/her personal health. Some Chain Reaction, later). An updated list of relatively common expanded screening panels screen for selected autosomal genetic conditions for which DNA-based prenatal diagnosis dominant and X-linked conditions, and likewise an indi- is available is kept on the Genetic Testing Registry website vidual may learn that he/she has one of these conditions (www.ncbi.nlm.nih.gov/gtr). that might affect his/her health. Referral to an appropriate specialist for medical management and genetic counsel- ing is indicated in such circumstances to review the inher- Sources of Fetal Genetic Material s0050 itance patterns, recurrence risks, and clinical features. EMBRYO BIOPSY s0055 o0080 8. It is important for parents to understand that over 30% of genetic disorders are the result of de novo genetic vari- Advancement of in vitro fertilization techniques has allowed p0170 ants. De novo variants arise in the germline or due to an optimization of methods to remove or biopsy small numbers error of somatic cell division. Therefore these variants of cells from an in vitro fertilized embryo for genetic assessment will not be detected on parental carrier screens. prior to implantation. The techniques available to retrieve pre- o0085 9. Downstream prenatal genetic testing on the offspring of implantation cells include biopsy of prefertilized carrier-positive parents is not available for all of the genes oocytes, biopsy of one or two cells (termed blastomeres) from offered on expanded carrier screening panels. Therefore the six- to eight-cell early-cleavage-stage embryo on day 3, or careful research on which laboratory can perform prenatal removal of 5 to 12 cells from the trophectoderm of the 5- to testing for a given condition is necessary prior to perform- 7-day blastocyst.20 In all cases, removal of the cells does not ing chorionic villus sampling (CVS) or amniocentesis. appear to cause any cellular damage, with continued develop- ment of the embryo and no increased risk for congenital s0040 Fetal Genetic Screening and Testing anomalies.21 p0155 ACOG and the Society for Maternal-Fetal Medicine (SMFM) recommend that all pregnant women be counseled, as early as NONINVASIVE APPROACH: CELL-FREE FETAL s0060 possible in their prenatal care, about the opportunities for pre- DNA IN THE MATERNAL CIRCULATION natal genetic assessment consisting of either aneuploidy screen- ing or diagnostic testing. This recommendation is not dependent Early attempts at noninvasive genetically based prenatal screen- p0175 on maternal age or other risk factors.9 Furthermore, the same ing were focused on isolation of intact fetal cells within the professional societies, in addition to the American College of maternal circulation. To date, this technology has proven Radiology, all recommend prenatal ultrasound for multiple unsuitable for clinical application due to multiple technological reasons, including accurate determination of gestational age, obstacles such as limited numbers of fetal cells, unreliable fetal number, cardiac activity, and placental localization and recovery of fetal cells, and evidence that these cells persist long diagnosis of major fetal anomalies.18 The identification of fetal after pregnancy, thus complicating specificity in the setting of structural anomalies or minor sonographic markers on ultra- subsequent pregnancies.22 sound increases the likelihood of aneuploidy, DNA microdele- In contrast, identification of fetal-derived cell-free smallp0180 tions, or other genetic syndromes.19 Prenatal genetic testing DNA fragments (<200 bp) in maternal plasma has been highly should be offered to further evaluate abnormal findings on successful. In 1997, Dr. Dennis Lo and colleagues published the prenatal ultrasound. first report of identifiable cell-free fetus-derived p0160 Comprehensive discussion of screening modalities and sequences in the plasma of pregnant women carrying male screening or testing indications for pregnancy is presented in fetuses.23 Subsequent work has established that approximately N Chapter 32. Here we focus on molecular genetic technologies, 5% to 20% of the cell-free DNA circulating in maternal plasma

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2 Molecular Genetic Technology 21

originates from the fetus (derived from the trophoblast) and single sample. In development the mosaicism becomes general- provides 25 times more fetal DNA present in a pregnant ized (present in the entire organism) or confined to a specific woman’s plasma than could be extracted from intact circulating compartment (placenta, fetus, specific fetal organ system), fetal cells.24 Massive parallel sequencing of cell-free DNA derived depending on whether the improper chromosome segregation from maternal plasma has led to the development of algorithms occurs in meiosis, early postzygotic mitosis, or late postzygotic to determine fetal ploidy (chromosome count) in noninvasive mitosis (also refer to Chapter 1 for review of mechanisms of prenatal screening (see details later). chromosome segregation). The most commonly encountered p0185 Circulating fetal DNA is predominantly a product of placen- aneuploidy in prenatal diagnostic testing is trisomy. Note that, tal apoptosis.25–27 This cell-free DNA consists of small fragments if trisomy originates in the zygote, the organism becomes (fewer than 200 bp), undergoes a rapid turnover, and may by way of trisomy rescue, or elimination of the extrane- appear in apoptotic bodies or nucleosomes.28 Fetal SRY gene ous chromosome copy. Generally speaking, the earlier in devel- sequences are present in the circulation as early as 18 days after opment that an abnormal chromosome segregation event such embryo transfer, before the definitive fetoplacental circulation, as or trisomy rescue occurs, the more wide- which is not established until 28 days after conception.29 Fetal spread the mosaicism may be in the differentiated organism DNA is continuously liberated into the maternal circulation, (i.e., more likely to affect both the chorion/placenta and the with a mean half-life estimated to be 16 minutes at term. Levels fetus). Later chromosome segregation events are more likely to increase until approximately 10 weeks’ gestation, remain stable be confined to specific cell types, giving rise to clinical findings between 10 and 21 weeks, and then continue to increase until such as confined placental mosaicism, which leads to discordant the third trimester.30 Fetal DNA levels are undetectable about 2 karyotypes found with CVS versus amniocentesis. By under- hours after birth.31 standing the developmental origin of the cells biopsied via CVS, amniocentesis, or postnatal karyotyping, the clinician can use appropriate testing methods to distinguish between placental s0065 INVASIVE PRENATAL GENETIC SAMPLES mosaicism and generalized mosaicism. p0190 For the goal of prenatal diagnosis (not screening), the most commonly employed forms of genetic testing are CVS and Laboratory Considerations for Amniocentesis s0075 amniocentesis to obtain cellular samples from the placenta and The cells in the amniotic fluid derive from extraembryonicp0205 fetus, respectively, for cytogenetic analysis and molecular genetic ectoderm, and include cells from the fetal skin, respiratory tract, testing (see also Chapter 32 for full discussion of indications urinary tract, gastrointestinal tract, and amnion. Amniotic fluid and techniques). ACOG recommends that all women, regard- after 16 weeks’ gestation may be analyzed directly by extraction less of age or risk, be offered biochemical and/or ultrasound of DNA from the amniocytes for hybridization-based methods screening and invasive testing.32 The decision to pursue invasive of DNA analysis such as microarray or PCR. Alternatively or in testing needs to incorporate considerations of level of risk that parallel, the amniocytes may be cultured for cytology-based the fetus is affected, level of risk associated with the procedure, methods of chromosomal analysis, such as karyotyping. and the patient’s impression of the impact of having an affected Cytology-based methods require the cells to proliferate in tissue child. culture. After 3 to 7 days of growth, sufficient mitoses are p0195 Of note, the risks involved with invasive testing may be much present for staining and karyotype analysis. Cells grown in lower than estimates that have been previously quoted since the flasks are harvested and analyzed together; those grown on cov- advent of invasive testing in the 1970s. Results of a recent meta- erslips are analyzed in situ as individual colonies.35,36 Amniocyte analysis, which included only contemporary large studies (pub- culture is quite reliable, with failure occurring in less than 1% lished after the year 2000, reporting greater than 1000 total of cases.37 Of note, hybridization-based analysis (microarray, procedures), demonstrated no significant difference in the risk PCR) can also be performed on DNA from cultured amnio- of prior to 24 weeks’ gestation for women undergo- cytes, and is customarily done to confirm any results from direct ing amniocentesis or CVS compared to those who do not have analysis. invasive testing.33 Procedure-related risk of miscarriage for amniocentesis was 0.1% and for CVS was 0.2%. Nevertheless, Amniocentesis: Mosaic Results s0080 rates of performance of these invasive procedures are signifi- Chromosomal mosaicism occurs in approximately 0.1% to p0210 cantly declining as screening tests with higher detection rates 0.3% of amniocentesis cases. The most common etiology is and lower false-positive rates have become available (see Non- pseudomosaicism,38 where the abnormality is evident in only invasive Prenatal Screening, later). Thus it will be more difficult one of several flasks or confined to a single colony on a cover- in coming years for practitioners to be adequately trained in slip. In this case the abnormal cells have arisen in vitro, are not these invasive techniques. It is important to note that the range present in the fetus, and are not clinically important. Even the of abnormalities that can be detected is far greater with invasive observation of multiple cell lines on more than one coverslip or testing than with any available noninvasive screening tests. in more than one flask in a sample does not necessarily mean that the fetus is mosaic, because the results are confirmed by s0070 Ontogenesis of Fetal Samples: repeat karyotyping at birth or pregnancy termination in only Origins of Mosaicism 70% of cases.39 Some mosaic results (e.g., trisomy 20) occur in p0200 To understand the clinical implications of abnormal chromo- the amniotic fluid relatively frequently, but are rarely confirmed somal content in some or all of the cells obtained from prenatal in the fetus.40 sampling (CVS or amniocentesis), it is important to review the True fetal mosaicism is rare, occurring in 0.25% of amnio- p0215 different cellular origins and early development of the fetus and centeses, but it can be clinically important, leading to pheno- placenta (Fig. 2.2).34 Chromosomal mosaicism refers to the typic or developmental abnormalities.38 This most frequently presence of two or more cell lines with different karyotypes in a results from postzygotic nondisjunction,38 but it can also occur N

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22 PART 1 Scientific Basis of Perinatal Biology

47 Potential origins of aneuploid cell lineage CHORIONIC VILLI (CVS) Placenta

47 Cytotrophoblast

Trophoblast Extraembryonic mesoderm (villus core)

47 AMNIOCENTESIS 47 Zygote Blastocyst Extraembryonic Cleavage ectoderm embryo (amniocytes)

47

Inner cell mass

f0015 Fetus Figure 2.2 Ontogenesis of fetal and placental tissues for prenatal testing: origins of mosaicism. Discrepancies in karyotype between the placenta and the fetus can occur because the cells contributing to the chorionic villi differentiate separately and distinctly from those forming the embryo in early development. Lineage differentiation that occurs at the blastocyst stage gives rise to the trophoblast cell line and the inner cell mass. The trophoblast becomes the cytotrophoblast and syncytiotrophoblast of the developing placenta. Cell subsets within the inner cell mass develop into the extraembryonic mesoderm (which makes up the core of the chorionic villi), the extraembryonic ectoderm, and the fetus proper. Mosaicism can occur through meiotic nondisjunction with subsequent trisomy rescue, or as a result of postzygotic mitotic nondisjunction. To under- stand the implications of abnormal karyotype analysis found on chorionic villus sampling (CVS) or amniocentesis, it is critical to understand the developmental origins of the cell populations sampled in each technique.

t0025 TABLE 2.4 Disorders Associated With Uniparental Disomy (UPD) Chromosome UPD Syndrome OMIMa 6 UPD(6)pat Transient #601410 7 UPD(7)mat Silver-Russell syndrome #180860 11 UPD(11)pat Beckwith-Wiedemann syndrome #130650 11 UPD(11)mat Silver-Russell syndrome #180860 14 UPD(14)pat Kagami-Ogata syndrome (uniparental disomy, paternal, ) #608149 14 UPD(14)mat Temple syndrome #616222 15 UPD(15)pat Angelman syndrome #105830 15 UPD(15)mat Prader-Willi syndrome #176270 20 UPD(20)pat Pseudohypoparathyroidism type Ib #603233 aOn the OMIM website, # indicates “Phenotype description, molecular basis known.” mat, Maternal; OMIM, Online Mendelian Inheritance in Man; pat, paternal.

from meiotic errors with trisomy rescue. An important consid- phenomenon in which certain genes are modified during game- eration for any prenatal mosaic results is to investigate the pos- togenesis or prior to fertilization such that they function differ- sibility of uniparental disomy (UPD) among euploid cells, or ently based on maternal or paternal inheritance. Uniparental the presence of two homologous chromosomes derived from inheritance of imprinted genes causes significant clinical syn- the same parent. If the mechanism of mosaicism is trisomy dromes, such as Prader-Willi syndrome caused by maternal rescue, theoretically one-third of the time the resulting diploid UPD of , and the converse, Angelman syn- cell line will have UPD.41 Clinical consequences of UPD depend drome caused by paternal UPD of chromosome 15. Table 2.4 on the chromosome involved and include loss of heterozygosity lists known imprinting disorders reported in the setting of and genomic imprinting. Loss of heterozygosity may result in UPD. expression of a recessively inherited variant when only one In many cases the question of whether amniotic fluid mosa- p0220 N parent is a carrier. Genomic imprinting is an epigenetic icism involves the fetus can be resolved by karyotyping fetal

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2 Molecular Genetic Technology 23

lymphocytes obtained by percutaneous umbilical blood sam- Direct cell analysis also minimizes the likelihood of maternal pling or cordocentesis.42 However, this approach may not be cell contamination because the maternal decidua has a low valid in all cases, because the mosaic cell line may involve other mitotic index. However, long-term tissue culture is better for fetal tissues but be excluded from the fetal hematopoietic com- generating a larger amount of material, if a small tissue sample partment, and therefore not present in a fetal blood sample.40 was recovered. Additionally, large series comparing CVS direct Certain chromosomes, such as 22, are notorious for exclusion and long-term culture method results to fetal karyotyping have from fetal blood and may require testing of additional fetal demonstrated a higher rate of inaccurate diagnosis by the direct tissues, such as the skin, or postnatal biopsy.43 method than by the long-term culture method.47,48 The false- p0225 Evaluation of mosaic results should include detailed ultra- positive rate for the direct method is estimated at 1 in 125, sound assessment to exclude structural anomalies. If both ultra- compared to 1 in 350 for the long-term culture method; and sound and fetal percutaneous umbilical blood sampling are the false-negative rate is estimated at 1 in 1000 for the direct normal, the parents can be generally reassured that in most method, compared to less than 1 in 5000 for the culture method. cases the fetus is unaffected.42 However, a small chance of fetal Given this low rate of false negatives overall, if a long-term involvement still exists, because the presence of an undetectable culture fails, a nonmosaic normal direct preparation result can but clinically significant abnormal cell line can never be abso- be considered conclusive, although rare cases of false-negative lutely excluded. Follow-up ultrasound examinations are usually rates for trisomy 21 and 18 have been reported.49,50 Ideally, both performed due to a higher chance of fetal growth restriction the direct and culture methods should be used to maximize associated with some mosaic results. Because of the complexity diagnostic accuracy because each evaluates slightly different of interpreting mosaic amniotic fluid results, consultation with tissue sources, namely cytotrophoblasts in the direct method a cytogenetics laboratory and a clinical geneticist or genetic and mesenchymal cells following long-term culture. If perfor- counselor is generally recommended. mance of both methods of analysis is not feasible or cost- effective, the long-term culture method is more likely to s0085 Laboratory Considerations for Chorionic accurately represent fetal karyotype. Villus Sampling Most biochemical diagnoses that can be made from amniotic p0245 p0230 Chorionic villi have three major components: an outer layer of fluid or cultured amniocytes can be made from chorionic villi hormonally active and invasive syncytiotrophoblast, a middle as well.51 In many cases the results are available more rapidly layer of cytotrophoblast from which syncytiotrophoblast cells and efficiently when villi are used rather than amniocytes, are derived, and an inner mesodermal core containing blood because sufficient enzyme is present to allow for direct analysis, capillaries and mesenchymal stroma. The cytotrophoblasts and rather than requiring products of tissue culture. However, for syncytiotrophoblasts derive from the trophoblast layer, which certain rare biochemical diagnoses, such as metachromatic leu- differentiates at the blastocyst stage. The villus core derives from kodystrophy, villi are not an appropriate or reliable diagnostic the extraembryonic mesoderm of the inner cell mass (see source.51 The affected enzyme in metachromatic leukodystro- Fig. 2.2). phy, arylsulfatase A, is expressed at very low levels in chorionic p0235 The average sample from a CVS procedure contains 15 to villi even in unaffected individuals. To ensure that appropriate 30 mg of villous material. The villi collected in the syringe are testing is possible for a specific diagnosis or condition, the labo- carefully and aseptically transferred for inspection and dissec- ratory should be consulted before CVS is performed. tion under a microscope to eliminate adherent decidua. The villi are then exposed to trypsin to digest and separate the Chorionic Villus Sampling: Maternal Contamination s0090 cytotrophoblast from the underlying mesodermal core. As with and Mosaicism amniocytes, DNA can be extracted from both cell types in the Genetic evaluation of chorionic villi provides a high degree of p0250 villi for direct analysis (without the need for culture) by hybrid- success and accuracy, particularly with regard to the diagnosis ization methods such as microarray or PCR. One notable of common .52 The US Collaborative Study revealed a advantage of chorionic villi over amniocytes is that the cytotro- 99.7% rate of successful cytogenetic diagnosis, with only 1.1% phoblast has a high mitotic index, with many spontaneous of the patients requiring a second diagnostic test such as amnio- mitoses available for immediate cytology-based chromosomal centesis or fetal blood analysis to further interpret the results.52 analysis. This direct chromosome preparation can give prelimi- In most cases the additional testing was required to delineate nary karyotype results within 2 to 3 hours.44,45 However, most the clinical significance of mosaic or other ambiguous results laboratories now use a short-term culture to improve karyotype (76%), although laboratory failure (21%) and maternal cell quality and thus report results within 2 to 4 days. Direct analysis contamination (3%) also required follow-up testing. results are generally confirmed with long-term culture-based Chorionic villus samples typically contain a mixture of pla- p0255 testing. The remaining villi are placed in tissue culture to allow cental villi and maternally derived decidua. Although specimens proliferation of cytotrophoblasts as well as mesenchymal cells are thoroughly washed and inspected under a microscope after from the mesodermal core. Cultured mesenchymal cells are collection, some maternal cells may remain and grow in the typically ready for harvest and cytology-based chromosomal culture. As a result, two cell lines, one fetal and the other mater- analysis within 1 week.46 As with amniocyte culture, DNA may nal, may be identified. In other cases the maternal cell line may be extracted from cultured cytotrophoblasts or villous mesen- completely overgrow the culture, thereby leading to diagnostic chymal cells as well, allowing for later application of errors, including incorrect sex determination53–55 and poten- hybridization-based DNA analysis (microarray, PCR). tially false-negative diagnoses. For this reason, a maternal blood p0240 The direct method of sample analysis has the advantage of sample should be provided along with the chorionic villus providing a rapid result and avoids cultural artifacts such as sample so that, if indicated, the level of maternal cell contami- culture-induced aneuploidy and overgrowth with cells of nation can be quantitated by genotyping informative microsat- certain that may have culture growth advantage. ellite markers in maternal and chorionic villus samples.56 Direct N

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24 PART 1 Scientific Basis of Perinatal Biology

preparations of chorionic villi are generally thought to prevent in a culture than in a direct preparation. The specific chromo- maternal cell contamination,44,53 whereas long-term culture has some involved also predicts the likelihood of fetal involve- a rate varying from 1.8% to 4%.54 Maternal cell contamination ment.62 Phillips and coworkers70 demonstrated that autosomal is much less likely to occur in cytogenetic laboratories that mosaicism involving common trisomies (i.e., 21, 18, and 13) process large numbers of prenatal samples and have experi- was confirmed in the fetus in 19% of cases, whereas uncommon enced technicians who can separate the two cell populations. trisomies involved the fetus in only 3%. When sex chromosome Interestingly, for reasons still uncertain, maternal cell contami- mosaicism was found in the placenta, the abnormal cell line was nation occurs more frequently in specimens retrieved by the confirmed in the fetus in 16% of cases. 54 transcervical route than by the transabdominal route. When placental mosaicism is discovered, amniocentesis can p0280 p0260 The second major source of potential diagnostic error asso- be performed to elucidate the extent of fetal involvement. When ciated with CVS is confined placental mosaicism. Due to dispa- mosaicism is limited to the direct preparation of CVS, subse- rate ontogenesis, or developmental origin, of fetal and chorionic quent amniocentesis correlates with fetal genotype in 100% of cells, chorionic villus tissue does not always reflect the fetal cases.70 When mosaicism is observed in long-term culture prep- genotype (see Fig. 2.2). There are two basic mechanisms by aration of CVS, the CVS result correctly predicts the true fetal which an aneuploid cell line would be confined to the placenta. karyotype in approximately 94% of cases, with both false- The first possibility is a trisomy rescue early in the cleavage- positive and false-negative results seen.70 Two cases were stage embryo, in which the aneuploid cell line becomes isolated reported of mosaic trisomy 21 on villus culture, and despite a to the trophoblast while the euploid (“rescued”) cell line is normal amniotic fluid analysis, a fetus or newborn was seen isolated to the inner cell mass. The other potential mechanism with mosaic aneuploidy.52 Therefore in cases of mosaicism is a mitotic error at the late cleavage or blastocyst stage that is present in long-term culture preparations of CVS, particularly isolated to the trophoblast or the portion of the inner cell mass involving common trisomies (21, 18, 13), amniocentesis should that gives rise to the extraembryonic mesoderm.34 Mosaic aneu- be offered, but the patient should be advised of the rare possibil- ploid results on CVS must therefore be interpreted with caution, ity of false-negative amniocentesis results. If amniocentesis is and follow-up testing is necessary to determine the full extent normal after a mosaic long-term culture CVS result, follow-up of cells that share the abnormal chromosome complement. may include detailed ultrasonography, fetal blood sampling, or p0265 Although initially there was concern that this phenomenon fetal skin biopsy to fully assess the risk of fetal mosaicism. At of confined placental mosaicism might invalidate CVS as a pre- present, the predictive accuracy of these additional tests is natal diagnostic tool, subsequent investigations have led to a uncertain and fetal skin biopsy is rarely performed. clearer understanding of villus biology, so that accurate clinical For CVS mosaicism involving sex chromosome abnor- p0285 interpretation is now possible. This understanding has also malities, polyploidy, marker chromosomes, structural rear- revealed new information about the etiology of pregnancy loss, rangements, and most uncommon trisomies, the patient can discovered a new cause of fetal growth restriction, and clarified generally be reassured that, if amniocentesis results are euploid the basic mechanism of UPD.41,57–59 Importantly, a CVS diagno- and detailed ultrasonographic examination is normal, the sis of confined placental mosaicism for trisomy 15 may be the mosaicism is unlikely to be present in the fetus. As described initial clue that UPD could be present and lead to an affected previously, in certain cases testing for UPD is indicated (see child.60,61 Therefore all cases in which trisomy 15 (either com- Table 2.4). plete or mosaic) is diagnosed in the placenta should be evalu- ated for UPD by amniotic fluid analysis. In addition to OTHER INVASIVE FETAL BIOPSY/DIAGNOSTIC s0095 chromosome 15, chromosomes 6, 7, 11, 14, and 20 contain PROCEDURES known imprinted regions and require similar follow-up (see 34,62 Table 2.4). On infrequent occasions analysis of other fetal tissues may be p0290 p0270 Confined placental mosaicism (unassociated with UPD) can required. Fetal skin biopsy can be helpful in the evaluation of alter placental function and lead to fetal growth restriction or fetal mosaicism for chromosomes (such as 22) known not to be perinatal death.57,59,63–67 This aneuploidy effect on placental manifested in fetal blood.43 Historically, fetal skin biopsy was function is generally limited to specific chromosomes. For also used to aid in the diagnosis of severe genetic skin blistering example, confined placental mosaicism for chromosome 16 disorders (ichthyosis) to allow for electron microscopy and leads to severe fetal growth restriction, prematurity, or perinatal immunohistochemistry of cell junction structures; however, the death, with less than 30% of pregnancies resulting in normal, availability of genetic testing for these disorders has since elimi- appropriate-for-gestational-age, full-term infants.64,68–71 Con- nated the need for fetal biopsy.73 Fetal muscle biopsy for dys- fined placental mosaicism involving chromosomes 2, 7 to 10, trophin analysis has been used to diagnose Duchenne muscular 13 to 18, 21, and 22 have also been associated with poor peri- dystrophy in a male fetus if DNA testing was not informa- natal outcomes; however, the exact mechanism by which abnor- tive.74,75 Fetal kidney biopsy has diagnosed congenital nephrosis mal cells in the placenta affect fetal growth or placental function in utero,76 and aspiration and analysis of fetal urine is impera- is poorly understood.41 tive in the pre-shunt evaluation of fetal renal function in cases 54,68,72 77 p0275 Mosaicism occurs in about 1% of all CVS samples but of bladder outlet obstructions. Fluid from a cystic hygroma is confirmed in the fetus in 10% to 40% of these cases. In most can be aspirated and accurately analyzed in lieu of amniocen- cases, if the mosaic results are confined to the placenta, fetal tesis or CVS. Each of these fetal biopsy techniques is performed development will be normal. If the mosaic cell line involves under ultrasound guidance. These procedures are only rarely the fetus, significant phenotypic consequences are possible. The required, so their use is usually confined to only a few regional probability of fetal involvement appears to be related to the referral centers (with the exception of cystic hygroma aspira- tissue source in which the aneuploid cells were detected; an tion, which is similar to amniocentesis) in hopes of limiting N abnormal result is more likely to reflect a true fetal mosaicism procedural risk.

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2 Molecular Genetic Technology 25

1 2 3 4 5

6789101112

13 14 15 16 17 18

19 20 21 22 XY f0020 Figure 2.3 A standard G-banded karyogram. There are approximately 550 bands in one haploid set of chromosomes in this karyogram. The sex karyotype is X,Y (male). A female karyogram would show two X chromosomes.

FISH probes are relatively short, fluorescence-labeled DNA p0305 s0100 Cytogenetic Testing: Detection of sequences that are hybridized to a known location on a specific Chromosomal or Subchromosomal chromosome, allowing the number and location of specific Abnormalities DNA sequences to be determined. Metaphase or interphase cells are evaluated by counting the number of discrete fluorescent s0105 KARYOTYPE signals from each probe. A normal diploid cell queried with a p0295 The predominant use for fetal cells obtained by amniocentesis probe for chromosome region in the long arm of chromosome or CVS has traditionally been cytogenetic analysis. Cells can be 21 would have two signals, whereas a trisomy 21 cell would have analyzed directly or after cell culture for approximately a week three (see Fig. 2.4D). to synchronize cells in metaphase for chromosomal Giemsa Prenatal interphase evaluation of uncultured amniotic fluid p0310 staining (G-banding) to determine karyotype. With this type of can detect aneuploidies caused by , complete tri- staining, each chromosome has a unique pattern (Fig. 2.3). The somies, trisomies associated with robertsonian translocations, stained chromosomes are visualized under light microscopy, triploidy, and other numerical chromosomal abnormalities. In and large deletions or rearrangements can be detected (resolu- standard practice, probes involving chromosomes 13, 18, 21, X, tion on the order of 5 to 10 Mb). Higher resolution or more and Y are used. This technology does not routinely detect cyto- specific testing for known disease-causing chromosome regions, genetic abnormalities such as mosaics, translocations, and rare such as 22q11, requires molecular cytogenetic technology such aneuploidies.78,79 Since 1993 the position of the American as fluorescence in situ hybridization (FISH) and chromosomal College of and Genomics (ACMG) has been microarray analysis (CMA). that prenatal FISH is investigational. In 1997 the US Food and Drug Administration cleared the specific FISH probes to enu- merate chromosomes 13, 18, 21, X, and Y for prenatal diagnosis. s0110 FLUORESCENCE IN SITU HYBRIDIZATION Subsequent studies have demonstrated an extremely high con- p0300 FISH, the most widely used molecular cytogenetic technology, cordance rate between FISH and standard cytogenetics (99.8%) takes advantage of the complementary nature of DNA. In this for the specific abnormalities that the assay is designed to approach, denatured DNA sequences labeled with a fluorescent detect.80–83 These performance characteristics support the use dye are hybridized (matched by complementary nucleotide of FISH for prenatal testing when a diagnosis of aneuploidy of sequence) onto denatured chromosomes that have been immo- , 18, 21, X, or Y is highly suspected by virtue of bilized onto a slide. The chromosomes are then viewed with a maternal age, positive maternal serum screening, or abnormal wavelength of light that excites the fluorescent dye (Fig. 2.4). ultrasound findings, although confirmation with amniocyte FISH is a powerful tool to confirm or diagnose syndromes that culture results is still recommended.84 are caused by microdeletions of segments of chromosomal At present, it is suggested that FISH analysis not be used as p0315 material (see Table 2.2). a primary screening test on all genetic amniocenteses because N

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26 PART 1 Scientific Basis of Perinatal Biology

A B

C D E f0025 Figure 2.4 Chromosomal aberrations detected by fluorescence in situ hybridization (FISH) analysis. (A) Two-color metaphase analysis reveal- ing an unbalanced chromosome rearrangement in a patient with normal karyotype. FISH analysis using probes mapped to the terminal 6p region (red signal) and a control probe, located at the long arm of chromosome 6 (green signal), detected an additional red signal on the short arm of chromosome 2 (arrow). (B) FISH analysis with the chromosome 2 locus-specific probes showing the red and green signals on the normal chromo- some 2. Red signal is absent on the second chromosome 2 (arrow), indicating a deletion on the terminal short arm. (C) A duplication involving the 17p11.2 region (red signal) is detected on interphase cells from a patient with Charcot-Marie-Tooth 1A disease. (D) FISH analysis of interphase amniotic fluid cells detecting three signals for (red signal) and two signals for chromosome 13 (green signal), indicating a fetus with trisomy 21. (E) Multicolor FISH analysis on paraffin-embedded tissue sections from a missed abortion showing three copies of (green signal) and a total of three sex chromosomes: two copies of the (aqua signal) and one copy of the Y chromosome (red signal), indicating a fetus with triploidy.

of its inability to detect structural rearrangements, mosaicism, diagnosis of 22q11.2 deletion syndrome, a phenotypically vari- marker chromosomes, and uncommon trisomies. Evans and able autosomal dominant deletion syndrome that commonly colleagues85 surveyed the results of almost 73,000 prenatal cases includes congenital heart defects, palate abnormalities, charac- from seven centers and reported that only 67% of chromosomal teristic facial features, immunodeficiency, learning disabilities, abnormalities would have been detected by routine FISH. This and other abnormalities requiring specialized medical care. interpretation may be misleading in that some of the missed Specialized FISH probes to the TUP1-like enhancer of split abnormalities would not have had an impact on fetal develop- protein 1 (TUPLE) region of hybridize within ment. Because all abnormalities would be detectable by tissue the deleted region. FISH results with only one hybridized probe culture, FISH analysis is not cost-effective. Most practitioners can diagnose a subchromosomal deletion that could not be order FISH to offer more rapid reassurance to patients with detected by conventional karyotype.87 an unusually high degree of anxiety, or to test fetuses at the highest risk, such as those with ultrasound anomalies. It is also CHROMOSOMAL MICROARRAY ANALYSIS s0115 beneficial when rapid results are crucial to subsequent manage- ment, such as with advanced gestational age. FISH on meta- CMA is a powerful technology with the ability to survey the p0320 phase chromosomes using probes for unique sequences has entire genome and to identify not only chromosomal abnor- greatly expanded the resolution of conventional chromosomal malities detected by conventional cytogenetic techniques but N analysis.86 One of the most commonly utilized examples is for also submicroscopic deletions and duplications (CNVs).88 This

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2 Molecular Genetic Technology 27

Test Control

1

2

3

C A B

pqpq 2.0

1.0 1.0 Deletion 0.0 0.0 Triplication Duplication –1.0 –1.0

1 copy 2 copies –2.0 2 copies 3 copies 4 copies –2.0

log2 log2 Position, Mb 26.0 27.0 28.0 29.0 36.0 38.0 40.0 42.0 44.0

f0030 DE Figure 2.5 Array comparative genomic hybridization (CGH). (A) A DNA microarray is usually a microscope slide with a set of short DNA frag- ments from selected regions of the genome spotted onto a surface. (B) A magnified view of the microarray surface after hybridization. (C) Genomic DNAs from the test (patient) and control (normal individual of the same gender) samples are differentially labeled using Cy3 and Cy5 cyanine dyes, mixed, and hybridized onto the array. Spots with an equal amount of Cy3 and Cy5 (spot 1) appear yellow, whereas spots with an extra amount of test DNA look green (spot 2) and spots where the amount of test DNA is decreased appear red (spot 3). (D) Array CGH plot showing a deletion. Probes (black and red dots) are aligned along the X axis according to the physical position on the chromosome (from the short to the long arm). The ratio between the intensity of Cy3 and Cy5 for each probe is calculated and values are placed onto a log2 scale (Y axis). Probes with an equal amount of the test and control DNA (black dots, ratio = 2/2, log2(2/2) = 0) are clustered around a “0” score on the log2 scale. A negative log2 score indicates deletion (red dots; ratio = 1/2, log2(1/2) = −1.0). (E) Array CGH plot showing a complex duplication/triplication rearrangement. Gain in DNA copy number is seen as positive log2 scores. Blue dots represent duplication (ratio = 3/2, log2(3/2) = 0.58). Green dots (ratio = 4/2, log2(4/2) = 1.0) depict a triplication (total four copies of DNA).

is a high-throughput technique to detect the relative “dose” of hybridized sequence can be quantitated (Fig. 2.5). Using this genetic material at thousands of points across the genome. A platform, a patient’s genome is compared to a normal control, microarray generally consists of a thin slice of glass or silicon and readout is expressed by comparative intensity between the about the size of a postage stamp on which threads of syn- patient and the control.89 The advantage of an oligonucleotide thetic nucleic acids are arrayed. Sample probes are added to array is less noise (variation generated by the experimental the chip, and matches are read by an electronic scanner. The method that does not represent true biological variation), and resolution of CMA is on the order of 10 to 400 kb, or more coverage of regions of the genome that do not contain SNPs. than 100-fold greater resolution than traditional G-banding The second popular platform for prenatal diagnosis is a pure p0330 karyotyping. SNP array. In an SNP array, probes are chosen from DNA loca- p0325 Three general microarray platforms are in common use for tions known to vary by a single base pair. A patient’s DNA is genome assessment: array comparative genomic hybridization hybridized to the array, and readout is by absolute intensity of (CGH), pure SNP array, and a combination platform that uses signal from bound DNA fragments (Fig. 2.6). This platform both oligonucleotides and SNPs. The array CGH platform is does not require a normal standard because the assay is designed used to measure differences in copy number or dosage of a to demonstrate the number of alleles the patient has at each particular chromosomal segment. In brief, two genomic librar- represented locus. Greater than or less than two alleles at any ies are mixed and hybridized to a panel of reference oligonucle- tested locus represents gain or loss of genetic material in that otides from across the genome such that relative “doses” of a region. This method can detect more abnormalities than just N

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28 PART 1 Scientific Basis of Perinatal Biology

Spot Patient Green label Genotype Allele A Allele B Hybridise AA AB 2 copies BB A- 1 copy B- -- 0 copies Computer analysis AAA and comparison to reference datafile AAB 3 copies ABB BBB 3 copies

2 copies 1 copy Intensity Fluorescence

BB Figure 2.6 Single nucleotide polymorphism (SNP) array. In an SNP array, a patient’s DNA is hybridized to the array and does not require a reference sequence, and readout is by absolute intensity of signal from bound B– DNA fragments. Resolution will depend on the probe density and may be limited by the nonrandom distribu- tion of SNPs in the genome. A major advantage of SNP arrays over other platforms is the ability to detect trip-

B Allele Frequency loidy, mosaicism, maternal cell contamination, and loss – – of heterozygosity. (From Karampetsou E, Morrogh D, Chitty L. Microarray technology for the diagnosis of fetal chromosomal aberrations: which platform should we use? J Clin Med. 2014;3:663–678. https://creativecommons. f0035 org/licenses/by-nc-sa/3.0/legalcode.)

copy number (e.g., UPD), and can determine , pater- fetal ultrasound. Several studies have demonstrated the incre- nity, degree of , parent of origin for a given mental diagnostic utility of CMA in the setting of a fetus with variant, and maternal cell contamination.90 A third type of plat- one or more anomalies on ultrasound but a normal karyotype. form combines both oligonucleotides and SNPs and provides A prospective study funded by the Eunice Kennedy Shriver the advantages of diminished noise and SNP information. Table National Institute of Child Health and Human Development 2.5 summarizes the relative benefits and drawbacks of the most identified clinically relevant CNVs by microarray in 6% of commonly employed microarray platforms. anomalous fetuses with a normal karyotype.93 Furthermore, the p0335 In neonatal and pediatric studies, microarray results have likelihood of identifying either pathogenic CNVs or CNVs of revealed underlying genetic etiologies for 15% to 20% of cases uncertain significance was more likely in fetuses with multiple with previously unexplained developmental delay, intellectual anomalies, whereas for isolated findings the greatest yield was disability, or congenital anomalies. Only about 3% of these in fetuses with cardiac and renal anomalies.94 Even in the cases would have been diagnosed by traditional karyotyping.91 absence of identified anomalies, CMA should be offered for any CMA is considered the first-line test of an individual with unex- patient undergoing invasive sampling, because CMA has been plained birth defects or mental retardation, or in unexplained demonstrated to detect a pathogenic or likely pathogenic CNV stillbirth.92 In contrast to neonatal studies, which have the in approximately 1% of patients with a normal ultrasound and advantage of correlating genomic findings with complete phys- a normal karyotype.95 ical examination and behavioral phenotype, prenatal applica- Clinicians and genetic counselors must continue to exercise p0340 N tions are limited to phenotypic findings that can be detected by reasonable caution when offering microarray analysis and when

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2 Molecular Genetic Technology 29

t0030 TABLE 2.5 Abnormalities Detected With Conventional Karyotype, CGH, and SNP Arrays Balanced Translocations Unbalanced AOH/ Technique Aneuploidy and Inversions Translocations Triploidy Consanguinity CNVs

Conventional karyotype + + + + − − CGH array + − + − − + SNP array + − + + + + AOH, Absence of heterozygosity; CGH, comparative genomic hybridization; CNVs, copy number variations; SNP, single nucleotide polymorphism. From Society for Maternal-Fetal Medicine (SMFM), Dugoff L, Norton ME, Kuller JA. The use of chromosomal microarray for prenatal diagnosis. Am J Obstet Gynecol. 2016;215(4):B2–B9.

interpreting results of microarray findings. Patients are entitled Although the full clinical effect of the patient’s CNV is not to professional genetic counseling before microarray testing to known, the pathogenic nature of the CNV is not in ques- appropriately discuss the risks of testing, including the possibil- tion. The laboratory should provide an explanation of ity of detecting consanguinity, nonpaternity, or genetic variants why the alteration is considered pathogenic, including a of uncertain significance (VUS). An excellent CNV database list of genes known to be dosage-sensitive that occur in exists through the International Standards for Cytogenomic the altered region. 91 Arrays Consortium. Many CNVs may have dose-dependent Uncertain clinical significance: This designation represents a u0105 phenotypic effects, or may be modified by the presence of other fairly broad category, including CNV findings that are genetic elements such as SNPs. later demonstrated to be either clearly pathogenic or p0345 Traditionally, prenatal screening and testing modalities clearly benign. Fortunately, additional information on the focused on diseases that significantly affect quality of life and classification of CNVs is accumulating rapidly, which will have a fetal, neonatal, or early childhood onset and well-defined reduce the number of findings with uncertain significance phenotypes. With microarray technology, genetic variant rec- in the future, and the current rate of uncertain significance ognition may outstrip our current ability to interpret some of classification is less than 5%. However, if, at the time of these alterations.96 Previously unreported and relatively rare reporting, insufficient evidence is available for unequivo- variants with unknown phenotypes may be identified, which cal determination of clinical significance and the CNV requires skilled genetic counseling and interpretation to help meets the reporting criteria established by the laboratory, patients decide what to do with this information. Informing the CNV should be reported as a CNV of uncertain clinical patients of VUS may generate significant anxiety and negative significance. This uncertainty should be clear in the report. anticipation. Learning that one’s fetus has a possible abnormal- Laboratories are encouraged to include available evidence ity with unquantifiable risks of mysterious consequences natu- for the likelihood that the CNV is pathogenic or benign. rally causes worry, may result in ambivalence about continuing Clinicians are encouraged to discuss uncertain variants the pregnancy, and has the potential to weigh on the parents’ with the reporting laboratory and to share clinical infor- minds with any perceived “abnormal” phenotype their child mation that might affect the interpretation. Referral for may exhibit. genetic counseling in these situations is encouraged. These categories do not cover all scenarios because each CNV s0120 Interpretation of Copy Number Variation has unique considerations requiring clinical judgment and cor- p0350 All practitioners offering prenatal CMA should be aware of the roboration with phenotypic findings. reporting criteria of the laboratory analyzing the sample. Guidelines recently provided by ACMG for postnatal CMA Limitations of Chromosomal Microarray Analysis s0125 interpretation are available and have been adapted for prenatal Although CMA is designed to assess genetic imbalance, it does p0375 use.97 These guidelines recommend that the interpreting labora- not reach the resolution of single nucleotide variants or very tory geneticist assign a CNV to one of four main categories of small CNVs below the level of detection. Likewise, balanced significance to facilitate unambiguous communication of clini- rearrangements, such as inversions and reciprocal or insertional cal significance: translocations, are not detected by CMA. Overall, these occur 98 u0090 Normal: No copy number changes of clinical significance in approximately 0.6% of individuals. One in 2000 prenatal were identified. Normal results may or may not include cases has an apparently balanced translocation (0.05%).99 In mention of copy number changes that were identified and most cases this does not lead to an altered fetal phenotype, but believed to be benign. the information will not be available for reproductive counsel- u0095 Benign: A CNV should be considered benign if it has been ing. Notably, CMA can detect some copy number changes near reported in multiple peer-reviewed publications or the chromosome breakpoint sites in rearrangements that appear curated databases as a benign variant, particularly if the to be balanced on a conventional karyotype. nature of the CNV has been well characterized, or if the CMA fails to detect pathogenic copy number changes in areas p0380 CNV represents a common polymorphism documented not represented on the specific array platform. Commercially in greater than 1% of the population. available arrays include probes that span the entire genome, with u0100 Pathogenic: The CNV is documented as clinically significant denser probe coverage in sequence regions known to have clinical in multiple peer-reviewed publications, even if penetrance relevance. Denser probe coverage allows for a larger volume of and expressivity of the CNV are known to be variable. output information, but is more prone to VUS. Less dense probe N

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30 PART 1 Scientific Basis of Perinatal Biology

coverage can minimize the chance of a VUS, but it also may fail for genes in which a high proportion of patients may have de to detect a rare or novel genomic imbalance. CMA used for pre- novo variants. natal diagnosis tends to have less dense probe coverage to focus results on areas of known genotype-phenotype correlation.88 HYBRIDIZATION TECHNIQUES: SOUTHERN s0140 p0385 CMA arrays that rely solely on oligonucleotide probes do not BLOT, POLYMERASE CHAIN REACTION routinely detect polyploidy, because there are no relative genomic imbalances. SNP arrays, however, can detect trip- The initial molecular diagnostic techniques to make use of the p0405 loidy.100 Importantly, neither CMA platform can provide infor- recombinant DNA revolution were based on restriction frag- mation about the chromosomal mechanism of a genetic ment length polymorphisms, generated by cutting amplified imbalance. For example, CMA does not differentiate between DNA sequences using specialized bacterial enzymes called nondisjunction and translocation, which may have important restriction endonucleases. These highly specific enzymes rec- implications for recurrence risk counseling.88 So, if a fetus is ognize and cut a unique sequence of nucleotides, usually a identified to have trisomy 21 by CMA, conventional karyotyp- palindrome of 4 to 8 bp in length. In a palindrome of double- ing is then needed as a follow-up to differentiate a full trisomy stranded DNA, complementary strands at the cut site will read from an unbalanced translocation; if the latter is found, parents the same when moving from the 5′ to the 3′ direction, such should be karyotyped to identify the balanced carrier. as GAATTC. Because of this sequence specificity, the pattern of DNA fragments resulting from restriction endonuclease s0130 Ethical Considerations of Genetic Diagnosis digestion is unique to each gene sequence. The resulting frag- in a Fetus ments are separated by gel electrophoresis, transferred (blotted) p0390 Commonly identified “pitfalls” of identifying genetic anoma- onto a membrane, and hybridized with a radioactively labeled lies, which lead to formidable counseling challenges, include probe with known sequence. This technique is called Southern VUS, identification of adult-onset disease or parental presymp- blotting,101 which requires substantial amounts of DNA and is tomatic disorders, revelation of nonpaternity, unsuspected con- relatively labor intensive and difficult to automate, making it sanguinity, incest, findings linked to diseases with variable expensive (Fig. 2.7). This approach is still used to identify large expressivity and penetrance, or microdeletions involving a gene repeat expansions for genes subject to dynamic variants, such linked to cancer development or progression.96 These restraints as in fragile X,102 or to identify large deletions within the dys- are amplified in the prenatal setting, where correlation of phe- trophin gene responsible for Duchenne and Becker muscular notypic data is significantly limited. The widespread use of dystrophies.103 CMA for genetic diagnosis in the pediatric population is The development of PCR by Mullis and coworkers in the mid- p0410 strongly informed by evaluation of a patient expressing a defi- 1980s allowed selective amplification of any desired sequence of nite abnormal phenotype. However, for a fetus, genetic variants DNA, and radically changed the power of DNA diagnostics in identified on CMA may have unclear penetrance or expressivity, terms of sample requirements and assay options.104,105 Synthetic which complicates counseling and prognostication. oligonucleotides are used to prime DNA synthesis so that the p0395 The weight of the evidence for the reported finding must be single-stranded product generated from each primer includes carefully considered before irrevocable action is pursued, such the sequence complementary to the other primer (Fig. 2.8). as pregnancy termination. The potential impact even goes Multiple temperature-dependent cycles of DNA denaturation, beyond the tested pregnancy. Incidental findings such as men- primer annealing, and elongation of DNA synthesis create an delian diseases with adult-onset symptoms or inherited variants exponential increase in copy number or amplification of the in cancer-associated genes raise the risk of labeling the unborn DNA sequence between the two primers. child as well as the parents or extended family with predestined The power of PCR lies in its ability to rapidly replicate any p0415 illness. Furthermore, these findings may cause considerable genomic sequence starting from minimal amounts of biological unfounded anxiety throughout pregnancy and into childhood, sample. This is useful for tracking known genetic variants, such depending on the strength of disease association. In light of as genotyping SNPs or identifying disease-associated alleles for these numerous important issues, pre- and posttest counseling single-gene disorders. Alternatively, PCR can identify previously is imperative for the parents to understand the scope and limi- unknown sequence variants that give rise to a clinical pheno- tations of the available genetic technologies. type. As an example, for genes in which no single variant is common among patients, such as the hereditary hearing loss gene GJB2, novel sequence variations can be identified by PCR s0135 Genotyping/Sequencing amplification of an affected patient’s DNA using flanking p0400 This section provides an overview of many methods to evaluate probes at the gene locus. The PCR reaction generates large genetic sequence variation and disease-causing gene character- amounts of identical DNA copies of the locus in question, and ization. These techniques can be applied to the gamete, embryo, the gene sequence for the affected patient can be analyzed. fetal, or adult genomic DNA to decipher the individual DNA When PCR is used to amplify specific DNA fragments, mul- p0420 sequence and compare it to known reference standards. DNA tiple diagnostic tests can be performed on minimal amounts of diagnostics in medicine can use either direct assays for a spe- starting material. Variations in length, such as found in dynamic cific variant or linkage analysis with polymorphisms linked to a variants in genes with expandable trinucleotide repeat sequences disease locus. Direct assays for specific variants are most useful (e.g., Huntington disease106), can be assayed directly by PCR when relatively few distinct variants account for most patients amplification followed by gel electrophoresis to determine the with a particular form of disease. As analysis methods have size of the PCR product. Anonymous marker loci (e.g., SNPs or become faster, cheaper, and more highly automated, perform- simple sequence length polymorphisms) can be assayed in the ing direct sequencing of a disease gene without knowing the same way for gene mapping and forensic studies. SNPs are N precise variant has become increasingly practical, particularly amplified singly or in a multiplex combination of loci and

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2 Molecular Genetic Technology 31

Target DNA sequence Genomic DNA

Restriction Denature, anneal, extend enzyme site Restriction enzyme cleavage

Denature, anneal, extend Electrophoresis

Transfer to 25 cycles nylon filter 106 - 107 copies of target sequence f0045 Figure 2.8 Polymerase chain reaction (PCR). Repeated synthesis of a specific target DNA sequence (upward arrows) results in exponential amplification. The reaction proceeds from the primers (blue squares, red squares) in the 3′ direction on each strand. The first two cycles of the PCR are shown. Hybridization with 32P-labeled probe full karyotype result within about 4 hours! Rapid karyotyping may be applied in situations with fetal anomalies identified on ultrasound, as a deciding factor as to whether to proceed with chromosomal microarray or if routine karyotype may be suf- X-ray film ficient for diagnosis.

f0040 DNA SEQUENCING s0145 Figure 2.7 Southern blotting. DNA is cleaved by a restriction Sequencing determines the complete nucleotide sequence, or p0430 enzyme, separated according to size by agarose gel electrophoresis, specific order of nucleotides in a gene. By listing the full genetic and transferred to a filter. After hybridization of the DNA to a labeled probe and exposure of the filter to x-ray film, complementary sequences code, variations from an accepted “normal” (reference or con- can be identified. sensus sequence) may be discovered. This has the potential to uncover pathogenic variants as well as benign variants, and given our limited understanding of how the genome is trans- detected by electrophoretic, hybridization, or spectroscopic lated, will potentially identify what are now VUS into a defined methods. Multiplex PCR amplifications can allow rapid and clinical phenotype. parallel analysis of many genetic loci simultaneously. The original technique of sequencing, called Sanger sequenc- p0435 p0425 Perhaps one of the most useful examples of rapid multiplex ing, involves synthesizing multiple copies of DNA that is com- PCR in prenatal applications is real-time quantitative PCR for plementary to a single-stranded template of interest using rapid karyotype analysis.107 Appropriately selected primers nucleotide-specific chain terminators, or dideoxynucleotide amplify representative genes from each chromosome. Assay triphosphates (ddATP, ddGTP, ddCTP, ddTTP). This generates parameters are carefully optimized in order to multiplex (simul- synthesized fragments of varying length that can be arranged taneously run) the full panel of PCR reactions. Moreover, “real- by size, and the reactions containing each terminating base (A, time” technology incorporates fluorescent markers at each G, C, or T) are kept separated. Then, by “reading” the terminat- round of replication such that tracking the exponential rate of ing base of the synthesized copies from smallest to largest, the fluorescence increase serves to measure the dose of starting sequence of the original single-stranded template is revealed. material that was in the reaction.108 The end result is a rapid This molecular method was revolutionary but very time con- measurement of the amount of each chromosome from as little suming, labor intensive, and limited to relatively short DNA starting material as a single cell—that translates to an estimated sequences. N

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32 PART 1 Scientific Basis of Perinatal Biology

p0440 Next-generation sequencing (NGS) has revolutionized molecular genetic diagnosis rate among all patients referred, genetic sequencing capabilities, and moved the fields of science and among patients in the study with an abnormal neurologic and medicine into the “post-genome” era. NGS involves prepa- phenotype, molecular diagnosis was abnormal in as high as ration of a full DNA library (no longer one small segment at a 33% of cases.110 Among the 62 patients with likely causative time) by amplifying (making many copies) and fragmenting the diagnosis, 26% were autosomal recessive, 16% were X-linked, DNA source of interest (genomic DNA, coding DNA). After 53% were autosomal dominant, and 50% were de novo variants. amplification and fragmenting, the DNA library consists of At present, the practical clinical application of sequencing is p0455 thousands or even millions of small overlapping DNA copy to search one or many genes (or the whole genome) for a caus- fragments, which are physically bound to a solid surface (plat- ative variant in the setting of a recognized abnormal phenotype. form specific, often beads or glass slides). The fragments are Small case series in the prenatal setting are accumulating, loaded into specialized multiplex machines for parallel sequenc- describing results of WES performed on prenatal samples from ing; in other words, the sequence of every fragment on the fetuses with congenital anomalies. These early data suggest that surface can be assayed simultaneously. Individual sequence WES has the potential to significantly increase the proportion reads are subsequently aligned (recall the expected overlap due of cases for which a genetic etiology can be identified.111 to random fragmentation of many identical copies of DNA) However, larger studies are needed for validation, and wide- using various bioinformatics platforms for comparison to a spread use of this technology is currently limited by very long reference sequence. The full set of aligned reads reveals the turnaround times, prohibitive cost, and generation of high entire sequence of the starting DNA product. Thus, NGS applies numbers of VUS. Moreover, prenatal genotyping by WES or the concept of Sanger sequencing to an entire genome with WGS is not easy to interpret in the setting of limited phenotyp- results produced in matter of hours. NGS is a very thorough ing offered by ultrasound. It is therefore prudent to postpone and powerful method of generating genetic sequence informa- such testing to the postnatal period, when more phenotyping is tion. Whereas CMA can simultaneously assess many snippets available to make the interpretation. of DNA sequence from representative regions across the genome, NGS can provide the entire sequence of the substrate DNA, in a matter of hours. This could be likened to quickly Special Applications s0150 finishing a book by reading only the first and last page of every NONINVASIVE PRENATAL SCREENING s0155 chapter (microarray) versus developing true speed-reading capability to read every single word within a matter of hours. Noninvasive prenatal screening (NIPS) refers to a class of p0460 p0445 Whole exome sequencing (WES), which includes only the genetic tests designed to detect fetal aneuploidy by quantitation protein-coding region of DNA, and whole genome sequencing of small fragments of cell-free fetal DNA (cffDNA) circulating (WGS) are currently in use mostly in the pediatric setting, and in the maternal plasma. The advent of NGS technologies rapidly are considered the “next frontier” in prenatal genetic diagnostic expanded the amount of data that can be gathered from fetal techniques. WES is designed to determine the DNA sequence DNA in maternal plasma in a time frame amenable to prenatal of the 20,000 to 25,000 genes that code for known proteins screening applications. As a result, cell-free DNA tests to screen (about 1.5% of the full genome). This coding portion, or exome, for fetal aneuploidy moved quickly through development and is said to contain up to 85% of the variants that have been into clinical use by 2011 in the US and Asia, followed by Canada demonstrated to cause genetic disorders19; however, it is likely and Europe in 2012.112 NIPS represents the first widespread that many of the variants in noncoding portions of the genome application of genomic testing in medicine. also play a significant role in human disease. By comparison, In 2008 two groups identified pregnan- p0465 WGS requires sequencing of the full genome, including non- cies113,114 by analyzing the cffDNA in the maternal plasma. Using coding regions and introns. Data generated from sequencing massively parallel shotgun sequencing, the beginning portions methods are vast. The primary limitation of both WES and of all circulating DNA fragments (both maternal and fetal) were WGS is that accurate clinical interpretation lags behind the sequenced and identified to determine their specific chromo- ability to generate sequence data. This is because sequencing is somal origin. If the fetus has a third chromosome 21, the per- high throughput and there are currently no similar high- centage of chromosome 21 fragments compared to disomic throughput assays of function to assess putative pathologic chromosomes will be slightly higher than expected. In a preg- sequence findings. This technology generates thousands of vari- nancy in which 10% of the cell-free DNA is fetal, a woman ants, and new databases, such as ClinVar and ClinGen as well carrying a trisomy 21 fetus should have about 1.05 times more as the Human Gene Mutation Database, allow for evolving DNA from chromosome 21 fragments than a woman carrying interpretation as more sequence data become available. Cura- a disomic 21 fetus. Trisomy 21 prediction is based on the ability tion of variants remains a problem and will be a challenge for to distinguish this difference by sequencing millions of frag- years to come. There is enough natural variation in the human ments, identifying their chromosomal origin, and then quanti- genome that any putative finding requires rigorous validation fying their relative proportion. at the level of sequence, molecular function, and interaction Following the initial success in aneuploidy detection and p0470 within the full biological system. Current recommendations widespread clinical application of NIPS technology, several dif- from ACOG and SMFM specify that the use of WES or WGS ferent platforms are now available to generate sequencing reads for prenatal diagnosis be limited to clinical trials until these of the cffDNA material; platform type determines the speed, techniques can be further validated in prenatal samples.109 cost, and resolution of the commercially available NIPS options. p0450 The first published large clinical series presented WES analy- Fig. 2.9 diagrams the key features of the different sequencing sis from 250 consecutive patients referred for phenotypes sug- and tabulation platforms.115,116 Each of the commercially avail- gestive of as-yet unidentified genetic causes (no prenatal cases able NIPS methods uses the same fundamental approach to N were included in this series). The study demonstrated a 25% detecting abnormal fetal DNA complement: (1) an NGS method

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2 Molecular Genetic Technology 33

cfDNA in maternal plasma

Maternal DNA

Fetal DNA Red blood cell

Plasma maternal DNA NGS of total DNA present Plasma in maternal plasma for fetal DNA aneuploidy detection Ligation probe A Random sequencing Targeted sequencing Ligation probe B

ABTag-counting approach SNP-based approach C Non–SNP-based approach

Genome-wide shotgun sequencing Multiplex PCR targeting polymorphic Selection of targeted regions SNPs on the chromosomes of interest CCTGA CCTGA Nontarget locus Target locus SNP1 ATGTT SNP2 CCTGA SNP1 ATGTT SNP1 SNP2 Counting of aligned reads across the genome Sequencing Adding locus-specific oligonucleotides ATGTT CCTGA CCTGA ATGGT ATGTT CCTGA ATGTT Multiple ATGGT CCTGA ATGTT regions ATGTT ATGGT CTAGG CCTGA ATGTT Coamplification of targeted ATGGT CTAGG CCTGA ATGTT

Read count Calculation of the distributions of regions and sequencing ATGGT CTAGG CCTGA ATGTT fetal- and meternal-specific alleles ATGGT CTAGG CCTGA ATGTT MatF: etal Chr1 ...Chr13 ...Chr18 ... Chr21 SNP1 2:1 G+C% correction SNP2 1:0 Sequencing

Apply counting statistics to Apply counting statistics to Apply counting statistics to calulate genome representation calulate genome representation calulate genome representation f0050 Figure 2.9 Noninvasive prenatal screening (cell-free fetal DNA) aneuploidy screening methods. Two major strategies are widely implemented in routine cell-free fetal DNA (cfDNA) aneuploidy testing: random sequencing and targeted sequencing. (A) Random sequencing applies counting statistics on the reads that map to the human reference genome. (B) In targeted sequencing, selected loci with single nucleotide polymorphisms (SNPs) (red and blue stars) are analyzed. The maternal (mat) genotypes are used to model the allelic distributions for every ploidy scenario and based on the actual SNP distributions the likelihood for each hypothesis is calculated. (C) In an alternative targeted sequencing approach, selected fragments are enriched, sequenced, and mapped to the human genome. A chromosome proportion metric is calculated by applying counting statistics. PCR, Polymerase chain reaction. (From Vermeesch JR, Voet T, Devriendt K. Prenatal and pre-implantation genetic diagnosis. Nat Rev Genet. 2016;17:643–656.)

is used to produce millions of simultaneous sequencing reads; proportion of the genome represented and the number of (2) generated sequences are hybridized to reference templates; sequence reads per amplification round; (2) the reference tags and (3) bioinformatics tabulation methods “count” relative rep- used for alignment of the sequence reads; and (3) the counting resentation of fetal- versus maternal-derived genetic segments. statistics applied to generate a readout of relative fetal sequence The key differences between the platforms include (1) the representation. Fig. 2.9A models a platform commonly called breadth and “depth” of sequencing, also expressed as the massively parallel sequencing, which is the most resource N

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34 PART 1 Scientific Basis of Perinatal Biology

intensive due to the depth and breadth of sequencing. Fig. 2.9B for a wider range of microdeletion/duplication syndromes may represents an SNP allelic ratio platform, and Fig. 2.9C represents be routine in the near future.127 Of note, ACOG, SMFM, and a non-SNP allelic ratio platform. ACMG have also stated that, in the interest of patient autonomy, p0475 The NIPS clinical performance statistics for detection of spe- NIPS should still be made available to all patients who desire cific aneuploidies (trisomy 21, 18, and 13) in a high-risk popu- additional testing beyond current professional recommenda- lation are far better than any other standard screening methods, tions. NIPS is superior to serum screening in detecting the although NIPS is still not considered diagnostic. A 2016 system- common aneuploidies, and studies among all risk individuals atic review and meta-analysis found that the sensitivity of NIPS indicate that it performs well in low-risk populations. NIPS can for detection of trisomy 21 was 97%, that for trisomy 18 was be used as a first-tier screening test for aneuploidy detection, 93%, and that for trisomy 13 was 95% in a combined high-risk followed by invasive testing where needed. cohort representing 10,000 pregnancies. The positive predictive values in this high-risk group were 91%, 84%, and 87% for Expanded Applications of Cell-Free Fetal Nucleic s0160 trisomy 21, 18, and 13, respectively. In the 100,000 tabulated Acid Testing low-risk pregnancies the positive predictive values of NIPS fell Applications of NIPS are rapidly broadening, although these p0490 to 82%, 37%, and 49% for trisomy 21, 18, and 13, respectively.117 “frontier” applications of cffDNA and cell-free fetal RNA will Although there are several different NIPS platforms, their diag- require rigorous validation before transitioning to routine nostic accuracy, sensitivity, specificity, and positive predictive use. There are promising data on accurate detection of fetal value are very similar. microdeletion syndromes such as 22q11.2 (DiGeorge), 5p p0480 There are several important caveats to the use of NIPS, which (cri du chat), and 1p36 (Noonan) using an SNP-based NIPS are critical for the provider to communicate to any patient approach.128 However, SMFM does not recommend routine considering this form of screening. As mentioned previously, screening for microdeletions with cffDNA at this time because while NIPS performs well clinically from the standpoint of a clinical validation with patient samples is currently lacking.112 screening test, false negatives do occur. Furthermore, for all Some commercial laboratories have marketed a cffDNA-based screen-positive cases, invasive diagnostic testing is recom- screening test designed to detect genome-wide gains or losses mended. Most commercial platforms offer standard screening that include DNA regions of 7 Mb or more (similar to resolu- for trisomy 21, trisomy 18, and trisomy 13. Noninvasive screen- tion of a conventional karyotype).129 Early reports have been ing for sex chromosome abnormalities has also become more published to serve as proof of principle for the derivation of commonly offered, but with a lower reported sensitivity and prenatal plasma methylomic and transcriptomic profiles.115 specificity than for trisomy 21.118–122 This reduced performance Researchers continue to expand the scope of fetal genetic testing is related to the differing guanine-cytosine content of the chro- by a noninvasive prenatal approach, promising to drastically mosomes, making sequencing less efficient. Furthermore, there enrich our capabilities of prenatal genetic surveillance. is active debate over how to interpret tests that either do not produce a result, or produce results discrepant from diagnostic PREIMPLANTATION GENETIC TESTING s0165 testing. About 5% of samples may initially be unacceptable for analysis for a number of reasons, including a low percentage of Over the past two decades, methods of in vitro fertilization p0495 fetal DNA (low fetal fraction). This rate is higher in patients (IVF) and embryo culture and transfer have become routine with a body mass index greater than 30, probably due to the clinical practice. Simultaneously, the introduction of increas- increased apoptosis of maternal cells reducing the percentage ingly sophisticated genetic diagnostic procedures now allows of fetal DNA.113,119,123,124 Interestingly, the aneuploidy rate is preimplantation diagnoses to be performed by testing a few much higher in samples with an inadequate fetal fraction com- cells of a developing preimplantation (usually day 5 or day 6) pared to the general population (2.7% versus 0.4%, P < .001), blastocyst embryo. This technique was developed initially to suggesting that “no readout” results should be considered a benefit patients at high risk for a fetal genetic disorder (parents form of positive screen.125,126 Other causes of inaccurate results with known variants). Preimplantation testing allows for selec- include confined placental mosaicism, true fetal mosaicism, tion of unaffected embryos for implantation, and avoids the maternal sex , organ transplant, emotional stress of having to terminate an affected pregnancy. co- demise, maternal chromosomal deletion, maternal Most of these diagnoses were for relatively rare mendelian neoplasm, maternal fibroids, and laboratory error.111 disorders. p0485 Current guidelines from multiple US and international pro- In recent years, more widespread utilization of embryo p0500 fessional societies recommend that the use of cffDNA screening testing began with screening preimplantation embryos for be limited to women with increased risk of fetal aneuploidy. aneuploidies to presumably improve implantation rates and This includes women of advanced maternal age (>35 years at take-home pregnancy rates.130 Initial indications for preimplan- delivery); women with soft markers or structural abnormalities tation genetic testing (PGT) have been to improve singleton associated with trisomy 21, 18, or 13; prior pregnancy history pregnancy rates by IVF, reduce twinning rates, improve out- of a fetus with trisomy 21, 18, or 13; a biochemical screening comes for patients with repetitive , and improve result positive for increased risk of aneuploidy; or known pregnancy rates for women of advanced maternal age. However, parental balanced robertsonian translocation with increased randomized controlled data supporting many of these indica- risk of trisomy 21, 18, or 13. In a low-risk patient cohort the tions are lacking. PGT is now divided into two general catego- scope of genetic abnormalities represented will not be as heavily ries: high-risk PGT (also known as preimplantation genetic skewed toward Down syndrome, which argues the need for a diagnosis [PGD]) and low-risk PGT (also known as preim- screening tool with a broader scope such as traditional first- plantation genetic screening [PGS]). High-risk PGT (or PGD) trimester screening. Nonetheless, cffDNA screening is an area refers to analysis carried out for patients at risk of transmit- N of active research and development, and detection capabilities ting a known genetic or chromosomal abnormality to their

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2 Molecular Genetic Technology 35

offspring. This includes single-gene defects, translocations, and During the two-, four-, and eight-cell stages of early embryo p0510 structural chromosomal aberrations. Low-risk PGT (or PGS) is development, mosaicism with normal and aneuploid cells reserved for infertility patients undergoing IVF, with the goal may be common, but only the small minority of normal cells of increasing take-home pregnancy rates by screening for aneu- will endure and give rise to the embryo. Other drawbacks of ploidy.131 PGT raises many ethical questions before conception, these studies included examination of only a select number of including whether to test for adult-onset disorders, whether to chromosomes, errors resulting from single-cell analyses, and use PGS for sex selection outside of medical indications, and embryo biopsy at the cleavage stage, which is a developmental whether to transfer embryos with identified pathogenic variants stage known to contain many aneuploid cells that are shed with due to family wishes. It is important to keep in mind that any further embryo growth. It is unlikely that molecular testing form of genetic testing of the embryo or parents should involve would be predictive at cleavage stage because naturally 50% of genetic counseling by the health care provider. There is little IVF blastomeres had chromosomal abnormalities and only 9% controversy regarding detecting embryos at risk for heritable of embryos were normal diploid in all blastomeres.136 mendelian disorders. However, there is significant controversy Negative results on this cleavage-stage randomized con- p0515 regarding aneuploidy screening in embryos to improve implan- trolled trial did not deter various groups from applying new tation and take-home pregnancy rates, with expert opinions technologies to biopsy embryos at a later stage of develop- ranging from offering PGS to all women undergoing IVF to ment. The current “PGS version 2.0” usually refers to embryo recommending that no PGS be offered until further clinical biopsy at the blastocyst stage (day 5 or 6), followed by genome- studies are completed to show its utility.132 wide aneuploidy detection via array CGH, quantitative PCR, or massive parallel sequencing, and then subsequent frozen elec- s0170 Preimplantation Genetic Screening for Aneuploidy tive single embryo transfer. The advantages of trophectoderm p0505 Aneuploidy screening by FISH for select chromosomes (1, 13, biopsy at a blastocyst stage include: (1) the ability to harvest 16, 17, 18, 21, X, and Y) and preselection of euploid embryos, several cells for more DNA to analyze; (2) the blastocyst biopsy particularly in women of advanced maternal age, has been sug- is less damaging to the embryo than blastomere biopsy; and gested to improve IVF implantation and pregnancy rates even (3) there is, in general, less overall aneuploid mosaicism at if no genetic indication for testing is present. This reasoning the blastocyst stage. Massive parallel sequencing (NGS tech- is supported by the very high number of aneuploid embryos nology) relies on the same principle as WES/WGS sequencing encountered in women over 35 years of age (>30%), a statistic (Fig. 2.10) and has only recently entered PGS.132 While array that rises exponentially to 60% by age 40. However, early enthu- CGH is considered a gold standard, early claims are being made siasm for this approach has been tempered by several clinical that massive parallel sequencing may detect clinically signifi- trials.133,134 One large multicenter, randomized, double-blind, cant mosaicism. However, such claims need to be tempered controlled trial conducted between 2003 and 2007 comparing against the facts that only a few cells are being analyzed, and IVF with PGS to standard IVF in 408 women (age range, 35 the site of biopsy may significantly alter detection of mosa- to 41 years) showed a decreased pregnancy success rate (25% icism; therefore further studies are necessary before massive versus 37%) in those receiving PGS compared to those who parallel sequencing can become a standard assay for mosa- did not.133 Aneuploidy screening may have failed to improve icism. A number of small randomized controlled studies have outcomes because many of the embryos that were rejected on shown 15% to 20% increased implantation and delivery rates the basis of aneuploidy by PGD FISH results would have been with PGS version 2.0.137–140 Other studies on PGS have shown ultimately normal embryos with successful pregnancy potential decreased miscarriage rates in women older than 37 years of (perhaps indicating that biopsy of a single blastomere is not age who used PGS, and no effect on individuals younger than representative of the karyotype of the conceptus).135,136 35 years.140

Deletion 9p, Male 0.5 0.0 –0.5

–1.5 12345671890111213141516171819202122X Y A

Trisomy 14 0.5 0.0 –0.5 Female –1.5 1234567189 01112131415161718 19 20 21 22 XY f0055 B Figure 2.10 Whole-genome profiling by oligonucleotide comparative genomic hybridization microarray performed on biopsy from a 6-day blastocyst and displayed by Agilent CytoGenomics software 4.0. Chromosomes are plotted in a horizontal orientation and are listed at the bottom of a plot. The Single Cell Recommended Analysis Method was used to detect aberrations with a minimum size of 5 Mb, and minimum log2 ratios of 0.35 for gains and −0.45 for losses. The moving average from an embryonic DNA was compared against the male reference sample (blue line) and the female reference sample (pink line). The gender of an embryo is determined by a copy number of sex chromosomes and by comparison to normal reference male and female DNA. (A) A loss in copy number for the short arm of chromosome 9 (9p deletion) and a gain of all chromo- some 16 probes (trisomy 16) were detected in a male embryo. (B) Chromosome 14 gain (trisomy 14) was observed in a female embryo. Arrows indicate location of abnormal loss and gain in copy numbers. N

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36 PART 1 Scientific Basis of Perinatal Biology

p0520 A number of indications have been proposed for PGS, found not to have the genetic variant are transferred into the including recurrent miscarriage, advanced maternal age, dimin- uterus. This is the method that has been most commonly used ished ovarian reserve, multiple failed IVF cycles, personal to date. Finally, PGD can be performed at the blastocyst stage reasons (sex selection), improvement of singleton pregnancy by sampling a portion of the trophectoderm (early placenta). IVF, reduction of twinning, and patient preference for transfer All three approaches appear to be safe, with studies showing no of a euploid embryo. Many couples at risk of carrying a child increased risk of birth defects or growth disorders in infants with a mendelian disorder undergo PGS in addition to PGD. born after PGD when compared to infants born after other Despite all of the reasons mentioned previously, there is signifi- assisted reproductive technologies. cant controversy regarding the effectiveness of PGS due to lack Each technique is associated with some limitations. Polar p0535 of larger multicenter randomized controlled trials, practical body testing can result in an erroneous diagnosis because of problems of obtaining significant number of blastocysts in crossing over occurring during meiosis. This limitation has older patients, reports of euploid deliveries from aneuploidy become rare as newer technologies of direct gene testing have embryos, and controversies regarding discrepant karyotype and been introduced, replacing earlier studies that used linkage mosaicism in inner cell mass versus trophectoderm.141 Claims markers. However, its major limitation for recessive disorders is of higher implantation rates after PGS are countered by the fact that polar body biopsy only determines the maternal contribu- that performing PGS is also associated with a subsequent lower tions to the embryo. A finding that the embryo will have the number of embryos for transfer and/or cryopreservation. The maternal variant does not differentiate between a carrier and lack of consensus regarding the overall clinical utility of PGS an affected embryo, thus decreasing the number of embryos will hopefully improve with outcomes of additional trials that that are “unaffected” to only those that receive the normal allele are underway.132 Nonetheless, the attractiveness of assessing and from the mother. Blastomere biopsy is subject to error relating only transferring seemingly euploid embryos has become com- to the requirement for DNA amplification from a single cell by monplace in the United States. PCR. Erroneous amplification of sperm DNA from the zona p0525 Careful counseling of couples regarding the pros and cons pellucida, allele dropout of one of the parental alleles during of embryo aneuploidy screening is very important. It is also amplification, or both may lead to false-negative results. critical to understand the limitations of PGS and to communi- However, current molecular technologies using SNPs and short cate these limitations to the couple considering this procedure. tandem repeats allow simultaneous variant detection and Errors in PGS can arise due to mislabeling of samples, contami- marker analysis, almost completely eliminating the risk of mis- nation with extraneous DNA, technical problems (amplifica- diagnosis by sperm contamination. Similarly, SNP microarrays tion failure, array noise), discrepancy between inner cell mass following whole genome amplification provide methods to karyotype and trophectoderm karyotype, and mosaicism in the overcome the allele dropout problem as well. Blastocyst biopsy trophectoderm sample. Moreover, PGS is a screening test, and has the benefit of providing more cells but requires a longer the detection rate is not 100%. Screening is limited to whole culture period. Because of the late stage when the biopsy is chromosome gain or loss and will not detect subchromosomal performed, any genetic testing must be done rapidly, within 24 deletions/duplications. PGS also does not test for whole chro- hours, or the biopsied blastocysts must be cryopreserved for mosome mosaicism, triploidy (three sets of each chromosome), later use. With improved embryo cultured conditions, many or genetic conditions caused by single-gene variants (such as groups are performing PGD on blastocyst embryos (embryo CF). The false-negative rate for microarray PGS is lower than day 5 or 6), and in many centers, PGD is usually performed in 1%. Every couple, regardless of their ethnic background and conjunction with PGS so that euploid and pathogenic variant- family history, has a 3% to 5% risk for birth defects with each free embryos are transferred. pregnancy, and even if the result of PGS is normal, the baby In order for PGD to be performed, carrier status of the p0540 could still have one or more birth defects or intellectual dis- parents and the precise coordinates of the pathogenic variants ability from causes not detected by PGS testing. PGS does not are required. It is important to understand that currently PGD replace prenatal testing such as CVS or amniocentesis. Stan- only detects inherited disorders, that is, pathogenic variants dard prenatal screening or testing should still be made available present in the parents and transmitted to the offspring. PGD to patients undergoing IVF, including patients who had PGS. will not detect de novo genetic disorders, variants that are not Women who do not desire to undergo diagnostic procedures detectable in parental blood but present in the offspring. Impor- due to associated risk of loss can be offered cffDNA screening tantly, de novo variants contribute as much as 40% to clinically test with all the caveats associated with such testing. significant phenotypes. It is critical for couples to understand the scope and limitations of current PGD testing. The most s0175 Preimplantation Genetic Diagnosis common monogenetic disorders evaluated by PGD are CF, s0180 PGD for Mendelian Disorders. PGD is used to describe β-thalassemia, and SMA among the autosomal recessive disor- p0530 genetic testing that occurs before an embryo implants in the ders; myotonic dystrophy, Huntington disease, and Charcot- uterus. Three approaches have been utilized for PGD to date. Marie-Tooth disease among the dominant disorders; and fragile The first approach was based on polar body removal, and the X syndrome, Duchenne or Becker muscular dystrophy, and genetic status of the oocyte was inferred from the results of the hemophilia among the X-linked disorders. polar body assay. In the circumstance in which the polar body has the mutated gene, the oocyte is inferred to be “normal,” and PGD for Carriers of Balanced Rearrangements. A parent s0185 therefore an embryo obtained by fertilization of this oocyte who is the carrier of a balanced rearrangement typically has a p0545 would be unaffected with the genetic condition of interest. A high risk of producing unbalanced embryos, leading to recur- second method of PGD is blastomere biopsy, in which one or rent miscarriages or a child with an unbalanced rearrangement. two blastomeres are removed from an eight-cell (day 3) embryo PGD permits the transfer of only those embryos with a normal N and analyzed for the genetic condition of interest. Only embryos or balanced chromosome complement, and it has been

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2 Molecular Genetic Technology 37

demonstrated to significantly improve outcomes.142,143 An nonhuman organ donor, or crops that carry variants to grow impressive report comes from Otani and colleagues,144 who faster and be disease resistant. Routine application of genome assessed PGD in 33 couples with repetitive miscarriages and no editing to humans is unlikely to occur soon due to various liveborn children, in which one member of the couple is a problems, including generation of mosaic embryos, off-target translocation carrier. After PGD (an average of 1.24 cycles per effects (genetic editing at nonprescribed sites) with unintended patient), only 18% of the total embryos were either balanced or consequences, and overall safety concerns inherent in new tech- normal: 20 of 88 embryos from robertsonian translocation car- nologies. Research in this arena should be allowed to proceed riers and 86 of 491 from reciprocal translocation carriers. Of because there are significant benefits that genetically affected the 19 subsequent pregnancies in which normal or balanced human embryos may derive in the future. embryos were implanted, just one (5%) miscarried; the other 18 pregnancies proceeded into the second trimester or culmi- Conclusion s0195 nated in a live birth. The past two decades have yielded revolutionary advances in p0560 s0190 Ethical Issues Surrounding Preimplantation reproductive genetic technology. Curation of human genome Genetic Diagnosis sequence information in the years since completion of the p0550 PGD has raised numerous ethical issues. It allows individu- Human Genome Project and expanding applications of NGS als with adult-onset disorders, such as carriers for the BRCA1 have provided seemingly endless genotype data, and work is variant, to carry embryos free of the pathogenic variants. PGD ongoing to translate that information to correlated phenotypes for adult-onset conditions is ethically justified both in cases and reduce the burden of VUS. Access to genetic material has when the condition is serious and for adult-onset conditions of also burgeoned, with improved understanding of embryonic lesser severity or penetrance.145 Significant counseling regard- development, results of embryo biopsy, and ongoing explora- ing PGD in such cases should include detailed review of the tion of data available from cell-free DNA. With rapidly devel- adult condition from which the parent suffers, the degree of oping technologies at our disposal, reproductive and obstetrics penetrance and expressivity of such condition, and medical professionals have a responsibility to connect patients with interventions to manage the condition, as well as the overall resources to ensure full understanding of the benefits and limi- residual risk of genetic disorders. Penetrance of many patho- tations of various screening and diagnostic options. Obtaining genic variants for individual conditions is below 100%, and genetic information prior to conception or birth is fundamen- intrafamily variation is significant and should be understood tal to making informed reproductive decisions, particularly for by the patient. Publicly funded databases such as ClinGen patients with a personal, family, or obstetric history signifying are striving to curate all the pathogenic variants and provide elevated risk for a genetic disorder. Often a combination of a clinicians with guidelines regarding phenotypes and medical long-standing diagnostic method (such as karyotyping) and a interventions. Another set of ethical considerations arises when more recently developed technology (array CGH) will be the individuals want to transfer embryos with genetic anomalies.146 best approach to generate clinically useful information. Exciting Such requests are rare, and may involve individuals who them- new technologies are in development, and should continue to selves are affected with conditions such as hearing loss and be held to high standards of scientific rigor and clinical valida- achondroplasia and would like to rear children with a similar tion before entering mainstream practice. phenotype. Extensive and highly individualized counseling in such situations is important, as for any PGD procedure, and Key Points s0200 should involve discussion about the condition; the full spec- trum of the phenotype expressivity and penetrance as well as • There is a significant amount of naturally occurringp0565 the potential lethality; and the emotional, physical, and finan- genetic variation in the human genome. Single nucleotide cial effects that such a condition may have on the family unit. polymorphisms (SNPs) introduce genetic variation at the If the provider is not willing to assist the patients with their level of individual base substitutions, while copy number requests, patients should be given the option to seek help else- variations (CNVs) represent variation in the “dose” of where. Overwhelming numbers of individuals with genetic a relatively large DNA segment (1000 to 500,000 bp or conditions are born to couples without infertility problems, more). The biological implications of genetic variants and therefore individuals who seek assisted reproductive ser- depend on the gene or genes affected by the change in vices should likewise have the same choices. Nonetheless, in DNA. circumstances in which a child is highly likely to be born with • The term mutation has been replaced by the term variant, u0115 a severe condition that is associated with severe handicap and which can be classified as benign, likely benign, of suffering, the physician can refuse to transfer such embryos. unknown significance, likely pathogenic, or pathogenic. p0555 Much controversy has also been generated surrounding the • Genetic disease ranges from abnormal numbers of whole u0120 use of newly available genome editing technologies, such as chromosomes, to phenotypes caused by loss or gain of clustered regularly-interspaced short palindromic repeats/ subchromosomal DNA segments containing several con- CRISPR-associated protein 9 (CRISPR/Cas9), to edit embryo tiguous genes (called microdeletions and microduplica- genomes. Genome editing theoretically can be used to revert a tions), to single-gene disorders (also called mendelian pathogenic variant in the embryo to a benign variant. Alterna- disorders). Human disease may also have a genetic basis tively, it could be used to “enhance” the embryo, with variants with a more complex genotype-phenotype correlation, that can, for example, prevent infection with human immuno- such as autism, which may involve multiple genes in addi- deficiency virus or provide other advantages. Genome editing tion to environmental influences. is now widely used in animals and plants for various purposes, • Genetic screening and testing should be accompanied by u0125 including pigs that carry variants that allow them to be a patient education, genetic counseling, timely disclosure of N

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38 PART 1 Scientific Basis of Perinatal Biology

test results to patients, and the availability of “next steps” transfer of the fragments to a membrane where they can (such as definitive prenatal genetic testing) in the event of be probed for the DNA segment of interest. This technique positive results. is commonly used to identify large repeat expansions for u0130 • Carrier screening refers to genetic testing of asymptomatic genes subject to dynamic variations, such as in fragile X individuals to determine if they carry one or more disease- syndrome. associated genetic variants. Ethnic-based carrier screening • When PCR is used to amplify specific DNA fragments,u0170 is targeted to specific ethnic populations known to be at multiple diagnostic genetic tests can be performed on increased risk for particular disorders. Pan-ethnic carrier minimal amounts of starting material. screening tests for a select panel of disorders in all patients, • Next-generation sequencing (NGS) is the revolutionary u0175 regardless of ethnicity. technology that changed the scale of genetic testing pos- u0135 • Genetic risk assessment before or during pregnancy may sibilities. NGS, or massively parallel sequencing, is an be indicated based on family history, prior obstetric automated technology that generates millions of simulta- history, parental age, or ethnic background. Additionally, neous sequencing reads. NGS can be used to sequence it is acceptable for any patient, regardless of risk, to choose entire genomes or constrained to specific areas of interest, diagnostic prenatal genetic testing after informed consent. including all protein-coding genes (a whole exome) or u0140 • Benefits of genetic testing include optimization of neona- small numbers of individual genes. tal outcomes by planning appropriate delivery staffing and • Current recommendations from ACOG and SMFM u0180 location, identification of disorders for which in utero specify that use of whole exome sequencing (WES) or treatment may provide benefit, the option of pregnancy whole genome sequencing (WGS) for prenatal diagnosis termination or preimplantation selection, and providing be limited to clinical trials until these techniques can be reassurance when results are normal. further validated in prenatal samples. u0145 • The fetus, amniocytes, and chorionic villi each develop • The diagnostic accuracy of molecular genetic techniques u0185 from different cell lines in early embryo development. depends on the source of DNA, the percentage of the total Thus it is possible for tissue samples from each of these genome that is assessed in any given technique and, for sources to have disparate karyotypes resulting from abnor- sequencing technology, the depth of sequence assessment mal chromosome segregation. The earlier in development (the number of overlapping sequence reads for a given that an abnormal chromosomal segregation event such as segment of DNA). nondisjunction or trisomy rescue occurs, the more wide- • Noninvasive prenatal screening (NIPS) uses NGS of cell- u0190 spread the mosaicism may be in the differentiated organ- free DNA derived from maternal plasma coupled with ism (i.e., more likely to affect both the chorion/placenta bioinformatics algorithms to determine fetal ploidy (chro- and the fetus). Later chromosomal segregation events are mosome count). The available tests have a high sensitivity more likely to be confined to specific cell types, giving rise and specificity for common trisomies (21, 18, 13); however, to clinical findings such as confined placental mosaicism, the predictive value of the tests is significantly different which leads to discordant karyotypes between chorionic depending on the prevalence of aneuploidy in the popula- villus sampling (CVS) and amniocentesis. Factors influ- tion being tested. NIPS is an excellent screening tool for encing the likelihood of fetal involvement for a mosaic common aneuploidies in high-risk populations; however, aneuploidy result include the specific chromosome limitations in predictive value should be discussed with involved and the tissue source where the aneuploidy was patients who desire additional testing beyond current pro- detected. fessional recommendations. u0150 • CVS and amniocentesis samples can be assessed by direct • Women whose reports from NIPS are indeterminate or u0195 analysis and by analysis following long-term culture uninterpretable have an increased risk of aneuploidy and (approximately 1 week). There are benefits and draw- should undergo comprehensive ultrasound evaluation backs to each method, but diagnostic accuracy is maxi- and genetic counseling with the option for diagnostic mized when both direct and culture methods are used genetic testing. concurrently. For both CVS and amniocentesis, long-term • Low-risk preimplantation genetic testing (also known as u0200 culture gives more accurate diagnostic capability for fetal preimplantation genetic screening [PGS]) is an option for karyotype. infertility patients undergoing in vitro fertilization (IVF), u0155 • The molecular resolution of a G-banded karyotype is with the goal of increasing take-home pregnancy rates by 5 Mb, which allows detection of chromosome number screening for aneuploidy. There is significant controversy changes and relatively large structural chromosome regarding the effectiveness of PGS due to lack of larger rearrangements. multicenter randomized controlled trials. u0160 • Chromosomal microarray analysis (CMA) is a technique • High-risk preimplantation genetic testing (also known as u0205 that samples across the whole genome with resolution preimplantation genetic diagnosis [PGD]) involves genetic down to a 50- to 100-kb level. CMA allows for detection testing on embryos prior to implantation, from parents of CNVs, including microdeletions and microduplications with identified risk of transmitting a known genetic or that would not be diagnosed on karyotype. Microarray is chromosomal abnormality to their offspring. PGD detects recommended for genetic analysis in cases with sonographic only inherited disorders, and will not detect de novo fetal anomalies, and can be considered in any patient who genetic variants, which contribute as much as 40% to chooses to undergo diagnostic prenatal testing. clinically significant phenotypes. u0165 • Southern blotting involves enzymatic digestion of DNA at

unique cut sites, separation of fragment sizes on a gel, and A full reference list is available online at ExpertConsult.com. p0670 N

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2 Molecular Genetic Technology 38.e1

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38.e2 PART 1 Scientific Basis of Perinatal Biology

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Wapner RJ, Martin CL, Levy B, et al. Chromo- 112. SMFM Consult Series #36. Prenatal aneu- natal diagnosis of congenital nephrosis of the somal microarray versus karyotyping for pre- ploidy screening using cell free DNA. Am J Finnish type (CNF) in the second trimester. Int natal diagnosis. N Engl J Med. 2012;367:2175. Obstet Gynecol. 2015;711. J Gynaecol Obstet. 1993;41:165. 94. Donnelly JC, Platt LD, Rebarber A, et al. Asso- 113. Fan HC, Blumenfeld YJ, Chitkara U, et al. 77. Johnson MP, Bukowski TP, Reitleman C, et al. ciation of copy number variants with specific Noninvasive diagnosis of fetal aneuploidy N In utero surgical treatment of fetal obstructive ultrasonographically detected fetal anomalies. by shotgun sequencing DNA from maternal uropathy: a new comprehensive approach to Obstet Gynecol. 2014;124:83. blood. Proc Natl Acad Sci USA. 2008;105:16266.

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2 Molecular Genetic Technology 38.e3

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ISBN: 978-0-323-47910-3; PII: B978-0-323-47910-3.00002-4; Author: Resnik; 00002

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