Part 10 — Thompson & Thompson Genetics and Genomics in Medicine

Chapter 18: Preconception and Prenatal Screening and Diagnosis

Ch18 · Pt1 chapter 18 Preconception and Prenatal Screening and Diagnosis Angie Child Jelin ▶

Ignatia B. Vanden Veyver The goal of preconception and prenatal screening and diagnosis is to inform women and couples about the risks for genetic disorders and birth defects in their fetus during a f...

Ch18 · Pt2 392 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Screening for Neural Tube Defects An estimated 95% of infants with NTDs are born into families with no prior history of this malformation. The first developed noninvasive serum analyte-based screening test measures the amount of maternal serum α-fetoprotein (MSAFP) to identify pregnancies at increased risk for a fetal open NTD, including open spina bifida, anencephaly, and encephalocele not covered by skin. These are associated with high amounts of AFP in the amniotic fluid. AFP is a fetal glycoprotein produced mainly in the liver, secreted into the fetal circulation, and excreted through the fetal kidneys (see also Amniocentesis, later). AFP also leaks into the amniotic fluid when the fetal skin is breached. Because AFP enters the maternal bloodstream via the placenta, membranes, and maternal-fetal circulation, MSAFP is also elevated, which is the basis for using MSAFP measurements at ~16 weeks (15–21 weeks) to screen for open NTDs. There is considerable overlap between the range of MSAFP concentrations in unaffected pregnancies and those where the fetus has an open NTD (Fig. 18.1), and the sensitivity of MSAFP screening to detect an increased risk for fetal open NTDs depends on statistically defined cutoff values. As shown in Fig. 18.1, if the cutoff for an elevated concentration is defined as 2.5 multiples of the median (Mo M) value in unaffected pregnancies (which is 1 Mo M), one can estimate that 80% of fetuses with open NTDs are detected and 20% remain undetected. However, lowering the cutoff to improve sensitivity would be at the expense of reduced specificity, thereby increasing the number of unaffected pregnancies that would be interpreted as high risk (false-positive rate). An elevated MSAFP concentration is not specific to a pregnancy with an open NTD. As listed in Table 18.1, there are many other causes of high MSAFP, most of which can be distinguished from open NTDs by fetal ultrasonography, which should be offered when MSAFP is increased. Combining MSAFP screening with detailed diagnostic ultrasonography approaches the accuracy for the detection of open NTDs of ultrasonography combined with amniocentesis to measure AFP in amniotic fluid. Thus it is acceptable to offer ultrasound examination (at 18 weeks) paired with an MSAFP assay to first-degree, second-degree, or more distant relatives of individuals with NTDs instead of amniocentesis. MSAFP measurement has now become integrated in a second trimester multiple serum analyte screen for fetal trisomies (see later), but far more women now have first trimester trisomy screening with serum analytes and ultrasound, or more recently with cell-free DNA analysis. Thus providers should remember to offer these women screening for open NTDs by ultrasound with or without MSAFP. Currently, in expert centers, NTDs are increasingly diagnosed by a screening anatomy ultrasound alone, without MSAFP. Screening for Down Syndrome and Other Aneuploidies Although the association between advancing maternal age and increased risk for major trisomies is well known, more than 70% of children with autosomal trisomies are born to women without risk factors. The 0.2 0.5 0.8 1 2 3 4 5 10 20 Unaffected Maternal serum AFP (multiple of normal median) Proportion of individuals Down syndrome Spina bifida Figure 18.1 Maternal serum α-fetoprotein (AFP) concentration, expressed as multiples of the median, in normal fetuses, fetuses with open neural tube defects, and fetuses with Down syndrome. (Redrawn from Wald NJ, Cuckle HS: Recent advances in screening for neural tube defects and Down syndrome. In Rodeck C, editor: Prenatal diagnosis, London, 1987, Bailliére Tindall, pp 649–676.) TABLE 18.1 Findings Associated With Elevated α-Fetoprotein Concentration Gestational age older than calculated Spina bifida* Anencephaly* Congenital skin defects* Abdominal wall defects* Gastrointestinal defects* Liver necrosis Cloacal exstrophy* Cystic hygroma* Fetal demise* Sacrococcygeal teratomas* Urinary obstruction* Polycystic kidney* Absent kidney* Congenital nephrosis* Other renal anomalies* Osteogenesis imperfecta* Fetal growth restriction* Oligohydramnios* Multiple gestation* Decreased maternal weight Fetal bleeding* All listed findings can result in elevated maternal serum α-fetoprotein (MSAFP). *Indicates causes of elevated AFP level that can be seen by ultrasonographic examination. ▶

392 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Screening for Neural Tube Defects An estimated 95% of infants with NTDs are born into families with no prior history of this malformation. T...

Ch18 · Pt3 CHAPTER 18 — Preconception and Prenatal Screening and Diagnosis 393 unexpected finding that the concentration of MSAFP, measured during the second trimester to screen for NTDs, was lower in women who carried pregnancies with autosomal trisomies, particularly trisomies 18 and 21, led to its investigation as a screening analyte for Down syndrome (trisomy 21); however, MSAFP concentrations of unaffected pregnancies and Down syndrome pregnancies overlap too much (see Fig. 18.1) for MSAFP to be a useful screening analyte on its own. Since then more sensitive and specific trisomy screening strategies have been developed that combine different serum analytes in the first or second trimester with specific ultrasound measurements. About 10 years ago analysis of cell-free DNA in maternal plasma was introduced. Cell-free DNA screening can be done at any gestational age after 10 weeks and is more sensitive and specific for aneuploidy screening than serum analyte screening. The most recent guidance states that all pregnant women should be informed about the option of cell-free DNA analysis along with other screening and testing, but the less costly serum analyte screening is still used for women at low or average risk for fetal aneuploidy. Thus all these different screening options will be explained in more detail later. First Trimester Screening First trimester screening is ideally performed between 11 and 13 completed weeks of gestation and relies on measuring the levels of pregnancy-associated plasma protein A (PAPP-A) and the hormone human chorionic gonadotropin (h CG), either as total h CG or as its free β subunit in maternal serum. PAPP-A levels are below the normal range in all trisomies; h CG (or free β-h CG) is higher in trisomy 21 but lower in the other trisomies (Table 18.2). These analyte measurements are combined with the ultrasonographic measurement of the nuchal translucency (NT), defined as the thickness of the echo-free space between the skin and the soft tissue overlying the dorsal aspect of the cervical spine caused by subcutaneous edema of the fetal neck (Fig. 18.2A). An increase in NT is commonly seen in fetuses with trisomies 21, 13, and 18 and in 45,X (see Fig. 18.2B). Sonographers performing NT measurements used for first trimester screening must obtain and maintain special certification, and the size must be determined with reference to gestational age. An enlarged NT in fetuses with a normal karyotype is associated with an increased risk for other genetic conditions and birth defects. The most common ones are listed in Table 18.3. TABLE 18.2 Performance of First and Second Trimester Screening Tests First Trimester Screen Second Trimester Screen NT PAPP-A Free β-h CG SPR DR u E3 AFP h CG Inhibin A SPR DR Trisomy 21 ↑ ↓ ↑ 5% 85–90% ↓ ↓ ↑ ↑ 5% 80% Trisomy 18 ↑ ↓ ↓ 5% 90–95%* ↓ ↓ ↓ — 5% 60–70% Trisomy 13 ↑ ↓ ↓ 5% 90–95%* ↓ ↓ ↓ — n/a n/a NTD — — — n/a n/a — ↑↑ — — 5% 80–85% Up and down arrows indicate direction of change in measurement compared to average. AFP, α-fetoprotein; β-h CG, human chorionic gonadotropin β subunit; DR, detection rate; NT, nuchal translucency; PAPP-A, pregnancy-associated plasma protein A; SPR, screen positive rate; u E3, unconjugated estriol. *Indicates combined trisomy 13/18 detection rate. A B Figure 18.2 First trimester nuchal translucency (NT) measurements. The NT is a dark, echo-free zone beneath the skin in an ultrasonographic sagittal section through the fetus and is marked by two yellow “+” signs. The average NT size is 1.2 mm at 11 weeks of gestation (95th percentile up to 2 mm) and 1.5 mm at 14 weeks of gestation (95th percentile up to 2.6 mm). (A) Normal NT measurement of 1.25 mm. (B) Increased NT measurement associated with a greatly increased risk for Down syndrome. (Images courtesy of Wesley Lee, MD, Baylor College of Medicine, Houston, Texas.) ▶

CHAPTER 18 — Preconception and Prenatal Screening and Diagnosis 393 unexpected finding that the concentration of MSAFP, measured during the second trimester to screen for NTDs, was lower in women who...

Ch18 · Pt4 394 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Second Trimester Screening Second trimester screening for trisomies 21 and 18 is usually accomplished by measuring h CG levels in combination with three other analytes: MSAFP, unconjugated estriol (u E3), and inhibin A. This battery of tests is referred to as a quadruple screen. MSAFP and u E3 are lower than average when the fetus has a trisomy 21 or 18, whereas h CG is higher with fetal trisomy 21 but lower with trisomy 18, and inhibin A is higher with fetal trisomy 21 but not significantly affected in trisomy 18 (see Table 18.2). These analyte levels are affected by other factors, including ancestry, smoking, maternal diabetes, multiple pregnancy, and pregnancy conceived by in vitro fertilization (IVF), and laboratories generally adjust for these variables. Extremely low levels of u E3 may be indicative of rare genetic conditions such as steroid sulfatase deficiency or Smith-Lemli-Opitz syndrome. Second trimester screening is generally reserved for low-risk patients who missed the window for first trimester screening. For standard first and second trimester screening a cutoff of screen positive rate of 5% results in the detection rates of first and second trimester screening shown in Table 18.2. Integrated Screening Strategies Different strategies for combining the results of first trimester and second trimester screening to increase the ability to detect pregnancies with autosomal trisomies, particularly trisomy 21, have also been developed. In one approach, integrated screening, women undergo first trimester screening with serum analytes with or without NT, followed by second trimester serum screening, and the results are combined to provide a more precise risk estimate, but only after the second trimester result is available. The integrated screening strategy has the highest overall sensitivity of analyte-based screening and can detect up to 95% of all Down syndrome cases with an ~5% false-positive rate. Sensitivity for other trisomies is in the 90% to 95% range, with a low false-positive rate of less than 1%. This strategy is less attractive to women because they have to wait until the second trimester for their screening result. More stepwise variations also exist, wherein women found to be at highest risk after the first trimester screen are offered diagnostic testing and those not at increased risk or more moderate risk are offered a second trimester screen, followed by combined interpretation of the first and second trimester screening results. Noninvasive Prenatal Screening by Analysis of Cell-Free Fetal DNA All individuals have fragmented DNA in their blood that is not contained in the nucleus of cells but free floating and can be assayed from plasma or serum. The discovery that during pregnancy maternal plasma contains fetal cell-free DNA derived from trophoblast cells of the placenta, which have the same genome as the fetus, has drastically changed the approach to prenatal screening for fetal chromosomal anomalies. After 10 weeks of gestation, the proportion of cell-free DNA in maternal blood that is derived from trophoblast, referred to as fetal fraction, is ~5% to 20%. The circulating cell-free DNA can be analyzed using high-throughput DNA sequencing technologies to noninvasively evaluate whether the fetus has aneuploidy. This led to the introduction and rapid expansion of cell-free DNA-based noninvasive prenatal screening (NIPS) (also known as noninvasive prenatal testing [NIPT]) for trisomies 21, 13, and 18, with sensitivities and specificities approaching 99% for trisomy 21 (Table 18.4). A growing number of commercial NIPS tests on the market assess variable combinations of testing for these common aneuploidies, combined with sex chromosome abnormalities (see Table 18.4), other rare autosomal aneuploidies, and selected microdeletions. A few providers also offer genome-wide analysis of copy number gains and losses. Cell-free DNA can also be used to detect Y chromosome sequences for the purposes of determining fetal sex. TABLE 18.3 Common Causes of Increased Nuchal Translucency Thickness Chromosomal aneuploidy: ▶

Ŋ Trisomies 21, 13, or 18 ŊMonosomy X Ŋ Other rare aneuploidies Triploidy Pathogenic copy number variants Congenital heart defects Rasopathies Skeletal dysplasias Other single-gene disorders TABLE...

Ch18 · Pt5 CHAPTER 18 — Preconception and Prenatal Screening and Diagnosis 395 Analyzing cell-free DNA for aneuploidy detection is done in different ways, but the common principle is to detect the small increased amount of total cell-free DNA from a particular chromosome if the fetus has trisomy. In one approach, referred to as the counting approach, total cell-free DNA is subjected to next generation sequencing, and millions of molecules of DNA are each mapped to its particular chromosome of origin (Fig. 18.3). The number of molecules that map to each chromosome is counted, without knowing which of the fragments are fetal and which are maternal. Because chromosome 21 constitutes ~1.5% of total DNA in the genome, ~1.5% of total fragments should be assigned to chromosome 21 if the fetus and mother have two normal copies of chromosome 21. If, however, the fetus has trisomy 21, more sequences than expected will map to chromosome 21, and this can be measured relative to the number of sequences that map to an appropriate reference chromosome or to the full set of chromosomes not including chromosome 21. Similar calculations can be used for the other autosomal trisomies and for sex chromosome aneuploidies. Other commonly used approaches evaluate not only the amount of cell-free DNA coming from each chromosome but also take into account differences in the nucleotide sequence between the maternal and fetal DNA (polymorphisms) to assign whether the sequenced DNA comes from the maternal or the fetal DNA. Although cell-free DNA provides a substantial improvement in sensitivity and specificity of screening for fetal trisomies (particularly trisomy 21), it remains a screening test, not a diagnostic test. A result that indicates the fetus is at increased risk for a chromosomal abnormality should be confirmed by diagnostic testing, either via CVS or amniocentesis (presented later in this chapter). If prenatal diagnostic testing is declined, it should be confirmed on a blood sample obtained from the infant after birth. Furthermore, the accuracy by which NIPS can predict that the fetus is affected by a chromosomal abnormality, calculated as the positive Chr 1 Chr 1 Number of sequences per chromosome Chr 2 Chr 3... Chr 21 Chr 22 Chr 2 Chr 3 Chr 21 Chr 22.................................................................. A G C G A T A C A T G T A C T A G A C G A A G T T T A G C C T C C A A G T T T T T G C C G C G C G G A G A C T T G C A C C G G A C G G A A G C A T G C T A A G T T C G G A G G C C T A G A T A C T G G A A C C T A G G A T A G C A G G C T A T C C C G C T T G A T T C A A A G C T C C T T A T G A G A T G C C A G C A A C A G G C G A A A A G A T C A A A C C C A A G T A C G G G C G Cell-free DNA in maternal serum Count the number of sequences assigned to each chromosome Align to human genome by computer and assign to individual chromosomes NGS Figure 18.3 Schematic diagram of noninvasive prenatal screening for trisomies by analysis of cell-free DNA in maternal blood. Fetal component of maternal plasma cell-free DNA is shown in red; maternal contribution is in blue. Millions of molecules of DNA are sequenced and assigned to each chromosome by computerized alignment against the human genome. Highly accurate measurements of small but significant increases in the fraction of molecules assigned to chromosome 13, 18, 21, or X compared to a reference indicate increased risk for trisomy of each of these chromosomes. ▶

CHAPTER 18 — Preconception and Prenatal Screening and Diagnosis 395 Analyzing cell-free DNA for aneuploidy detection is done in different ways, but the common principle is to detect the small increas...

Ch18 · Pt6 396 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE predictive value (PPV) varies. The PPV depends on the prevalence of the condition tested for, and PPV for common trisomies is lower for younger women but is not affected by age for monosomy X (see Table 18.4). The accuracy by which NIPS correctly predicts that a fetus is unaffected, the negative predictive value (NPV), is greater than 99% for all aneuploidies, as there are far more unaffected fetuses in the population in all age groups. PPVs are typically lower for rarer conditions, such as rare autosomal trisomies and microdeletions and duplications. Therefore the current recommendation in the United States and many other countries is that NIPS is not recommended for screening for conditions other than the common aneuploidies (trisomies 21, 13, 18). As the technology continues to improve and more data are accumulated, this guidance may change in the future. For example, newer data on screening performance for 22q11.2 deletions are promising. Other Current and Future Applications of Cell-Free Fetal DNA Analysis Cell-free fetal DNA in maternal plasma is also used to genotype the fetus at the RH locus and to determine fetal sex. In some countries, noninvasive tests for a growing number of single-gene disorders in high-risk pregnancies are already available (Table 18.5), and noninvasive cell-free DNA sequencing tests for small panels of genes have been introduced already but with still limited validation, and cell-free DNA-based sequencing of the entire fetal genome has been explored on a research basis. Refinements in the analysis of cell-free DNA will likely make noninvasive testing for many other genetic disorders available in the future. Prenatal Detection of Fetal Congenital Anomalies by Ultrasonography High-resolution, real-time ultrasonography is widely used for assessment of fetal viability, fetal number, fetal size, gestational age, amniotic fluid volume, and evaluation of fetal and placental morphology. Most basic ultrasonography exams are done via two-dimensional (2D) ultrasound imaging, but ultrasound in three dimensions (3D) (Fig. 18.4) and four dimensions (4D) (which is 3D over time) is also possible and allows more detailed examination of the fetal anatomy such as, for example, for fetal echocardiography (targeted ultrasound exam on the fetal heart). These are usually done in advanced imaging centers and are reserved for better definition of suspected congenital anomalies detected by 2D sonography. Fetal magnetic resonance imaging (MRI) is also increasingly used in specialized centers for high-resolution imaging of the fetus when there is suspicion for conditions that are difficult to detect by ultrasound or need more detailed evaluation (see Fig. 18.4). Studies investigating the safety indicate that prenatal ultrasonography and fetal MRI are not harmful to the fetus or mother. As equipment and techniques used by ultrasonographers continue to improve, the detection of many malformations by routine ultrasonography in the second trimester (optimally around 18–20 weeks of gestation), and increasingly also in the late first trimester continues to improve (Fig. 18.5; see also Fig. 18.4). Prenatal Ultrasonographic Findings with Fetal Chromosomal Abnormalities A number of findings on prenatal ultrasonography are associated with chromosomal aneuploidy, including trisomies 21, 18, and 13; 45,X; and many other abnormal karyotypes (Tables 18.6 and 18.7). Some are soft sonographic markers that are more common in fetuses with common aneuploidies, and others are major congenital anomalies (see Table 18.6). Many of these can also be present as isolated findings in a chromosomally normal fetus or in fetuses with other genetic conditions. The likelihood of a chromosomally abnormal fetus increases dramatically when more than one fetal abnormality is detected. When any of these findings are detected, referral for further specialized prenatal imaging and genetic counseling with the offer of diagnostic genetic testing, usually by amniocentesis, are indicated (see later). If no chromosomal abnormalities are identified, single-gene disorders or multifactorial etiologies for the congenital anomalies should be considered. TABLE 18.5 Cell-Free DNA Assays Developed for Single-Gene Disorders Clinically Available* Achondroplasia Apert syndrome Congenital adrenal hyperplasia Crouzon syndrome Cystic fibrosis Duchenne and Becker muscular dystrophy Blood group genotyping (RHD/RHCE; Kell) Thanatophoric dysplasia Torsion dystonia Spinal muscular atrophy Selected familial known mutation analysis cf DNA screening tests for small panels of genes** Examples of Reported Assay Development† Fraser syndrome Hemoglobinopathies (sickle cell, thalassemias) Hemophilia A and B Huntington disease Leber congenital amaurosis Polycystic kidney disease Propionic acidemia Methylmalonic acidemia Retinitis pigmentosa *Only in certain countries, primarily United Kingdom. †Incomplete list. **Commercially available in some countries, limited clinical validity data. Modified from Van den Veyver IB, Chitty LS: Noninvasive prenatal diagnosis and screening for monogenic disorders using cell-free DNA. In Milunsky A, Milunsky JM, editors, Genetic disorders and the fetus: Diagnosis, prevention and treatment, ed 8, New York, 2021, John Wiley & Sons, Ltd. ▶

396 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE predictive value (PPV) varies. The PPV depends on the prevalence of the condition tested for, and PPV for common trisomies is lower for youn...

Ch18 · Pt7 CHAPTER 18 — Preconception and Prenatal Screening and Diagnosis 397 A B G F J E I D H C Figure 18.4 Examples of advanced imaging of fetal anomalies that can be present with chromosomal disorders. (A–C) Some features of trisomy 21: (A) atrioventricular septum defect of the heart; (B) double bubble sign with duodenal atresia; (C) cystic hygroma (also high risk of 45,X). (D–H) Some features of trisomy 13: (D, E) 2D and 3D views of cleft lip; (F, G) 2D and 3D views of proboscis in a fetus with holoprosencephaly; (H) monoventricle in holoprosencephaly. (I–K) Some features of trisomy 18: (I) ventricular septum defect; (J) large omphalocele containing liver. (Arrowheads point to defects in each panel.) (Images courtesy Wesley Lee, Baylor College of Medicine, Houston, Texas.) Prenatal Ultrasonography for Single-Gene and Multifactorial Disorders Prenatal ultrasonography can detect certain features that are highly suggestive of specific single-gene disorders. This can be useful when prenatal DNA testing is not possible because the patient declines the amniocentesis procedure or when a sample or specific prenatal genetic test is otherwise unavailable. For example, skeletal dysplasias such as osteogenesis imperfecta or thanatophoric dysplasia can present with distinct features on prenatal ultrasound. Recognizing the prenatal presentation of common genetic syndromes can also be helpful in deciding which genetic test—a specific gene test, gene panel, or whole exome analysis—to offer, based on the combined anomalies that are detected. However, ultrasonography cannot identify disorders with phenotypes that either develop only after birth or that are not detectable by imaging, such as metabolic disorders or syndromes that present primarily with intellectual disability. Ultrasound examination can be used to determine fetal sex as early as 13 weeks of gestation. This may help with the prenatal diagnosis of certain X-linked recessive disorders (e.g., hemophilia) for women who are carriers and at increased risk of having an affected son (see Chapter 7). Fetal chromosomal sex can now also be screened by cell-free DNA analysis as early as 10 weeks of gestation. Ultrasonography can also identify isolated abnormalities that may recur in families and are believed to have multifactorial inheritance, including NTDs (see Fig. 18.5), cleft lip and palate (see Fig. 18.4), congenital heart defects (Fig. 18.5), and others. Fetal ▶

CHAPTER 18 — Preconception and Prenatal Screening and Diagnosis 397 A B G F J E I D H C Figure 18.4 Examples of advanced imaging of fetal anomalies that can be present with chromosomal disorders. (A–...

Ch18 · Pt8 398 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE A B F E D C Figure 18.5 Additional examples of advanced imaging of fetal anatomy in other disorders. (A) Narrow chest in fetus with lethal osteogenesis imperfecta. (B, C) 2D and 3D views of polydactyly of feet. (D) Sagittal view of lumbar spine with meningomyelocele sac. (E) Large echogenic kidneys can be seen in fetuses with infantile polycystic kidney disease. (F) Cardiac rhabdomyomas can be seen in fetuses with tuberous sclerosis. (Images courtesy Wesley Lee, Baylor College of Medicine, Houston, Texas.) TABLE 18.7 Some Examples of Indications for Fetal Echocardiography* Maternal Indications (% Risk for Congenital Heart Defect) Insulin-dependent diabetes mellitus (3–5%) Phenylketonuria (15%) Teratogen exposure Thalidomide (10% if 20–36 days postconception) Phenytoin (2–3%) Alcohol (25% with fetal alcohol syndrome) Maternal congenital heart disease (5–10% for most lesions) Pregnancy conceived by in vitro fertilization Fetal Indications Abnormal general fetal ultrasound examination results Arrhythmia Chromosome abnormalities Nuchal thickening Nonimmune hydrops fetalis Familial Indications Mendelian syndromes Paternal congenital heart disease (2–5%) Previously affected child with congenital heart lesion (2–4%, higher for certain lesions) *This list is not comprehensive, and indications vary between centers. TABLE 18.6 Typical Major Congenital Anomalies Visible on Prenatal Ultrasonography of Fetuses With Common Aneuploidies Trisomy 21 Trisomy 13 Trisomy 18 Monosomy X 50% have findings Cystic hygroma CHD (VSD, AVSD) Duodenal atresia Wide gap between first and second toe 80–90% have findings Cystic hygroma CHD Polydactyly Holoprosencephaly Omphalocele Cleft lip / palate 80–90% have findings Cystic hygroma CHD (polyvalvular disease) Clenched fist Omphalocele Rocker bottom feet Fetal growth restriction Up to 90% have findings Cystic hygroma CHD (HLHS, aortic coarctation) Renal anomalies (horseshoe kidney) Hydrops Foot edema Other defects are also commonly found, but those listed are more typical or can be seen at higher frequency. AVSD, Atrioventricular septum defect; CHD, congenital heart defect (multiple types of CHD possible; more typical ones are listed in parentheses); HLHS, hypoplastic left heart syndrome; VSD, ventricular septum defect. echocardiography is also available at many centers for a detailed assessment of pregnancies at risk for a congenital heart defect. Table 18.7 shows some common indications for prenatal echocardiography. PRENATAL DIAGNOSTIC PROCEDURES To perform definitive prenatal diagnosis, diagnostic procedures that retrieve fetal cells are required. The two most common ones are amniocentesis to retrieve amniotic fluid, performed from 15 weeks of gestation onwards, and CVS to retrieve a small sample of placental villi, usually done between 11 and 13 completed weeks of gestation. When amniocentesis is not technically possible, or for specific indications, fetal cord blood sampling or late CVS or placental biopsies can be done instead. Amniocentesis Technique During amniocentesis a needle is inserted transabdominally under continuous ultrasound visualization into the amniotic sac to remove a sample of amniotic fluid (Fig. 18.6A). The amniotic fluid contains fetal cells that can be cultured or from which DNA can be prepared without prior culture and used for diagnostic tests. Before amniocentesis, ultrasonography is used to assess fetal viability, gestational age (by measuring biometric parameters such as head circumference, abdominal circumference, and femur length), the number of fetuses, ▶

398 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE A B F E D C Figure 18.5 Additional examples of advanced imaging of fetal anatomy in other disorders. (A) Narrow chest in fetus with lethal o...

Ch18 · Pt9 CHAPTER 18 — Preconception and Prenatal Screening and Diagnosis 399 volume of amniotic fluid, fetal anatomic structures, and position of the fetus and placenta to allow the optimal approach for needle insertion. Amniocentesis is typically performed between 16 and 20 weeks of gestation but can be done any time after 15 weeks. Different types of tests can be done on amniotic fluid. Fetal chromosome analysis by karyotype and chromosomal microarray analysis (CMA) are the standard genetic tests when there is an increased risk for chromosomal conditions based on screening or presence of fetal congenital anomalies. When there is an increased risk for single-gene disorders based on family history or because of distinct findings on prenatal ultrasound, sequencing of genes, gene panels, or the fetal exome can also be done (see Laboratory Studies, later). When an amniocentesis is done between 16 and 20 weeks, the concentration of AFP in amniotic fluid (AFAFP) can be measured by a relatively simple and inexpensive immunoassay to detect fetal open NTDs that can be an added test on second trimester amniotic fluid samples retrieved for other indications. If the AFAFP level is above the normal range for a particular gestational age, targeted ultrasound is recommended to look for an open NTD and other causes of high AFAFP (see Table 18.1). When the AFAFP assay is used together with ultrasonography at 18 to 19 weeks of gestation, ~99% of fetuses with open spina bifida and virtually all fetuses with anencephaly are identified. Other tests sometimes done on amniotic fluid samples include studies to detect viral infections or, less frequently, metabolic studies. Complications The major complication associated with midtrimester amniocentesis at 16 to 20 weeks of gestation is a 1 in 909 risk for inducing miscarriage over the baseline risk of pregnancy loss of ~1% to 2% for any pregnancy at this stage of gestation. Other complications are rare, including leakage of amniotic fluid, infection, and injury to the fetus by needle puncture. Early amniocentesis performed between 10 and 14 weeks is no longer recommended because of an increased risk for amniotic fluid leakage, a threefold increased risk for spontaneous abortion, and an approximately six- to sevenfold increased risk for talipes equinovarus (clubfeet), over the 0.1% to 0.3% population risk. Early amniocentesis has now been replaced by CVS. Chorionic Villus Sampling Technique For CVS a small amount of placental villi (5–40 μg) is removed between weeks 10 and 14 of pregnancy (see Fig. 18.6B). Chorionic villi consist of a mesenchymal core that contains capillaries and are covered by a layer of trophoblast cells, which are derived from the extraembryonic part of the early developing embryo (Fig. 18.7) and are a ready source of fetal tissue. As with amniocentesis, ultrasonographic scanning is used before CVS to determine the best approach, and the procedure is performed under continuous ultrasound visualization. CVS can be performed transabdominally with a needle or transcervically with a flexible catheter that is advanced into the placenta. The major advantage of CVS over amniocentesis is that results are available at an early stage of pregnancy, thus reducing the period of uncertainty and allowing termination, if it is elected, to be performed earlier. However, AFAFP cannot be assayed at this stage, and evaluation for a possible open NTD thus must be done by other methods, including MSAFP screening and ultrasonography. The success of chromosome analysis by karyotype or CMA is the same as with amniocentesis (>99%). Scanner Pubic bone Placenta Uterine wall Amniotic cavity Urinary bladder Cannula Vagina Rectum Uterine cavity Chorionic cavity Villous chorion Amniotic cavity Spinal needle Scanner A B Figure 18.6 Prenatal diagnostic procedures. (A) Amniocentesis. A needle is inserted transabdominally into the amniotic cavity, and a sample of amniotic fluid (usually ~20 m L) is withdrawn by syringe for diagnostic studies (e.g., chromosome studies, enzyme measurements, or DNA analysis). Ultrasonography is routinely performed before or during the procedure. (B) Chorionic villus sampling. Two alternative approaches are drawn: transcervical (by means of a flexible cannula) and transabdominal (with a spinal needle). In both approaches, success and safety depend on use of ultrasound imaging. ▶

CHAPTER 18 — Preconception and Prenatal Screening and Diagnosis 399 volume of amniotic fluid, fetal anatomic structures, and position of the fetus and placenta to allow the optimal approach for needle...

Ch18 · Pt10 400 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE However, ~1% of CVS yield ambiguous results because of chromosomal mosaicism (including true mosaicism and pseudomosaicism; described later in this chapter). In these situations, follow-up with amniocentesis may be recommended to establish whether the fetus has a chromosomal abnormality. Complications In prenatal diagnostic centers experienced with CVS, the rate of procedure-related fetal loss is about 1 in 450, only slightly increased over the baseline risk of 2% to 5%, and approximating the risk of amniocentesis. Although there were initial reports of an increase in the frequency of birth defects, particularly limb reduction defects, after CVS, this has not been confirmed in large series of CVS procedures performed after 10 weeks of gestation by experienced physicians. Indications for Prenatal Diagnosis by Amniocentesis or Chorionic Villus Sampling There are several well-accepted indications for prenatal testing by amniocentesis or CVS (see Box 18.1). The most common indication for invasive prenatal diagnosis is to test for Down syndrome (trisomy 21) and the more severe autosomal trisomies, 13 and 18. For this reason, advanced maternal age is a common reason for referral for prenatal diagnosis by amniocentesis or CVS. Other reasons include increased risk for an affected fetus because of a family history of a specific genetic condition, a positive maternal screening test result, or other well-defined risk factors. Current clinical guidelines no longer support using only maternal age as an indication for invasive testing for aneuploidies. The American College of Obstetricians and Gynecologists (ACOG) has now recommended that amniocentesis or CVS be made available to all women regardless of age along with screening options by the noninvasive methods described earlier. It is also considered appropriate that if a woman elects to undergo testing for fetal chromosomal abnormalities by amniocentesis or CVS, to perform not only karyotype but also CMA (see Chapter 5 and later in this chapter) on the extracted fetal DNA. There are also numerous single-gene disorders for which testing is available. Couples known to be at risk for any of these disorders in their fetus can be offered genetic counseling and prenatal testing by amniocentesis or CVS. Whether or not a couple considers the risk for a genetic condition in their fetus sufficiently burdensome to justify an invasive procedure is a personal decision each woman makes for herself. It is important to stress in counseling that invasive prenatal diagnosis cannot be used to rule out all possible fetal abnormalities. LABORATORY STUDIES Methods to Detect Fetal Chromosomal Abnormalities Karyotype Analysis Either amniocentesis or CVS can provide fetal cells for karyotyping (see Chapter 5). Preparation and analysis of chromosomes from cultured amniotic fluid cells or cultured chorionic villi require 10 to 14 days, although chorionic villi can also be used for karyotyping after short-term incubation. This short-term incubation using rapid metaphase analysis of villous cytotrophoblast tissue provides a result more quickly, but it has lower resolution and a higher rate of mosaicism (see later) that can render interpretation difficult. With long-term culture, the cultured cells from which the karyotype is obtained come from the mesenchymal core of the villus (see Fig. 18.7), which is embryologically more closely related to the developmental lineages that give rise to the fetus. Some Maternal sinusoid Maternal blood Intervillous space Tertiary villus Cytotrophoblastic shell Connective tissue Syncytiotrophoblast Cytotrophoblast cells Capillaries A B Figure 18.7 Development of the tertiary chorionic villi and placenta. (A) Cross-section of an implanted embryo and placenta at ~21 days. (B) Cross section of a tertiary villus showing establishment of circulation in mesenchymal core, cytotrophoblast, and syncytiotrophoblast. (From Moore KL: The developing human: Clinically oriented embryology, ed 4, Philadelphia, 1988, WB Saunders.) ▶

400 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE However, ~1% of CVS yield ambiguous results because of chromosomal mosaicism (including true mosaicism and pseudomosaicism; described later...

Ch18 · Pt11 CHAPTER 18 — Preconception and Prenatal Screening and Diagnosis 401 laboratories use both techniques, but if only one is used, long-term culture is therefore the technique of choice. The resolution of chromosome spreads prepared from prenatal samples is lower than from other tissues, but segmental abnormalities of 7 to 10 Mb and larger should be readily visible, depending on the region involved. Chromosomal abnormalities are detected in 10% to 30% of pregnancies with fetal congenital anomalies, and this number is higher when multiple malformations are present. The karyotypes most often seen in fetuses ascertained by abnormal ultrasonographic findings are the common autosomal trisomies (21, 18, and 13) and 45,X (Turner syndrome). The presence of a cystic hygroma is associated with aneuploidy in more than 50% of cases, most commonly 45,X, but it can also occur in Down syndrome and trisomy 18, or in fetuses with normal karyotypes. Fluorescence In Situ Hybridization Fluorescence in situ hybridization (FISH) (see Chapter 5) makes it possible to rapidly screen interphase nuclei in fetal cells for the common aneuploidies of chromosomes 13, 18, 21, X, and Y immediately after amniocentesis or CVS, with a result available usually in 1 to 2 days. This can be useful when rapid information is needed if time- sensitive decisions regarding pregnancy management and delivery planning could be affected by a trisomy diagnosis (e.g., when a growth-restricted fetus is suspected to have trisomy 18). Because FISH only provides limited information, it should always be followed by a more definitive test, karyotype, or CMA. Although still offered, FISH is now less commonly used because CMA can provide more definitive results with only a slightly longer turnaround time of 5 to 7 days. In some countries outside the United States, rapid aneuploidy testing on CVS or amniotic fluid samples is done by quantitative polymerase chain reaction (PCR) amplification of unique regions on chromosomes 13, 18, and 21. Chromosomal Microarray Analysis CMA (see Chapter 5) is increasingly replacing karyotyping for prenatal diagnosis. Copy number variants (CNVs), including chromosomal aneuploidy and segmental imbalances, such as duplications, triplications, deletions, or marker chromosomes (see Chapter 4), can be detected at much higher resolution by CMA than can be accomplished even with high-resolution karyotyping. Although ACOG has advised that CMA, rather than karyotyping, should be the first-line test when a fetal abnormality is detected by ultrasonography and recommends that all women having invasive testing be given the option to have CMA, the Society of Obstetricians BOX 18.1 PRINCIPAL INDICATIONS FOR PRENATAL DIAGNOSIS BY AMNIOCENTESIS OR CHORIONIC VILLUS SAMPLING The pregnant woman or couple wishes diagnostic testing Although limited at one time to a pregnant woman with no increased risk other than advanced maternal age, some current professional guidelines call for diagnostic testing (amniocentesis or CVS) to be offered to all couples. Increased risk as determined by maternal serum screening, ultrasound examination, and noninvasive prenatal screening test of cell-free DNA Genetic assessment and further testing are recommended when fetal abnormalities are suspected based on routine screening by maternal serum screening and fetal ultrasound examination. Previous child with de novo chromosomal aneuploidy or other genomic imbalance Although the parents of a child with chromosomal aneuploidy may have normal chromosomes themselves, in some situations there is still an increased risk for a chromosomal abnormality in a subsequent child. For example, if a woman at 30 years of age has a child with Down syndrome, her recurrence risk for any chromosomal abnormality is ~1 per 100, compared with the age-related population risk of ~1 per 390. Parental mosaicism is one possible explanation for the increased risk, but in most cases the mechanism of the increased risk is unknown. Presence of structural chromosomal or genome abnormality in one of the parents The risk for a chromosome abnormality in a child varies according to the type of abnormality and sometimes the parent of origin. The greatest risk, 100% for Down syndrome, occurs only if either parent has a 21q21q robertsonian translocation (see Chapter 6). Family history of a genetic disorder that may be diagnosed or ruled out by biochemical or DNA analysis Most of the disorders in this group are caused by single- gene defects with 25% or 50% recurrence risks. Cases in which the parents have been diagnosed as carriers after a carrier screening test, rather than after the birth of an affected child, are also in this category. Mitochondrial disorders pose special challenges for prenatal diagnosis. Family history of an X-linked disorder for which there is no specific prenatal diagnostic test When there is no alternative method, the parents of a boy affected with an X-linked disorder may use fetal sex determination to help them make decisions about their pregnancy because the recurrence risk may be as high as 50% for male children. For X-linked disorders for which prenatal diagnosis by DNA analysis is available, DNA analysis is the preferred method of testing. Note that if familial increased risk is known before pregnancy, preimplantation genetic testing (see later) with the transfer to the uterus of only those embryos determined to be unaffected for the disorder in question is an option. Risk for a neural tube defect (NTD) First-degree relatives (and second-degree relatives at some centers) of patients with NTDs are eligible for amniocentesis because of an increased risk for having a child with NTD. However, as described in this chapter, most open NTDs can be detected by ultrasound and amniocentesis is no longer commonly done to confirm or exclude NTDs by assaying AFP levels but instead is done to determine whether a fetus with NTD has an associated chromosomal abnormality. ▶

CHAPTER 18 — Preconception and Prenatal Screening and Diagnosis 401 laboratories use both techniques, but if only one is used, long-term culture is therefore the technique of choice. The resolution o...

Ch18 · Pt12 402 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE and Gynaecologists of Canada still recommends CMA as a second-tier test following a normal karyotype. For some findings a karyotype is still needed (e.g., to determine whether a copy number gain for chromosome 21 is the result of a trisomy or an unbalanced robertsonian translocation). CMA also does not detect balanced translocations and balanced inversions, but these are more rarely the cause of fetal congenital anomalies or syndromes. The current data support that a prenatal CMA can identify a clinically significant CNV about 1% to 1.7% of the time, and when a CMA is done for fetal congenital anomalies this number goes up to 6% to 7% overall and to over 10% when there are multiple congenital anomalies. About 1% to 2% of the time CMA can identify variants of uncertain significance (VUS), or findings that indicate presence of a condition in the fetus that was not suspected. These can make counseling complex and are discussed in more detail later. Sequencing to Detect Chromosomal Abnormalities Some laboratories outside the United States are beginning to use low-coverage whole genome sequencing with counting of fragments aligned to each chromosome to determine the copy number of entire chromosomes (aneuploidy) or chromosomal segments as a low-cost, high-throughput alternative method to CMA. This is not used in the United States for prenatal diagnosis but is the method of choice for preimplantation genetic testing for aneuploidy (see later). Fetal DNA Sequencing and Fetal Genome Analysis As the molecular basis for an increasing number of genetic disorders is determined (see Chapter 12), many conditions that were not previously detectable prenatally by other means can now be diagnosed by analyzing fetal DNA. Any technique used for direct variant analysis can be used for prenatal diagnosis on a fetal DNA sample extracted from amniotic fluid or CVS samples or from cell cultures derived from these samples. Three main modalities that can be used are single-gene testing, either by sequencing or targeted analysis for a known variant, gene panel sequencing, or sequencing of the exome or genome. Because not 100% of the exome (all exons) or genome (all genomic DNA) can be sequenced, we refer to these as exome sequencing (ES) and genome sequencing rather than whole exome or whole genome sequencing. Single-Gene Testing and Gene Panel Sequencing for Prenatal Diagnosis When there is a known familial pathogenic variant for which the fetus is at risk, or a recognizable fetal condition, such as thanatophoric dysplasia, a lethal skeletal dysplasia caused by only a few different pathogenic variants, a specific targeted molecular test can be done to determine if that variant is detected in the fetal DNA. These tests are highly accurate, but such clinical presentations are relatively rare. More commonly, fetal anomalies detected by ultrasonography are suggestive for a class of genetic disorders. In those situations, a gene panel sequencing test, which analyzes a variable number of genes that have been associated with that class of disorders (e.g., a broad skeletal dysplasia panel), can be done. These panels have limitations. They are usually designed based on gene-disease relationships that are recognized postnatally, but the prenatal phenotypes for the same conditions may not be well known or may differ from those observed postnatally. In addition, the gene content of these panels needs to be kept up to date with the rapidly growing knowledge of gene-disease relationships. Participation of medical geneticists or genetic counselors, whose role it is to stay informed about these rapid changes, is essential for counseling and selection of such tests for prenatal diagnosis. This type of genetic testing is reserved for pregnancies found to be at increased risk and not for routine screening or testing. Exome Sequencing and Genome Sequencing for Prenatal Diagnosis In addition to the above mentioned limitations of gene panels for sequencing of DNA from fetuses diagnosed with congenital anomalies, it is increasingly recognized that the genetic basis for many fetal anomalies, in particular those that lead to fetal or neonatal demise, are not yet known. Furthermore, some prenatally diagnosed fetal anomalies may be caused by variants in genes known to cause a different phenotype postnatally, which is referred to as prenatal phenotype expansion. For these reasons and because of its success in diagnosing genetic conditions postnatally, multiple studies have investigated the benefit of prenatal ES of fetal DNA for pregnancies complicated with fetal anomalies for which standard testing by CMA has not yielded a diagnosis. A few large studies have demonstrated that in these circumstances the diagnostic rate is between 8.5% and 13%, but in some more selected series that include pregnancies with stronger suspicion for a single gene disorder, it is higher, ranging from ~20% to 40%, or up to 80% in very selected series with fetal skeletal dysplasias. Much more research is needed on the impact of prenatal exome sequencing on the care of pregnancies and newborns, but professional societies are now supporting its clinical use for selected pregnancies when a diagnosis cannot otherwise be made. This test should be offered by genetics providers who are familiar with prenatal genetics and the complexities of such testing and who are skilled at counseling pregnant individuals and their partners about its benefits and limitations. With exome sequencing there is a substantially higher chance of detecting unwanted findings such as VUS, incidental and secondary findings, including possible diagnosis of adult-onset disorders, and unexpected paternity (see ▶

402 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE and Gynaecologists of Canada still recommends CMA as a second-tier test following a normal karyotype. For some findings a karyotype is stil...

Ch18 · Pt13 CHAPTER 18 — Preconception and Prenatal Screening and Diagnosis 403 upcoming discussion). The analysis of exome sequences is more complex and time consuming, and it may take longer to obtain results than for prenatal CMA and karyotypes. To speed up variant interpretation, prenatal exome sequencing is primarily done in trios, where the parental DNAs are also sequenced and analyzed to help interpret the variants found in the fetal DNA. More recently, trio genome sequencing is also being studied for prenatal diagnosis. While it is more comprehensive, evaluating both noncoding and coding regions of DNA as well as including the possibility of copy number analysis, the information obtained from genome sequencing is even more complex. Prenatal genome sequencing is a rapidly evolving area that bears watching closely in the years ahead, with increasingly important ethical and policy implications for the practice of fetal medicine and prenatal genetics. Biochemical Assays for Metabolic Diseases Although any disorder for which the genetic basis and responsible genetic variant(s) are known can be diagnosed prenatally by DNA analysis, more than 100 metabolic disorders can also be diagnosed by biochemical analysis of chorionic villus tissue or cultured amniotic fluid cells. A few rare conditions can even be identified directly by assaying a substance in amniotic fluid. Most metabolic disorders are rare in the general population but have a high recurrence risk since most are autosomal recessive conditions. Because each condition is rare, the experience of the laboratory performing the prenatal diagnostic testing is important and it should be done at specialized centers. Whenever possible, a biochemical assay on directly sampled chorionic villus tissue (as opposed to cultures) is preferred to avoid misinterpretation of results due to the expansion in culture of contaminating maternal cells. Access to a cultured cell line from an affected individual in the family is highly advisable so that the laboratory can confirm its ability to detect the biochemical abnormality in the proband before the assay is attempted in CVS or amniotic fluid cells from the pregnancy at risk. Many metabolic disorders cannot be diagnosed prenatally by enzyme assays because the enzyme is not expressed in amniocytes or chorionic villi or a reliable biochemical assay is not available. For these, DNA sequencing should be performed. Biochemical tests have one advantage over DNA: they can detect abnormalities caused by any mutant allele that has a significant effect on the protein function. This is particularly significant for disorders with a high degree of allelic heterogeneity, genes in which pathogenic variants occur in regions that are not routinely sequenced, or by a high proportion of new mutations (see Chapter 12). In addition, biochemical testing may be the only option for prenatal diagnosis if the causative mutations in the family are unknown. Problems in Prenatal Chromosome Analysis and Gene Sequencing Mosaicism Mosaicism refers to the presence of two or more cell lines in an individual or tissue sample (see Chapter 7). Because invasive prenatal techniques, particularly CVS, sample extraembryonic tissues of the placenta, and not the fetus itself, mosaicism found in cultured fetal cells may be difficult to interpret. The prenatal geneticist must determine if the fetus is truly mosaic and understand the clinical significance of any apparent mosaicism. Cytogeneticists distinguish three levels of mosaicism in amniotic fluid or CVS cell cultures based on the number of cells with mosaicism and the number of colonies from which they arise. Mosaicism detected in multiple colonies from several different primary cultures is considered true mosaicism. Postnatal studies have confirmed that true mosaicism in culture is associated with a high risk that mosaicism is present in the fetus. The probability varies with different situations. However, mosaicism for structural aberrations of chromosomes, for example, is hardly ever confirmed. Mosaicism involving several cells or colonies of cells from a single primary culture is difficult to interpret, but it is generally thought to reflect pseudomosaicism that has arisen in culture. When mosaicism is restricted to only a single cell, it is also considered to reflect pseudomosaicism and is typically disregarded. Maternal cell contamination (MCC; see later) can explain some cases of apparent mosaicism in which both XX and XY cell lines are present. This is more common in products of conception from miscarriages and in long-term CVS cultures than in amniotic fluid cell cultures because chorionic villi and maternal tissue are anatomically closely associated (see Fig. 18.6). In CVS studies, mosaic discrepancies between the karyotypes found in the cytotrophoblast, villous stroma, and fetus have been reported in 1% to 2% of pregnancies studied at 10 to 11 weeks of gestation. Confined placental mosaicism (CPM) is mosaicism that is present in the placenta but not in the fetus (Fig. 18.8). CPM can be the result of a trisomic cell line arising postzygotically in the placenta, in which case the fetus will always be diploid. Another mechanism for CPM is trisomy rescue (see Chapter 6), where the zygote has trisomy, but postzygotically during one of the cell divisions one of the copies of the trisomic chromosome is lost, establishing diploid normal cell lineages, alongside the trisomic cells. Occasionally, a liveborn infant or fetus with nonmosaic trisomy 13 or trisomy 18 has been reported in a pregnancy with placental mosaicism for the trisomic cell line and a normal diploid cell line. It has been proposed that the placental diploid cell line improves the probability of intrauterine survival of a trisomic fetus. When trisomy rescue results in a diploid fetus, it raises the concern that the fetus could have retained two copies of a chromosome from the same parent, resulting ▶

CHAPTER 18 — Preconception and Prenatal Screening and Diagnosis 403 upcoming discussion). The analysis of exome sequences is more complex and time consuming, and it may take longer to obtain results t...

Ch18 · Pt14 404 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE in uniparental disomy (UPD) (see Chapter 6). This can affect all chromosomes but is particularly concerning for chromosomes 7, 11, 14, or 15, which contain imprinted genes. For example, two maternal copies of chromosome 15 cause Prader-Willi syndrome, and two paternal copies are associated with Angelman syndrome (see Chapter 6). Thus when there is CPM for these chromosomes, tests should be done to exclude UPD. Because CMA uses pooled DNA from tissues or cultured cells and does not examine individual cells the way karyotyping does, it is less sensitive for detection of mosaicism. Mosaicism in which 10% of the cells are aneuploid is difficult to detect as a copy number change by CMA, whereas 10% mosaicism will be detected with greater than 99% probability when 50 cells are examined by karyotype. CMA is even less sensitive for detecting mosaicism for a CNV of only a segment of a chromosome unless it is present in more than 20% to 25% of the cells. Confirmation and interpretation of apparent mosaicism are difficult challenges in genetic counseling for prenatal diagnosis when mosaicism is identified during an amniocentesis because clinical outcome information on the different types and extents of mosaicism can be limited. Further studies such as cordocentesis (fetal blood sampling) may provide some guidance, but the interpretation can remain uncertain. If mosaicism is identified at the time of CVS, parents can be reassured if follow-up amniocentesis results is normal and UPD is excluded (see earlier), particularly if the prenatal ultrasound demonstrates normal growth and no congenital anomalies are visualized. Parents should be counseled in advance of the possibility for mosaicism and that its interpretation could be uncertain. After birth, an effort should be made to verify any abnormal chromosome findings suspected on the basis of prenatal diagnosis. Confirmation of mosaicism, or lack thereof, may prove helpful with respect to medical management as well as for genetic counseling of the specific couple and other family members. Culture Failure and Maternal Cell Contamination Prenatal diagnosis is time sensitive, and culture failure, which is fortunately very rare, can be a concern. When a CVS culture fails to grow, there is time to repeat the chromosome study with amniocentesis. If an amniotic fluid cell culture fails, either repeated amniocentesis or cordocentesis could be offered, depending on fetal age. MCC is another potential risk of prenatal sampling. During cell culture, contaminating maternal cells could outgrow the fetal cells. MCC can be suspected when there are XX cell lines with a male pregnancy and is common in CVS cultures as a consequence of the intimate association between chorionic villi and the maternal tissue (see Fig. 18.6). To minimize the risk for MCC, maternal decidua present in a CVS must be carefully dissected and removed, but this does not always eliminate every cell of maternal origin. A maternal blood sample can also be utilized to confirm or refute MCC through parallel genotyping of the DNA from the maternal and fetal sample with use of polymorphisms. Unexpected Findings: Variants of Uncertain Significance, Incidental and Secondary Findings On occasion, prenatal chromosome analysis performed primarily to rule out aneuploidy reveals some other unusual chromosome finding (e.g., a rare chromosomal rearrangement, a marker chromosome [see Chapter 5], Diploid Aneuploid A B C D Figure 18.8 The different types of mosaicism that may be detected by prenatal diagnosis. (A) Generalized mosaicism with diploid (yellow) and abnormal aneuploid (blue) cell lineages affecting both the fetus and placenta. (B) Confined placental mosaicism with diploid and aneuploid cells in the placenta and a diploid fetus. (C) Mosaicism with only aneuploid cells in the placenta and a diploid fetus. (D) Mosaicism confined to the fetus with a diploid placenta. (Modified from Kalousek DK: Current topic: Confined placental mosaicism and intrauterine fetal development, Placenta 15:219–230, 1994.) ▶

404 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE in uniparental disomy (UPD) (see Chapter 6). This can affect all chromosomes but is particularly concerning for chromosomes 7, 11, 14, or 15...

Ch18 · Pt15 CHAPTER 18 — Preconception and Prenatal Screening and Diagnosis 405 and now more commonly a VUS on CMA or exome sequencing and genome sequencing). Unbalanced or de novo structural rearrangements may cause serious fetal abnormalities (see Chapter 6). If a parent carries a balanced structural rearrangement (e.g., a balanced translocation) that is present in unbalanced form in the fetus (unbalanced translocation), the consequences for the fetus can be serious. If a fetus has a structural chromosomal rearrangement that is also present in one of the parents, it is more likely to be a benign change without untoward consequences, but there are exceptions to this. They include variable expressivity and involvement of a region of the genome that contains imprinted genes. CMA has a higher chance of detecting VUS than a karyotype analysis, which is one reason why it has been more selectively used for pregnancies with fetal anomalies rather than as a first-line test. As experience and knowledge of CNV in the human genome improves (see Chapter 4), the medical relevance of an increasingly greater fraction of CNVs will become clearer. The incidence of VUS with CMA has dropped to levels close to what is seen with a karyotype, justifying replacing fetal karyotyping by CMA for nearly all indications. Exome sequencing and genome sequencing remain tests for second-line evaluation of pregnancies with fetal anomalies for which a genetic etiology is not identified by CMA. They both have a higher chance for detecting VUS, which can be mitigated by limiting prenatal analysis to genes relevant for the sonographic phenotype or family history. When a VUS is identified, analysis of parental data from trio sequencing may help with interpretation. Incidental findings of pathogenic or likely pathogenic variants that are unrelated to the fetal phenotype but may cause a different serious condition can occasionally be discovered in the sequence of the fetal or parental samples. It is recommended that significant incidental findings related to serious childhood disorders in the fetal sample are reported. However, the reporting of parental findings (e.g., a variant that increases the risk for late-onset conditions such as cancer) often involves the option for parents to opt out of obtaining parental results. Secondary findings are pathogenic and likely pathogenic variants in a list of genes curated by the ACMG to be associated with diseases for which the discovery of these variants could result in health care measures that benefit the individual. In general, patients should be given the option to opt out of receiving secondary findings. PRECONCEPTION GENETIC SCREENING AND TESTING Parental Carrier Screening for Autosomal Recessive and X-Linked Disorders Preconception screening refers to the evaluation of parents for the risk for genetic disorders in their future children before the pregnancy. This is the optimal time to offer and perform carrier screening, but it is infrequently done preconceptionally. Therefore risks for autosomal recessive and X-linked single-gene disorders and carrier screening should also be addressed in prenatal counseling. The approach to carrier screening has evolved over time. It initially relied on targeted screening for a few diseases, based on individual risk factors assessed through family history, such as a prior affected child, the presence of consanguinity, and information on the ancestry of the prospective parents. With the currently available high-throughput laboratory methods, cost- effective rapid sequencing of many genes is now available and allows for a more equitable approach to carrier screening with larger panels of genes. The ACMG now recommends that carrier screening is offered to all preconception or pregnant patients and their reproductive partners in an ancestry and population neutral fashion. They recommend using a panel with genes for 97 autosomal recessive and 16 X-linked conditions with a carrier frequency at or greater than 1/200 to be offered to all pregnant patients while reserving larger panels with additional disease genes for patients with possible consanguinity or when warranted based on family or medical history. Couples who are carriers for pathogenic or likely pathogenic variants that increase the risk for inherited recessive or X-linked disease in their current or future pregnancy should be offered appropriate counseling about reproductive options and residual risk. Preconceptionally, they can consider preimplantation genetic testing for monogenic disorders (PGT-M) (see later), use of donor eggs or sperm, adoption, or the option to forgo having children. They can also choose to conceive and have CVS or amniocentesis during the pregnancy or testing of the infant at the time of birth. Preimplantation Genetic Testing PGT, formerly referred to as preimplantation genetic diagnosis (PGD), is performed on in vitro fertilized embryos prior to embryo transfer (Fig. 18.9). PGT offers couples at significant risk for a specific genetic disorder or aneuploidy in their offspring an option to manage reproductive risks that avoid pregnancy termination. In the most commonly used approach, blastocyst biopsy (see Fig. 18.9), women have to undergo IVF. They first undergo ovarian stimulation and retrieval of ~10 or fewer oocytes in the stimulated cycle. The oocytes are then fertilized in vitro and cultured for 5 to 6 days until the blastocyst stage. At that time, 5 to 10 cells are retrieved from the trophectoderm, which develops into the future placenta, without disrupting the inner cell mass, which will develop into the fetus (see Chapter 15). PGT was initially done by blastomere biopsy on eight- cell–stage embryos, but trophectoderm biopsy on blastocysts, which provides more cellular material and apparently greater accuracy, is now preferred. Usually, ▶

CHAPTER 18 — Preconception and Prenatal Screening and Diagnosis 405 and now more commonly a VUS on CMA or exome sequencing and genome sequencing). Unbalanced or de novo structural rearrangements may c...

Ch18 · Pt16 406 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE biopsied embryos are frozen while the molecular diagnosis proceeds. One of the embryos that are found not to carry the genetic abnormality in question can then be transferred and allowed to implant in a future cycle, as is routinely done after IVF. Affected embryos are discarded. Data currently available suggest that there are no detrimental effects to embryos that have undergone biopsy. There are three types of PGT: PGT monogenic (M) evaluates for single-gene disorders, PGT aneuploidy (A) evaluates for whole chromosome abnormalities, and PGT structural rearrangements (SR) is used to detect structural chromosomal abnormalities such as translocations. PGT-M is utilized to detect known pathogenic variants in specific genes that were previously found in a family member with a genetic disorder or through parental carrier screening. It involves PCR amplification followed by a method to detect the variant. Because of the small amount of starting material, analysis of linked polymorphic markers is often included to improve accuracy. Initially, FISH and later microarray analysis were used to detect chromosome abnormalities, but more recently laboratories have switched primarily to next generation sequencing methods to detect CNVs for PGT-A and unbalanced translocations (see Chapters 4 and 5). PGT-A is now offered to women undergoing IVF to increase the live birth rate by only selecting euploid embryos for transfer. The American Society of Reproductive Medicine recommends offering PGT-A to all infertile women, but research is ongoing to fully establish the clinical utility of PGT-A. Despite technologic improvements in recent years, there are limitations to PGT. These include mosaicism and challenges with detecting de novo mutations, microdeletions, and duplications. Because with PGT diagnoses are made on small numbers of cells, women who have PGT-M and PGT-SR should be offered confirmatory CVS or amniocentesis during ensuing pregnancies. Because of the small risk (~2–3%) for false-positive and false-negative results with PGT-A, it is considered good practice to offer prenatal aneuploidy screening for pregnancies conceived by IVF with PGT-A. Although PGT was developed to avoid the ethical, religious, and psychological difficulties with pregnancy terminations, ethical dilemmas remain related to the disposition of affected embryos and remaining frozen healthy embryos after parents have completed their family. FETAL SURGICAL AND MEDICAL INTERVENTIONS The number of conditions for which fetal surgical or medical interventions are available has expanded significantly in the last 4 decades and is frequently impacted by ethical considerations related to the underlying genetic etiology of the fetal condition in need of treatment. Fetal therapy began in the 1960s with intrauterine transfusion for anemia, particularly in the setting of fetal Rh alloimmunization. This was followed by insertion of shunts to drain excess fluid from the fetus into the amniotic cavity. Shunts are still used for some cases of fetal uropathy (e.g., to drain urine when there is bladder outlet obstruction or to drain large pleural effusions). Laser ablation of communicating placental vessels for twin-twin transfusion syndrome started in the 1990s, and laser procedures for this condition have now become standard of care. For some cases of evolving Inner cell mass Trophoblast Blastocyst Affected Unaffected X X X X X X X X Affected Unaffected Implant Do not implant Genetic testing Figure 18.9 Preimplantation genetic diagnosis. After ovarian stimulation, oocytes are retrieved and fertilized in vitro. The fertilized embryos are incubated for 5 to 6 days, to the blastocyst stage, and about 5 to 10 cells are removed from the trophectoderm, which will develop into the placenta. These cells will be used for genetic testing for chromosomal abnormalities or single-gene disorders. In this example, the embryo (labeled X) will not be transferred. Only embryos that are unaffected will be transferred into the patient’s uterus to establish a pregnancy. ▶

406 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE biopsied embryos are frozen while the molecular diagnosis proceeds. One of the embryos that are found not to carry the genetic abnormality i...

Ch18 · Pt17 CHAPTER 18 — Preconception and Prenatal Screening and Diagnosis 407 hypoplastic left heart syndrome, aortic valvuloplasty and now balloon septostomy and atrial septostomy are done in expert centers. After a multicenter randomized controlled trial, the Management of Myelomeningocele Study (MOMS) showed improved outcomes after intrauterine surgical repair of fetal myelomeningocele (spina bifida); this option is now available at selected centers. In recent years the approach has transitioned from open to laparoscopic repair. A subset of fetuses with congenital diaphragmatic hernia have improved outcomes after a balloon is placed inside the trachea with the goal to improve growth and expansion of the fetal lungs. The balloon is removed prior to controlled delivery. Finally, investigation is underway to examine the value of serial amnioinfusions to treat early-onset anhydramnios for fetuses with nonfunctioning kidneys. In addition to these mechanical fetal procedures, a number of in utero medical therapies are being adopted. Fetal arrhythmias are treated with maternally administered antiarrhythmia medications such as digoxin and sotalol. Sirolimus has established efficacy in the treatment of severe cases of fetal rhabdomyomas. In utero stem cell transplantation and gene therapy trials are underway for conditions such as osteogenesis imperfecta and hemoglobinopathies. The disorders and congenital anomalies for which these fetal interventions can provide benefit can be caused by genetic defects. Therefore multidisciplinary approaches to evaluation and decision making that include prenatal genetic counseling and assessment are recommended prior to any such procedures. Debate remains as to the depths of investigation that should be undertaken, and a fetal karyotype is recommended as the minimum requirement for some procedures, which contrasts with general recommendations for CMA as a primary genetic test for major congenital anomalies. Post-test counseling after genetic test results are available should include multidisciplinary discussion about the risks, benefits, and alternatives of pursuing a fetal procedure. GENETIC COUNSELING FOR PRECONCEPTION AND PRENATAL DIAGNOSIS AND SCREENING Most prenatal and reproductive genetic counselors practice in the setting of a prenatal diagnosis program. The professional staff of a prenatal diagnosis program (physician, nurse, and genetic counselor) must obtain an accurate family history and determine whether other previously unsuspected genetic problems should be considered on the basis of family history or ancestry. Pretest and posttest counseling is recommended when genetic screening or testing is considered, including carrier screening, PGT, and prenatal screening and diagnosis. For carrier screening and testing, until recently, ancestry was utilized to assess the need for carrier testing for a small number of X-linked or autosomal recessive conditions that are more prevalent in certain populations. Such disorders include thalassemias in individuals of Mediterranean or Asian background, sickle cell anemia in people of African descent, and various disorders that are more prevalent in people of Ashkenazi Jewish ancestry. Because it is becoming increasingly difficult to assign a single background to each individual, use of broader carrier screening panels, in which individuals are tested for a large array of genetic disorders irrespective of apparent or stated ancestry, is now recommended. The complexities posed by the availability of so many different prenatal screening and testing options include the distinction between screening and diagnostic testing, the many different and distinctive indications for testing, the subtleties of interpretation of test results, and the personal, ethical, religious, and social considerations that enter into reproductive decision making. They make providing prenatal genetic services challenging, and prospective parents considering prenatal genetic screening or diagnosis should be provided with understandable information that will allow them to make informed decisions about which conditions to test or screen and whether they give or withhold consent for diagnostic procedures. Prenatal genetic counseling for women considering CVS or amniocentesis usually addresses several different points (see Box 18.2). Screening tests avoid the risk of a diagnostic procedure but do not give a yes/no diagnostic answer. They provide a risk estimate for a disorder relative to the background risk. The cutoff for a positive screen is set to balance sensitivity and specificity, and screening tests generally allow higher false-negative rates than would be acceptable for a diagnostic test to keep false-positive rates to a reasonable level, generally below 5%. Advances in laboratory technology, both for screening and diagnostic tests, have led to testing options that are both more accurate and more expansive, providing information for a growing number BOX 18.2 COUNSELING FOR CHORIONIC VILLUS SAMPLING OR AMNIOCENTESIS The reason/indication for testing and alternative options (screening) The risk that the fetus will be affected with the condition tested The nature and prognosis of the genetic condition (or categories of conditions) tested The risks and limitations of the procedures to be used The possible need for a repeated procedure in the event of a failed attempt The type of genetic testing performed The chance that the test will find the genetic cause The time required before a report can be issued The possibility of an uninterpretable result or a variant of unknown significance The possibility of incidental and/or secondary findings and the option to opt out of being informed of these findings ▶

CHAPTER 18 — Preconception and Prenatal Screening and Diagnosis 407 hypoplastic left heart syndrome, aortic valvuloplasty and now balloon septostomy and atrial septostomy are done in expert centers. A...

Ch18 · Pt18 408 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE of genetic conditions. This has made prenatal genetic counseling and parental decision making on which option to choose more complex. Although it is clear that cell-free DNA-based screening for Down syndrome is far more sensitive than maternal serum screening, we now know that CVS or amniocentesis coupled with CMA, a test that surveys the entire genome for aneuploidy and smaller imbalances (deletions and duplications), can detect a significant chromosomal abnormality in 1% to 1.7% of all pregnancies and in 6% of pregnancies with structural fetal abnormalities found on prenatal ultrasound. The latest data also support that amniocentesis and CVS are safer than previously thought. This has led to recommendations that this prenatal diagnostic test be offered to all women. Thus prenatal genetic counseling must inform women of all options and support them in decisions that balance their desire to know genetic information about their pregnancy with their willingness to undergo a procedure and the decisions they would make with this information. As with counseling for any genetic test, in addition, the couple must be advised that if a result is difficult to interpret, further tests and consultation may be required. After they are available, genetic counselors review the laboratory results and may seek clarification as indicated by clinical cytogeneticists or molecular geneticists. Result disclosure requires informing the patient of the implications of the results, recommending any additional testing that is needed to clarify the results, and discussing implications for other family members. Variants of uncertain clinical significance are often reported, along with the possibility of future reclassification. Incidental and secondary findings are reported if the patient did not opt out of receiving these results. Genetic counselors also discuss in generalized terms the implications of the results on future management of the pregnancy and infant after birth. For certain conditions, the availability of fetal therapy can be discussed, along with suggestions for appropriate subspecialist referrals depending on the specific findings. Providers can address options regarding continuation or termination of the pregnancy within the legal limitations of their location of practice while always respecting the personal thoughts of patients on whether this is an acceptable path for them. For parents at increased risk for a genetic condition in their children, the principal goal and benefit of PGT and prenatal genetic diagnosis is to be able to consider pregnancies that they might otherwise not have considered. PGT offers them a means to avoid pregnancies with an affected fetus, while through prenatal diagnosis they can learn early in a pregnancy if the fetus has the condition, allowing them to make an informed decision about whether to continue the pregnancy. For parents at low or average risk of having a child with a genetic disorder, the great majority of prenatal genetic screens and follow-up diagnostic tests ultimately end in reassurance. The primary objective of prenatal diagnosis is to determine whether the fetus is affected or unaffected with the disorder in question. Irrespective of the reason why testing is pursued, parents should be informed about all available options in the event of an abnormal result. Diagnosis of an affected fetus will allow parents to prepare emotionally and medically for the management of a newborn with a disorder. Termination of pregnancy is one choice they can make, but it is important parents understand that by undertaking prenatal diagnosis there is no implied obligation to terminate a pregnancy in the event of an abnormal result. In closing, prenatal genetic screening and diagnosis is a rapidly evolving discipline. Standards of care in this field will continue to be modified and refined because of the fast-paced technologic advances in methods available for assessing the fetus and the fetal genome, the ongoing discussions on social and ethical norms, and the varying governmental policies concerning prenatal diagnosis in different cultures and countries around the globe. GENERAL REFERENCES Gardner RJM, Amor DJ: Gardner and Sutherland’s chromosome abnormalities and genetic counseling, ed 5, New York, 2018, Oxford University Press. Milunsky A, Milunsky J: Genetic disorders and the fetus: Diagnosis, prevention, and treatment, ed 8, Chichester, West Sussex, England, 2021, Wiley-Blackwell. Norton M, Kuller J, Dugoff L: Perinatal genetics, ed 1, St. Louis, 2019, Elsevier. SPECIFIC REFERENCES Adzick NS, Thom EA, Spong CY, et al: A randomized trial of prenatal versus postnatal repair of myelomeningocele, N Engl J Med 364:993–1004, 2011. American College of Obstetricians and Gynecologists Committee on Practice Bulletins–Obstetrics: Practice Bulletin No. 187: Neural tube defects, Obstet Gynecol 130:e 279–e 290, 2017. American College of Obstetricians and Gynecologists Committee on Genetics: Committee Opinion No. 799: preimplantation genetic testing, Obstet Gynecol 135:e 133–e 137, 2020. American College of Obstetricians and Gynecologists: Practice Bulletin No. 162: Prenatal diagnostic testing for genetic disorders, Obstet Gynecol 127:e 108–e 122, 2016. American College of Obstetricians and Gynecologists Committee on Practice Bulletins–Obstetrics: Committee on Genetics; Society for Maternal-Fetal Medicine: Practice Bulletin No. 226: Screening for fetal chromosomal abnormalities, Obstet Gynecol 136:e 48–e 69, 2020. Armour CM, Dougan SD, Brock JA, et al: Canadian College of Medical Geneticists: Practice Guideline: Joint CCMG-SOGC recommendations for the use of chromosomal microarray analysis for prenatal diagnosis and assessment of fetal loss in Canada, J Med Genet 55:215–221, 2018. Bianchi DW, Chiu RWK: Sequencing of circulating cell-free DNA during pregnancy, N Engl J Med 379:464–473, 2018. Bianchi DW, Parker RL, Wentworth J, et al: DNA sequencing versus standard prenatal aneuploidy screening, N Engl J Med 370:799– 808, 2014. Deprest J, Benachi A, Gratacos E, et al: Randomized trial of fetal surgery for moderate left diaphragmatic hernia, N Engl J Med 385:119– 129, 2021. Deprest J, Nicolaides K, Benachi A, et al: Randomized trial of fetal surgery for severe left diaphragmatic hernia, N Engl J Med 385:107– 118, 2021. ▶

408 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE of genetic conditions. This has made prenatal genetic counseling and parental decision making on which option to choose more complex. Althou...

Ch18 · Pt19 CHAPTER 18 — Preconception and Prenatal Screening and Diagnosis 409 Fan HC, Gu W, Wang J, et al: Non-invasive prenatal measurement of the fetal genome, Nature 487:320–324, 2012. Grati FR, Ferreira J, Benn P, et al: Outcomes in pregnancies with a confined placental mosaicism and implications for prenatal screening using cell-free DNA, Genet Med 22:309–316, 2020. Gregg AR, Aarabi M, Klugman S, et al: ACMG Professional Practice and Guidelines Committee: Screening for autosomal recessive and X-linked conditions during pregnancy and preconception: A practice resource of the American College of Medical Genetics and Genomics (ACMG), Genet Med 23:1793–1806, 2021. Gregg AR, Edwards JG: Prenatal genetic carrier screening in the genomic age, Sem Perinatol 42:303–306, 2018. Harris S, Gilmore K, Hardisty E, et al: Ethical and counseling challenges in prenatal exome sequencing, Prenat Diagn 38:897–903, 2018. Kardon G, Ackerman KG, Mc Culley DJ, et al: Congenital diaphragmatic hernias: From genes to mechanisms to therapies, Dis Model Mech 10:955–970, 2017. Kitzman J, Snyder M, Ventura M, et al: Noninvasive whole-genome sequencing of a human fetus, Sci Transl Med 4(137):137ra 76, 2012. Lord J, Mc Mullan D, Eberhardt R, et al: Prenatal exome sequencing analysis in fetal structural anomalies detected by ultrasonography (PAGE): A cohort study, Lancet 393(10173):747–757, 2019. Malone FD, Canick JA, Ball RH, et al: First-trimester and second- trimester screening, or both, for Down’s syndrome, N Engl J Med 353:2001–2011, 2005. Mc Arthur SJ, Leigh D, Marshall JT, et al: Blastocyst trophectoderm biopsy and preimplantation genetic diagnosis for familial monogenic disorders and chromosomal translocations, Prenat Diagn 28: 434–442, 2008. Monaghan KG, Leach NT, Pekarek D, et al: The use of fetal exome sequencing in prenatal diagnosis: A points to consider document of the American College of Medical Genetics and Genomics (ACMG), Genet Med 22:675–680, 2020. Nassr AA, Erfani H, Fisher JE, et al: Fetal interventional procedures and surgeries: A practical approach, J Perinat Med 46:701–715, 2018. Petrovski S, Aggarwal V, Giordano J, et al: Whole-exome sequencing in the evaluation of fetal structural anomalies: A prospective cohort study, Lancet 393(10173):758–767, 2019. Pratt M, Garritty C, Thuku M, et al: Application of exome sequencing for prenatal diagnosis: A rapid scoping review, Genet Med 22: 1925–1934, 2020. Vossaert L, Chakchouk I, Zemet R, et al: Overview and recent developments in cell-based noninvasive prenatal testing, Prenat Diagn 41:1202–1214, 2021. Wapner RJ, Martin CL, Levy B, et al: Chromosomal microarray versus karyotyping for prenatal diagnosis, N Engl J Med 367:2175–2184, 2012. Zhang J, Li J, Saucier J, et al: Non-invasive prenatal sequencing for multiple mendelian monogenic disorders using circulating cell-free fetal DNA, Nat Med 25:439–447, 2019. PROBLEMS 1. Match the term in the top section with the appropriate comment in the bottom section. a. Cell-free DNA b. 10th week of pregnancy c. Cordocentesis d. Mosaicism e. 16th week of pregnancy f. α-fetoprotein in maternal serum g. Aneuploidy h. Cystic hygroma i. Amniotic fluid ________ method of obtaining fetal blood for karyotyping ________ usual time at which amniocentesis is performed ________ increased level when fetus has NTD ________ contains fetal cells viable in culture ________ major cytogenetic problem in prenatal diagnosis ________ ultrasonographic diagnosis indicates possible Turner syndrome ________ risk increases with maternal age ________ earliest time at which CVS can be performed ________ used to screen for aneuploidy starting at 10 weeks of gestation 2. A couple has a child with Down syndrome, who has a 21q21q translocation inherited from the mother. Could prenatal diagnosis be helpful in the couple’s next pregnancy? Explain. 3. Cultured cells from a chorionic villus sample show two cell lines: 46,XX and 46,XY. Does this necessarily mean the fetus is abnormal? Explain. 4. What two main types of information about a fetus can be indicated (although not proven) by assay of AFP, h CG, and u E3 in maternal serum during the second trimester? 5. A young woman consults a geneticist during her first pregnancy. Her brother was previously diagnosed with Duchenne muscular dystrophy and had since died. He was the only affected person in her family. The woman had been tested biochemically and found to have elevated creatine kinase levels, indicating she is a carrier of the disease. Unfortunately, no DNA analysis had been conducted on the woman’s brother to determine what type of altered DMD gene he had. a. What other testing can be done on her to evaluate her risk for a child with DMD? b. Can information from that test be used to diagnose her pregnancy? 6. Discuss the relative advantages and disadvantages of the following diagnostic procedures, and cite types of disorders for which they are indicated or not indicated: amniocentesis, CVS, first trimester maternal serum screening, second trimester screening, noninvasive screening of cell- free fetal DNA (NIPS). 7. A second trimester anatomy ultrasound exam is performed on a 30-year-old primigravida and reveals the following findings: Congenital diaphragmatic hernia, short femur at the 5th percentile, small ventricular septal defect. No other anomalies are found. You counsel her about the risk for chromosomal abnormalities and she agrees to have an amniocentesis. CMA and karyotype results are unremarkable. a. What follow-up test can you offer at this time? Discuss the next level of testing and consider what you should tell her about the benefits, detection rates, potential risks associated with this level of testing? Does the patient have options about disclosing certain types of information? b. What treatment options can you counsel her about for this defect, and how would you consider genetic testing results in the decision for treatments? ▶

CHAPTER 18 — Preconception and Prenatal Screening and Diagnosis 409 Fan HC, Gu W, Wang J, et al: Non-invasive prenatal measurement of the fetal genome, Nature 487:320–324, 2012. Grati FR, Ferreira J...

Chapter 19: Application of Genomics to Medicine and Individualized Health Care

Ch19 · Pt1 chapter 19 Application of Genomics to Medicine and Individualized Health Care Ronald Doron Cohn ▶

Iris Cohn The last several chapters have been dedicated to introducing various aspects of the applications of modern genomics to the practice of medicine. In Chapter 16 we described powerful new genom...

Ch19 · Pt2 412 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Many of the general issues concerning genetic screening are highlighted by newborn screening programs. A determination of the appropriateness of newborn screening for any particular condition is based on a standard set of criteria involving clinical validity and clinical utility (see Box 19.1). The design of newborn screening tests includes keeping false-negative rates low so that true-positive cases are not missed, without making the test so nonspecific as to drive the false-positive rate unacceptably high. False-positive results cause 600 500 400 300 200 100 0 30 35 45 65 40 55 60 50 70 Age (yr) No family history Family history Threshold for screening Cumulative incidence (cases/10,000) Figure 19.1 Cumulative incidence (per 10,000) of colon cancer versus age in individuals with and without a family history of the disease. Data from Fuchs CS, Giovannucci EL, Colditz GA, et al: A prospective study of family history and the risk of colorectal cancer, N Engl J Med 331:1669–1674, 1994. BOX 19.1 GENERAL CRITERIA FOR AN EFFECTIVE NEWBORN SCREENING PROGRAM Analytic Validity A rapid and economic laboratory test is available that detects the appropriate metabolite. Clinical Validity The laboratory test is highly sensitive (no false negatives) and reasonably specific (few false positives). Positive predictive value is high. Clinical Utility Treatment is available. Early institution of treatment, before symptoms become manifest, reduces or prevents severe illness. Routine observation and physical examination will not reveal the disorder in the newborn – a test is required. The condition is frequent and serious enough to justify the expense of screening; that is, screening is cost effective. The public health system infrastructure is in place to inform the newborn’s parents and physicians of the results of the screening test, to confirm the test results, and to institute effective treatment and counseling. unnecessary anxiety to the parents and increase costs, because more unaffected infants have to be recalled for retesting. At the other extreme, false-negative results vitiate the purpose of a screening program. In deciding whether to institute screening for any given condition, consideration must be given to the ability of the public health system infrastructure to handle the care of affected newborns so identified through such screening. The prototype condition that satisfies all of these criteria is phenylketonuria (see Chapter 13). For decades, finding elevated levels of phenylalanine in a spot of blood on filter paper obtained soon after birth has been the mainstay of neonatal screening for phenylketonuria and other forms of hyperphenylalaninemia. Currently, this applies throughout North America and Europe, most of Latin America and much of Asia Pacific (see Chapter 13). A positive screen result, followed by definitive confirmation of the diagnosis, leads to the institution of dietary phenylalanine restriction early in infancy, thereby preventing irreversible intellectual disability. Two other conditions that are widely targeted for newborn screening are congenital hearing loss and congenital hypothyroidism. Newborn screening for hearing loss is mandated throughout the United States and Canada. Approximately half of all congenital deafness is due to single-gene defects (Case 13). Infants found to have hearing impairments by newborn screening are offered intervention with sign language, cochlear implants, and other communication aids early in life, meant to improve their long-term language skills and intellectual abilities beyond those seen if the impairment is discovered later in childhood. Screening for congenital hypothyroidism, a disorder whose genetic basis is known in only 10 to 15% of cases, but is easily treatable, is universal in the United States and Canada and routine in many other countries. Thyroid hormone replacement therapy started early in infancy completely prevents the severe and irreversible intellectual disability caused by congenital hypothyroidism. Thus, both hypothyroidism and congenital hearing loss easily fulfill the criteria for newborn screening. A number of other disorders, such as galactosemia, sickle cell disease (Case 42), biotinidase deficiency (see Chapter 13), severe combined immunodeficiency, and congenital adrenal hyperplasia (see Chapter 6), are part of many or most neonatal screening programs. Which disorders should be the target of newborn screening varies among jurisdictions. In the United States, Recommended Universal Screening Panel (RUSP) is a national guideline listing conditions for which the U. S. Secretary of Health and Human Services recommends all newborns be tested. Standards for newborn screening differ widely around the globe. Which disorders should be the target of newborn screening varies from province to province in Canada without a national consensus. As of 2022, the United Kingdom’s national program to screen ▶

412 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Many of the general issues concerning genetic screening are highlighted by newborn screening programs. A determination of the appropriatenes...

Ch19 · Pt3 CHAPTER 19 — Application of Genomics to Medicine and Individualized Health Care 413 newborns across all jurisdictions included just nine disorders. Tandem Mass Spectroscopy For many years, most newborn screening was performed by a test specific for each individual condition. For example, phenylketonuria screening was based on a microbial or a chemical assay that tested for elevated phenylalanine level. This situation has changed dramatically with the application of the technology of tandem mass spectrometry (TMS). Not only can a neonatal blood spot be examined accurately and rapidly for an elevation of phenylalanine, with fewer false positives than with the older testing methods, but TMS analysis can simultaneously detect a few dozen other biochemical disorders. Some of these, such as homocystinuria (see Chapter 13) or maple syrup urine disease, were already being screened for by individual tests (Table 19.1). TMS, however, does not replace the disease- specific testing methods for other disorders currently included in some newborn screening, such as galactosemia, biotinidase deficiency, congenital adrenal hyperplasia, and sickle cell disease. TMS also provides a reliable method for newborn screening for some disorders that fit the criteria for screening but had no reliable newborn screening program in place. For example, medium-chain acyl-Co A dehydrogenase (MCAD) deficiency is a disorder of fatty acid oxidation that is usually asymptomatic but manifests clinically when the patient becomes catabolic. Detection of MCAD deficiency at birth can be lifesaving. Affected infants and children are at very high risk for life-threatening hypoglycemia in early childhood during the catabolic stress caused by an intercurrent illness, such as a viral infection; nearly 25% of children with undiagnosed MCAD deficiency will die with their first episode of hypoglycemia. The metabolic derangement can be successfully managed if it is treated promptly. In MCAD deficiency, alerting parents and physicians to the risk for metabolic decompensation is the primary goal of screening. The children are healthy between attacks and do not require daily management other than avoidance of prolonged fasting. TMS provides a rapid test for many disorders for which newborn screening is already being done or TABLE 19.1 Disorders Detectable by Tandem Mass Spectrometry A. Amino Acid Disorders Classical phenylketonuria (PKU) Variant PKU Guanosine triphosphate cyclohydrolase 1 (GTPCH) deficiency (biopterin deficiency) 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency (biopterin deficiency) Dihydropteridine reductase (DHPR) deficiency (biopterin deficiency) Pterin-4α-carbinolamine dehydratase (PCD) deficiency (biopterin deficiency) Argininemia/arginase deficiency Argininosuccinic acid lyase deficiency (ASAL deficiency) Citrullinemia, type I/argininosuccinic acid synthetase deficiency (ASAS deficiency) Citrullinemia, type II (citrin deficiency) Gyrate atrophy of the choroid and retina Homocitrullinuria, hyperornithinemia, hyperammonemia (HHH) Homocystinuria/cystathionine beta-synthase deficiency (CBS deficiency) Methionine adenosyltransferase deficiency (MAT deficiency) Maple syrup urine disease (MSUD) Prolinemia Tyrosinemia, types I, II, III, and transient Ornithine transcarbamylase deficiency (OTC deficiency) Remethylation defects (MTHFR, MTR, MTRR, Cbl D v 1, Cbl G deficiencies) B. Organic Acid Disorders 2-methyl-3-hydroxybutyryl-Co A dehydrogenase deficiency 2-methylbutyryl-Co A dehydrogenase deficiency 3-hydroxy-3-methylglutaryl-Co A lyase deficiency (HMG Co A lyase deficiency) 3-methylcrotonyl-Co A carboxylase deficiency (3MCC deficiency) 3-methylglutaconic aciduria (MGA), type I (3-methylglutaconyl-Co A hydratase deficiency) Beta-ketothiolase (BKT) deficiency Ethylmalonic encephalopathy (EE) Glutaric acidemia type-1 (GA-1) Isobutyryl-Co A dehydrogenase deficiency Isovaleric acidemia (IVA) Malonic aciduria Methylmalonic acidemia, mut – Methylmalonic acidemia, mut 0 Methylmalonic acidemia (Cbl A, B) Methylmalonic acidemia (Cbl C, D) Multiple carboxylase deficiency (MCD) Propionic acidemia (PA) C. Fatty Acid Oxidation Disorders Carnitine transporter deficiency Carnitine-acylcarnitine translocase deficiency (CAT deficiency) Carnitine palmitoyltransferase deficiency-type 1 (CPT-1 deficiency) Carnitine palmitoyltransferase deficiency-type 2 (CPT-2 deficiency) Long chain hydroxyacyl-Co A dehydrogenase deficiency (LCHAD deficiency) Medium chain acyl-Co A dehydrogenase deficiency (MCAD deficiency) Medium/short chain L-3-hydroxy acyl-Co A dehydrogenase deficiency (M/SCHAD deficiency) Multiple acyl-Co A dehydrogenase deficiency (MAD deficiency)/glutaric acidemia type-2 (GA-2) Short chain acyl-Co A dehydrogenase deficiency (SCAD deficiency) Trifunctional protein deficiency (TFP deficiency) Very long chain acyl-Co A dehydrogenase deficiency (VLCAD deficiency) Formiminoglutamic acid (FIGLU) disorder Cbl, Cobalamin; MTHFR, methylene tetrahydrofolate reductase; MTR, 5-methyltetrahydrofolate-homocysteine methyltransferase; MTRR, methionine synthase reductase. Modified from California Newborn Screening Program, http://www.cdph.ca.gov/programs/nbs/Documents/NBS-Disorders Detectable 011312.pdf. ▶

CHAPTER 19 — Application of Genomics to Medicine and Individualized Health Care 413 newborns across all jurisdictions included just nine disorders. Tandem Mass Spectroscopy For many years, most newbor...

Ch19 · Pt4 414 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE can easily be justified. TMS also identifies infants with inborn errors—such as ethylmalonic acidemia—that have not generally been the targets of newborn screening because of their rarity and difficulty of providing definitive therapy to prevent the progressive neurologic impairment. TMS can also identify abnormal metabolites whose significance for health are uncertain. For example, short-chain acyl-Co A dehydrogenase (SCAD) deficiency, another disorder of fatty acid oxidation, is most often asymptomatic, although a few individuals may have difficulties with episodic hypoglycemia. Thus, a positive TMS screen result is not particularly predictive of developing symptomatic SCAD later in life. Although TMS can identify many metabolic disorders, does the benefit of detecting disorders such as SCAD deficiency outweigh the negative impact of raising parental concern for most newborns whose test result is positive but who will never be symptomatic? Thus not every disorder detected by TMS fits the criteria for newborn screening. Some public health experts argue that only those metabolites of proven clinical relevance should be reported to parents and physicians. PHARMACOGENOMICS One area of medicine that is receiving a lot of attention for potential application of genomics to individualized medical care is pharmacogenomics: the study of how genetic variation among individuals affects the response to medication therapy. The development of a genetic profile that predicts efficacy, toxicity, or an adverse drug reaction is likely to have clinical significance. It allows health care professionals to choose a drug from which the patient will benefit—by reducing the risk for an adverse event—or to decide on a dosage that ensures adequate therapy and minimizes complications. The US Food and Drug Administration (FDA) has recognized the importance of pharmacogenetic variation in individual response to drug treatment by including pharmacogenetic information on the labels that come with a broad range of pharmaceuticals (Table 19.2). As with all other aspects of personalized medicine, however, the cost effectiveness of such testing must be proved if it is to become part of accepted medical care. There are two ways that genetic variation affects drug therapy. The first is the effect of variation on pharmacokinetics; that is, the rate at which the body absorbs, transports, metabolizes, or excretes drugs and/or their metabolites. The second is the variation affecting pharmacodynamics; that is, differences in the way the body responds to a drug. This can involve biochemical, physiologic, and molecular effects of drugs on the body and can include receptor binding (including sensitivity) and chemical interactions. The terms pharmacogenetics and pharmacogenomics can be used interchangeably, although historically TABLE 19.2 Gene-Drug Combinations for Which There Is Pharmacogenetic Information in Their US Food and Drug Administration Package Inserts* Gene Drug(s) CYP2C19 Clopidogrel, voriconazole, omeprazole, pantoprazole, esomeprazole, diazepam, nelfinavir, rabeprazole CYP2C9 Celecoxib, warfarin CYP2D6 Atomoxetine, venlafaxine, risperidone, tiotropium bromide inhalation, tamoxifen, timolol maleate, fluoxetine, cevimeline, tolterodine, terbinafine, tramadol and acetaminophen, clozapine, aripiprazole, metoprolol, propranolol, carvedilol, propafenone, thioridazine, protriptyline, tetrabenazine, codeine DPYD Capecitabine, fluorouracil G6PD Rasburicase, dapsone, primaquine, chloroquine HLA-B*1502 Carbamazepine HLA-B*5701 Abacavir (Case 1) NAT Rifampin, isoniazid, and pyrazinamide; isosorbide dinitrate and hydralazine hydrochloride TPMT Azathioprine, thioguanine, mercaptopurine UGT1A1 Irinotecan, nilotinib VKORC1 Warfarin *Constitutional variants only; chemotherapy whose usage is affected by somatic variants are not included. pharmacogenetics referred to variations in a single gene influencing drug response and pharmacogenomics referred to the sum total of all relevant genetic variation that determine drug behavior. Variation in Pharmacokinetic Response Variation in Drug Metabolism: The Example of Cytochrome P-450 The human cytochrome P-450 proteins are a large family of at least 57 different functional enzymes, each encoded by a different CYP gene. The cytochromes P-450 are grouped into 18 families according to amino acid sequence homology. They code for enzymes. Three of these families, CYP1, CYP2, and CYP3, are particularly active in the detoxification of exogenous chemicals (xenobiotics), such as drugs. Four cytochrome P-450 genes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5) are especially important because the enzymes they encode are responsible for the metabolism of about 75 to 80% of all commonly used drugs (Fig. 19.2). For many drugs, the action of a cytochrome P-450 is to begin the process of detoxification through a series of reactions (oxidation) that render the drug less active and easier to excrete. Some drugs, however, are themselves inactive prodrugs whose conversion into an active metabolite by a cytochrome P-450 is required for the drug to have any therapeutic effect. Many of the CYP genes important for drug metabolism (including CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) are highly polymorphic, with ▶

414 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE can easily be justified. TMS also identifies infants with inborn errors—such as ethylmalonic acidemia—that have not generally been the targe...

Ch19 · Pt5 CHAPTER 19 — Application of Genomics to Medicine and Individualized Health Care 415 alleles that result in absent, decreased, or increased enzyme activity. Variants affect the rate at which many drugs are metabolized, with real functional consequences for how individuals respond to drug therapy. As one example, CYP2D6, the primary cytochrome in the metabolism of more than 70 different drugs, has dozens of reduced, absent, or increased activity alleles, leading to normal, poor, intermediate, or ultrafast metabolism (see table on metabolizer phenotypes). Missense variants decrease the activity of this cytochrome; alleles with no activity are caused by splicing or frameshift variants. In contrast, the CYP2D6*1XN allele is actually a series of copy number variant alleles in which the CYP2D6 gene is present in three, four, or more copies on one chromosome. Predictably, these larger copy number variants produce high levels of the enzyme. There are dozens more alleles that do not affect the function of the protein and are considered to be wild type. Various combinations of these four classes of alleles produce quantitative differences in metabolizing activity, resulting in four main phenotypes: normal (also called extensive) metabolizers, intermediate metabolizers, poor metabolizers, and ultrafast metabolizers (Fig. 19.3). Depending on whether a drug is itself an active compound or is a prodrug that requires activation by a cytochrome P-450 enzyme to have its pharmacologic effect, poor metabolizers may either accumulate toxic levels of the drug or fail to have therapeutic efficacy because of poor activation of a prodrug. In contrast, ultrafast metabolizers are at risk for being undertreated by a drug with doses inadequate to maintain blood levels in the therapeutic range, or they may suffer overdose due to too rapid conversion of a prodrug to its active metabolite. For example, codeine is a weak narcotic drug that exerts most of its analgesic effect on conversion to morphine, a bioactive metabolite with a 10-fold higher potency. This conversion is carried out by the CYP2D6 enzyme. Poor metabolizers—quite common in some populations, carrying loss-of-function alleles at the CYP2D6 locus fail to convert codeine to morphine, thereby receiving little therapeutic benefit; in contrast, ultrafast metabolizers can become rapidly intoxicated with low doses of codeine. A number of children have died from codeine overdoses due to having an ultrafast metabolizer phenotype. As with many forms of genetic variation (see Chapter 10), the frequency of many of the alleles in the cytochromes P-450 differs among different populations (Table 19.3). For example, a slow metabolizing phenotype for CYP2D6 that is present in 1 in 14 individuals of European ancestry is rare in Asia and nearly absent in Native Americans and Pacific Islanders. Similarly, slow metabolizing alleles at CYP2C19 show striking population variability, with 1 in 33 individuals of European descent but nearly 1 in 6 Asians having slow metabolism. These differences in the frequency of poor and ultrarapid metabolizers are important for the delivery of individualized genetic medicine in heterogeneous populations. Other 4% CYP2D6 12% CYP2C19 13% CYP2C9 17% CYP1A2 8% CYP1A1 3% CYP3A4 43% CYP3A4 CYP1A1 CYP1A2 CYP2C9 CYP2C19 CYP2D6 Other Figure 19.2 Contribution of individual cytochrome P-450 enzymes to drug metabolism. Modified with permission from Guengerich F: Cytochrome P450s and other enzymes in drug metabolism and toxicity, AAPS J 8:E101–E111, 2006. Time A: POOR METABOLIZER Therapeutic range Time B: NORMAL METABOLIZER Drug level (plasma) Time C: ULTRAFAST METABOLIZER Drug level (plasma) Drug level (plasma) Figure 19.3 Serum drug levels after repeated doses of a drug (arrows) in three individuals with different phenotypic profiles for drug metabolism. (A) Poor metabolizer accumulates drug to toxic levels. (B) Normal (extensive) metabolizer reaches steady-state levels within the therapeutic range. (C) Ultrafast metabolizer fails to maintain serum levels within the therapeutic range. ▶

CHAPTER 19 — Application of Genomics to Medicine and Individualized Health Care 415 alleles that result in absent, decreased, or increased enzyme activity. Variants affect the rate at which many drugs...

Ch19 · Pt6 416 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE TABLE 19.3 Frequency of Poor CYP2D6 and CYP2C19 Metabolizers in Various Population Groups Population Frequency of Poor Metabolizers (%) Origin of Population CYP2D6 CYP2C19 Sub-Saharan Africa 3.4 4.0 Native American 0 2 Asian 0.5 15.7 European 7.2 2.9 Middle Eastern/North Africa 1.5 2.0 Pacific Islander 0 13.6 Data from Burroughs VJ, Maxey RW, Levy RA: Racial and ethnic differences in response to medicines: towards individualized pharmaceutical treatment, J Natl Med Assoc 94(Suppl):1–26, 2002. Metabolizer Phenotypes Arising from Various Combinations of CYP2D6 Alleles Metabolizer Status Alleles and Activity Anticipated Response (compared to average world population) Normal (extensive) Two normal activity alleles One normal activity allele and one decreased activity allele One increased activity allele and one decreased functional allele Typical metabolism Intermediate One normal activity allele and one nonfunctional activity allele Two decreased activity alleles One decreased activity allele and one non-functional activity allele Decreased metabolism Poor Two nonfunctional activity alleles Little or no metabolism Ultrarapid Two increased activity alleles Increased metabolism Clinical pharmacogenetics—the use of genetic data to guide drug therapy decisions, is supported by professional societies such as the Clinical Pharmacogenetics Implementation Consortium (CPIC), the Royal Dutch Association for the Advancement of Pharmacy– Pharmacogenetic Working Group (DPWG), the Canadian Pharmacogenomic Network for Drug Safety, and others. They established guidance on pharmacogenes to provide actionable recommendations for the use of genomic information in a consistent manner. Additionally, the FDA has recognized the importance of pharmacogenetic variation in individual response to drug treatment by including pharmacogenetic information on the labels that come with a broad range of pharmaceuticals (see Table 19.2). As with all other aspects of individualized medicine, further cost-effectiveness studies of such testing must be provided if it is to become part of accepted medical care. Variation in Pharmacodynamic Response Malignant Hyperthermia Malignant hyperthermia is a rare autosomal dominant condition in which there may be a dramatic adverse response to the administration of many commonly used inhalational anesthetics (e.g., halothane) and depolarizing muscle relaxants (e.g., succinylcholine). Soon after induction of anesthesia, a patient develops life-threatening fever, sustained muscle contraction, and attendant hypercatabolism. The fundamental physiologic abnormality in the disease is an elevation of the level of ionized calcium in the sarcoplasm of muscle. This increase leads to muscle rigidity, elevation of body temperature, rapid breakdown of muscle (rhabdomyolysis), and other abnormalities. The condition is an important, if not a common cause of death during anesthesia. The incidence is 1 in 50,000 adults undergoing anesthesia but for unknown reasons is 10-fold higher in children. Malignant hyperthermia is most frequently associated with pathogenic variants in a gene called RYR1, encoding an intracellular calcium ion channel. However, variants in RYR1 account for only approximately half of cases of malignant hyperthermia. At least five other loci have now been identified, one of which is the CACNA1S gene, which encodes the α1 subunit of a dihydropyridine- sensitive calcium channel. Precisely why the abnormalities in calcium handling in muscle found with RYR1 or CACNA1S variants make the muscle sensitive to inhalation anesthetics and muscle relaxants and precipitate malignant hyperthermia is unknown. The need for special precautions when at-risk persons require anesthesia is obvious. Cooling blankets, muscle relaxants, and cardiac antiarrhythmics may all be used to prevent or reduce the severity of the response if an unsuspected attack occurs, and alternative anesthetics can be given to patients at risk. Pharmacogenomics guidelines are available to help guide clinical decision making. Adverse Drug Reactions The majority (75–80%) of adverse drug events result from predictable, nonimmunologic drug toxicities such as overdoses caused by medication errors, renal or hepatic disease, or drug-drug interactions. Most nonpredictable adverse drug events are thought to have a genetic component related to drug-gene interactions that contribute to about one-third of potential major or substantial drug interactions that occur in patients. Of these, ~25 to 50% are true Ig E-mediated drug hypersensitivity reactions (HSRs), including life- threatening anaphylaxis characterized by sudden onset of laryngeal edema, leading to occlusion of the airway, marked hypotension, and cardiac arrhythmias. The remaining 50 to 75% of adverse drug reactions are genetically determined nonallergic immune reactions ▶

416 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE TABLE 19.3 Frequency of Poor CYP2D6 and CYP2C19 Metabolizers in Various Population Groups Population Frequency of Poor Metabolizers (%) Orig...

Ch19 · Pt7 CHAPTER 19 — Application of Genomics to Medicine and Individualized Health Care 417 (i.e., HSR). These manifest as widespread damage to skin and mucous membranes, referred to as Stevens-Johnson syndrome (SJS) and (in its more serious extreme form), toxic epidermal necrolysis (TEN) (Case 1). Although rare, TEN is a very serious adverse drug reaction that causes denuding of large areas of skin and carries a mortality rate of 30 to 40%. There is a strong correlation between particular drugs and certain human leukocyte antigen (HLA) alleles in the major histocompatibility complex (see Chapter 9) that result in SJS and TEN. For example, individuals who take the retroviral drug abacavir and carry the HLA-B*5701 allele have a 50% risk for SJS or TEN, leading to the introduction of HLA-B*5701 typing as a standard of care screening tool prior to prescribing abacavir. Because ~5 to 8% of Europeans carry the HLA-B*5701 allele, the risk for a severe drug reaction in abacavir-treated patients from this population is especially significant. HLA-B*5701 screening has a negative predictive value of 100% and a positive predictive value of 47.9% for immunologically confirmed HSR (i.e., positive result on epicutaneous patch testing 6 to 10 weeks after clinical diagnosis), as demonstrated by one study. A similar situation exists with the use of the antiseizure medication carbamazepine and HLA-B*1502, which is present in (see Table 19.3). PHARMACOGENOMICS AS A COMPLEX TRAIT The examples of pharmacogenomics provided in this chapter primarily involve variation at single genes and its effect on drug treatment. In truth, most drug response is a complex trait. A drug may have its effect directly or through more active metabolites, each of which may then be metabolized by different pathways and exert its effects on various targets. Thus, variants at more than one locus may interact, synergistically or antagonistically, either to potentiate or to reduce the effectiveness of a drug or to increase its toxic side effects. Further research is required to create a comprehensive pharmacogenomic profile that takes into account multiple genetic variants as well as other factors, to offer precise and predictive information to guide drug therapy. Other factors include environmental effects, interactions within the biologic system, disease state, and drug interactions, The ultimate goal is for a patient to receive the best drug at the right dose and avoid potentially dangerous side effects. We expect pharmacogenomics to become increasingly important in the delivery of individualized, precision medicine in the years ahead. SCREENING FOR GENETIC SUSCEPTIBILITY TO DISEASE Genetic Epidemiology Epidemiologic studies of risk factors for disease rely on population studies that measure disease prevalence or incidence and determine whether certain risk factors (e.g., genetic, environmental, social) are more prevalent in individuals with disease than those without. Genetic epidemiology is concerned with how genotypes and environmental factors interact to increase or decrease susceptibility to disease. Epidemiologic studies generally follow one of three different strategies: case-control, cross-sectional, and cohort design (see Box 19.2). BOX 19.2 STRATEGIES USED IN GENETIC EPIDEMIOLOGY Case-control: Individuals with and without the disease are selected, and the genotypes and environmental exposures of individuals in the two groups are determined and compared. Cross-sectional: A random sample of the population is selected and divided into those with and without the disease, and their genotypes and environmental exposures are determined and compared. Cohort: A sample of the population is selected and observed for some time to ascertain who does or does not develop disease, and their genotypes and environmental exposures are determined and compared. The cohort may be selected at random or may be targeted to individuals who share a genotype or an environmental exposure. Cohort and cross-sectional studies not only capture information on the relative risk conferred by different genotypes but, if they are random population samples, also provide information on the prevalence of the disease and the frequency of the various genotypes under study. A randomly selected cohort study, in particular, is the most accurate and complete approach, in that phenotypes that take time to appear have a better chance of being detected and scored; they are, however, more expensive and time consuming. Cross-sectional studies, on the other hand, suffer from underestimation of the frequency of the disease. First, if the disease is rapidly fatal, many of those with disease and carrying a risk factor will be missed. Second, if the disease shows age-dependent penetrance, some individuals carrying a risk factor will not be scored as having the disease. Case-control studies, on the other hand, allow researchers to efficiently target individuals, particularly with relatively rare phenotypes for which very large sample sizes would be needed in a cross-sectional or cohort study. However, unless a study is based on complete ascertainment of individuals with a disease (e.g., in a population register or surveillance program) or uses a random sampling scheme, a case-control study cannot capture information on the population prevalence of the disease. Disease Association A genetic disease association is the relationship in a population between a susceptibility or protective genotype and a disease phenotype (see Chapter 10). The ▶

CHAPTER 19 — Application of Genomics to Medicine and Individualized Health Care 417 (i.e., HSR). These manifest as widespread damage to skin and mucous membranes, referred to as Stevens-Johnson syndr...

Ch19 · Pt8 418 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE susceptibility or protective genotype can be an allele (in either a heterozygote or a homozygote), a genotype at one locus, a haplotype containing alleles at neighboring loci, or even combinations of genotypes at multiple unlinked loci. Whether a disease association between genotype and phenotype is statistically significant can be determined from standard statistical tests, such as the chi-square test; whereas, how strongly associated the genotype and phenotype are is given by the odds ratio or relative risk, as discussed in Chapter 10. The relationship between some of these concepts is best demonstrated by means of a 2 × 2 table. Determination of the Predictive Value of a Test Disease Genotype Affected Unaffected Total Susceptibility genotype present a* b a+b Susceptibility genotype absent c d c+d Total a+c b+d a+b+c+d=N Frequency of the susceptibility genotype = (a + b)/N Disease prevalence = (a + c)/N (with random sampling or a complete population survey) Relative Risk: = + + a/(a b) c/(c d) RR Disease prevalence in carriers of susceptibility genotype Dise = ase prevalence in noncarriers of susceptibility genotype Sensitivity: Fraction of individuals with disease who have the susceptibility genotype = a/(a + c) Specificity: Fraction without disease who do not have the susceptibility genotype = d/(b + d) Positive predictive value: Proportion of individuals with the susceptibility genotype who have or will develop a particular disease = a/(a + b) Negative predictive value: Proportion of individuals without the susceptibility genotype who do not have or will not develop a particular disease = d/(c + d) *The values of a, b, c, and d are derived from a random sample of the population, divided into those with and without the susceptibility genotype, and then examined for the disease (with or without longitudinal follow-up, depending on whether it is a cross-sectional or cohort study) (see later). Clinical Validity and Utility Finding the genetic contributions to health and disease is of obvious importance for research into underlying disease etiology and pathogenesis, as well as for identifying potential targets for intervention and therapy. In medical practice, however, whether to screen individuals for increased susceptibilities to illness depends on the clinical validity and clinical utility of the test. That is, how predictive of disease is a positive test, and how useful is it to have this information? Clinical Validity Clinical validity is the extent to which a test result is predictive for disease. Clinical validity is captured by the two concepts of positive predictive value and negative predictive value. The positive predictive value is the frequency with which a group of individuals who test positive have or will develop the disease. For mendelian disorders, the positive predictive value of a genotype is the penetrance. Conversely, the negative predictive value is the frequency with which a group of individuals who test negative are free of disease and remain so. When faced with a unique patient, the practitioner of individualized genetic medicine needs to know more than just whether there is an association and its magnitude (i.e., relative risk or odds ratio). It is important to know clinical validity (i.e., how well the test predicts the presence or absence of disease). Susceptibility Testing Based on Genotype The positive predictive value of a genotype that confers susceptibility to a particular disease depends on the relative risk for disease conferred by one genotype over another and on the prevalence of the disease. Fig. 19.4 provides the positive predictive value for genotype frequencies ranging from 0.5% (rare) to 50% (common), which confer a relative risk that varies from low (2-fold) to high (100-fold), when the prevalence of the disease ranges from relatively rare (0.1%) to more common (5%). As the figure shows, the value of the test as a predictor of disease increases substantially when one is dealing with a common disorder due to a relatively rare susceptibility genotype that confers a high relative risk, compared with the risk for individuals who do not carry the genotype. The converse is also clear; testing for a common genotype that confers a modest relative risk is of limited value as a predictor of disease. We will illustrate the use of the 2 × 2 table in assessing the role of susceptibility alleles in a common disorder, colorectal cancer. Shown in the following Box are data from a population-based study of colorectal cancer risk conferred by common variant in the APC gene (see Chapter 16) that changes isoleucine to lysine at position 1307 of the protein (p. Ile 1307Lys). This variant has an allele frequency of ~3.1% among those of Ashkenazi Jewish ancestry, which means that ~1 in 17 such individuals is a heterozygote (and 1 in 1000 are homozygous) for the allele. The prevalence of colon cancer among this population is 1%. The common p. Ile 1307Lys variant confers a 2.4-fold increased risk for colon cancer relative to individuals without the allele. However, the small positive predictive value (≈2%) means that an individual who tests positive for this allele has only a 2% chance of developing colorectal cancer. If this had been a cohort study that allowed complete ascertainment of everyone in whom colorectal cancer was going to develop, the penetrance would, in effect, be only 2%. Clinical Utility The clinical utility of a test is more difficult to assess than clinical validity because it has different meanings for different people. In its narrowest sense, the clinical ▶

418 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE susceptibility or protective genotype can be an allele (in either a heterozygote or a homozygote), a genotype at one locus, a haplotype cont...

Ch19 · Pt9 CHAPTER 19 — Application of Genomics to Medicine and Individualized Health Care 419 utility of a test is that the result is medically actionable; that is, the result will change medical care for an individual and, as a consequence, will improve the outcome of care, both medically and economically. At the other end of the spectrum is the broader definition as any piece of 100 90 80 70 60 50 40 30 20 10 0 Relative risk 0.5% Relative risk 5% Genotype frequency Positive predictive value Disease prevalence Relative risk 50% 100 20 2 100 20 2 5% 0.1% 1% 100 20 2 Figure 19.4 Theoretical positive predictive value calculations for a susceptibility genotype for a disease, over a range of genotype frequencies, disease prevalences, and relative risks for disease conferred by the genotype. BOX 19.3 THE P. ILE1307LYS ALLELE OF THE APC GENE AND COLON CANCER Colon Cancer Allele Affected Unaffected Total Lys 1307 7 310 317 Ile 1307 38 4142 4180 Total 45 4452 4497 Relative Risk Disease prevalence in allele carriers Diseasep = revalence in non-carriers / / = = 7 317 38 4180 2 4. Sensitivity: Fraction of individuals with colon cancer who have the Lys 1307 allele = 7/45 = 16% Specificity: Fraction without colon cancer who do not have the Lys 1307 allele = 4142/4452 = 93% Positive predictive value: Fraction of individuals with the Lys 1307 allele who develop colon cancer = 7/317 = 2% Negative predictive value: Fraction of individuals without the Lys 1307 allele who do not develop colon cancer = 99% Data from Woodage T, King SM, Wacholder S, et al: The APC I1307K allele and cancer risk in a community-based study of Ashkenazi Jews, Nat Genet 20:62–65, 1998. information an individual might wish to have, for any reason, including simply for the sake of knowing. In a person who tests positive for the APC Ile 1307Lys allele, how does a positive predictive value of 2% translate into clinical utility for medical practice? (see Box 19.3) One critical factor is a public health economic one: can the screening be shown to be cost effective? Is the expense of the testing outweighed by improving health outcomes while reducing health care costs, disability, and loss of earning power? In the example of screening for the APC p. Ile 1307Lys allele in those of Ashkenazi ancestry, more frequent screening or the use of different approaches to screening for colon cancer may be effective. Screening methods (occult stool blood testing vs fecal DNA testing, or sigmoidoscopy vs full colonoscopy) differ in expense, sensitivity, specificity, and potential for hazard; deciding which regimen to follow has important implications for the person’s health and health care costs. Even with demonstrable clinical validity and actionable clinical utility, to demonstrate that testing improves health care is not always straightforward. For example, 1 in 200 to 1 in 250 individuals of European ancestry are homozygous for a p. Cys 282Tyr variant in the HFE gene associated with hereditary hemochromatosis: a disorder characterized by iron overload that can silently lead to extensive liver damage and cirrhosis (Case 20, Chapter 7, Chapter 11). A simple intervention – regular phlebotomy to reduce total body iron stores – can prevent hepatic cirrhosis. The susceptibility genotype is common, and 60 to 80% of p. Cys 282Tyr homozygotes show biochemical evidence of increased body iron ▶

CHAPTER 19 — Application of Genomics to Medicine and Individualized Health Care 419 utility of a test is that the result is medically actionable; that is, the result will change medical care for an in...

Ch19 · Pt10 420 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE stores. This suggests that screening would be a reasonable and cost-effective measure to identify asymptomatic individuals who should undergo further testing and, if indicated, the institution of regular phlebotomy. However, most p. Cys 282Tyr homozygotes (>90–95%) remain clinically asymptomatic, leading to the argument that the positive predictive value of HFE gene testing for liver disease in hereditary hemochromatosis is too low to justify population screening. Nonetheless, some of these largely asymptomatic individuals do have signs of clinically occult fibrosis and cirrhosis on liver biopsy, indicating that the Cys 282Tyr homozygote may actually be at a higher risk for liver disease than previously thought. Thus, some argue for population screening to identify individuals in whom regular prophylactic phlebotomy should be instituted. The clinical utility of such population screening remains controversial and will require additional research to determine the natural history of the disease and whether the silent fibrosis and cirrhosis seen on liver biopsy represent the early stages of a progressive illness. As of 2019, guidelines recommend screening of transferrin levels to look for any cause of iron overload, rather than HFE testing, due to the very low penetrance. APOE testing in Alzheimer disease (AD) (Chapter 13) (Case 4) is another example of the role of a careful assessment of clinical validity and clinical utility in applying genetic testing to individualized medicine. Heterozygotes for the ε4 allele of the APOE gene are at two- to three-fold increased risk for development of AD, compared with individuals without an APOE ε4 allele. APOE ε4/ε4 homozygotes are at eight-fold increased risk. An analysis of both the clinical validity and clinical utility of APOE testing, including calculation of the positive predictive value for asymptomatic and symptomatic individuals, is shown in Table 19.4. As can be seen from these positive predictive values for asymptomatic people aged 65 to 74 years, a single ε4 allele is not a strong predictor of whether AD will develop, despite the three-fold increased risk conferred for the disease. Thus, most individuals heterozygous for an ε4 allele identified through APOE testing as being at increased risk will not develop AD. Even with two ε4 alleles, which occurs in ~1.5% of the population and is associated with an eight-fold increased risk relative to genotypes without ε4 alleles, the chance is still less than one in four to develop AD. APOE testing for the ε4 allele is, therefore, not recommended in asymptomatic individuals but is used by some practitioners in the evaluation of individuals with symptoms and signs of dementia. The utility of testing asymptomatic individuals at their APOE locus to assess risk for AD is also controversial. First, knowing that one is at increased risk for AD through APOE testing does not lead to any preventive or therapeutic options. Thus, under a strict definition of clinical utility – that is, the result is actionable and leads to changes in medical management – there would be little value in APOE testing for AD risk. There may be, however, positive and negative outcomes of testing that are psychological or economic in nature and more difficult to assess than the purely clinical factors. For example, testing positive for a susceptibility genotype could empower individuals with knowledge of their risks as they make important life decisions. On the other hand, it has been suggested that knowing of an increased risk through APOE testing might cause significant emotional and psychological distress. However, careful studies of the impact of receiving APOE genotype information have shown little harm in appropriately counseled individuals with a family history of AD who wished to know this information. Finally, individuals who test negative for the ε4 alleles could be falsely reassured that they are at no increased risk for the disorder, despite having a positive family history or other risk factors for dementia. Balancing all of these considerations, APOE testing is still not recommended in asymptomatic individuals, even in light of such a strong genotype-disease association, because of the low positive predictive value and lack of clinical utility, rather than because such information is harmful. As in all of medicine, the benefits and costs for each component of individualized genetic medicine need to be clearly demonstrated and continually reassessed. The requirement for constant reevaluation is obvious: imagine how the recommendations for APOE testing, despite its low positive predictive value, might change TABLE 19.4 Clinical Validity and Utility of APOE Population Screening and Diagnostic Testing for Alzheimer Disease Population Screening Diagnostic Testing Clinical validity Asymptomatic individuals aged 65–74 yr Individuals aged 65–74 yr with symptoms of dementia Population prevalence of AD = 3% Proportion of dementia patients with AD = ≈60% PPV given ε2/ε4 or ε3/ε4 = 6% PPV given ε2/ε4 or ε3/ε4 = ≈75% PPV given ε4/ε4 = 23% PPV given ε4/ε4 = ≈98% Clinical utility No intervention possible to prevent disease Increases suspicion that another, potentially treatable cause of dementia may be present Psychological distress for most people with ε4 alleles who are not likely to develop AD Reduces unnecessary testing False reassurance for those without ε4 alleles Positive predictive value (PPV) calculations are based on a population prevalence of Alzheimer disease (AD) of ~3% in individuals aged 65–74 years, an allele frequency for the ε4 allele in those of European ancestry of 10–15%, a relative risk of ~3 for one ε4 allele, and a relative risk of ~20 for two ε4 alleles. ▶

420 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE stores. This suggests that screening would be a reasonable and cost-effective measure to identify asymptomatic individuals who should under...

Ch19 · Pt11 CHAPTER 19 — Application of Genomics to Medicine and Individualized Health Care 421 if a low-risk and inexpensive medical intervention were discovered that could prevent or significantly delay the onset of dementia. Heterozygote Screening In contrast to screening for genetic disease in newborns or for genetic susceptibility in individuals, screening for carriers of mendelian disorders has, as its main purpose, the identification of individuals who are themselves healthy but are at substantial risk (25% or higher) to have children with a severe autosomal recessive or X-linked illness. The principles of heterozygote screening are shown in the accompanying (see Box 19.4). Until recently, heterozygote screening programs focused on particular population groups in which the frequency of variant alleles is high. In contrast to newborn screening, as discussed previously in this chapter, heterozygote screening is voluntary and focuses on individuals who identify themselves as members of particular high-risk groups. Heterozygote screening has been used extensively for a battery of disorders for which carrier frequency is relatively high: Tay-Sachs disease (Case 43) (the prototype of carrier screening) (see Chapter 13), Gaucher disease, and Canavan disease in the Ashkenazi Jewish population; sickle cell disease (Case 42) in the black population of North America; and β-thalassemia (Case 44) in high-incidence areas, especially in Cyprus and Sardinia, or in extended consanguineous families from Pakistan (see Chapter 12). Carrier screening for cystic fibrosis (Case 12) has become standard of care for couples contemplating a pregnancy. As discussed in Chapter 13, more than 2000 different disease-causing variants have been described in the CFTR gene. Although the vast majority of disease- causing variants in CFTR can be readily detected with greater than 99% sensitivity when the entire gene is sequenced, such an approach for every couple seeking preconception carrier testing would be expensive if carried out on a population-wide basis, particularly in individuals with low prior probability of carrying a variant. Current recommendations are to report pathogenic and likely-pathogenic variants identified by sequencing or targeted testing. Targeted testing panels range from the most frequent 23 variants found in persons of European ancestry—as proposed by the American College of Medical Genetics and Genomics (ACMG), to considerably more extensive panels with more than 60 distinct variants that include those identified in populations with lower frequencies of disease, such as of African or Asian ancestry. Because this approach is intended to find only the most frequent variants, their sensitivity is around (88 to 90%) in individuals of European descent and 64 to 72% among those of African descent. The classification-based reporting technique involves giving individuals comprehensive cystic fibrosis testing that includes an evaluation of all the exonic coding regions and +/−2 bp proximal splice junctions of the CFTR gene, as well as reporting on all pathogenic and likely-pathogenic variants for classic cystic fibrosis. Sanger sequencing has long been employed in medical laboratories for the study of CFTR because of its accuracy, precision, and simplicity of use. The analysis of CFTR using next-generation sequencing approaches is now successful, although there is still a danger of false negatives and positives. Furthermore, certain regions may need Sanger sequencing to detect variants. Multiplex ligation-dependent probe amplification (MLPA see Chapter 5) continues to be an efficient method to detect large deletions and duplications in the CFTR gene, and commercial reagents exist. Regardless of the test indication, all CFTR variants should be classified using ACMG sequence variant classification criteria. Information from CFTR variant databases can be used to inform those variant classifications. As the cost of variant detection using next-generation sequencing has fallen, it has become much less compelling to restrict carrier screening to a small number of alleles common in certain ancestral groups in genes that are known to be associated with disease. It is possible now to expand carrier screening beyond disorders common to particular groups, such as cystic fibrosis, sickle cell trait, or thalassemia, to include carrier status for more than 400 autosomal recessive and X-linked disorders. With sequencing instead of allele-specific detection methods, there is no longer any limit to which genes and which alleles in these genes can (theoretically) be detected. Rare variant alleles in genes associated with known disease will be found, thereby raising the sensitivity of carrier detection methods. Sequencing, however, can uncover variants—particularly missense changes, of uncertain significance in genes whose role in the disease may be known or unknown. Unless great care is taken in assessing the clinical validity of rare variants detected by sequencing, the frequency of false-positive carrier test results will increase. The impact of carrier screening in lowering the incidence of a genetic disease can be dramatic. Carrier screening for Tay-Sachs disease in the Ashkenazi Jewish population began in some communities in 1969. Screening followed by prenatal diagnosis, when indicated, has already lowered the incidence of Tay-Sachs BOX 19.4 CRITERIA FOR HETEROZYGOTE SCREENING PROGRAMS High frequency of carriers, at least in a specific population Availability of an inexpensive and dependable test with very low false-negative and false-positive rates Access to genetic counseling for couples identified as heterozygotes Availability of prenatal or preimplantation genetic diagnosis Acceptance and voluntary participation by the population targeted for screening ▶

CHAPTER 19 — Application of Genomics to Medicine and Individualized Health Care 421 if a low-risk and inexpensive medical intervention were discovered that could prevent or significantly delay the on...

Ch19 · Pt12 422 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE disease by 65 to 85% in this group. In contrast, attempts to screen for carriers of sickle cell disease in the US Black community have been less effective, with little impact on the incidence of the disease. The success of carrier screening programs for Tay-Sachs disease, as well as the relative failure for sickle cell anemia, underscores the importance of community consultation, community engagement, and the availability of genetic counseling and prenatal or preimplantation genetic diagnosis as critical requirements for an effective program. INDIVIDUALIZED GENOMIC MEDICINE More than a century ago, British physician-scientist Archibald Garrod proposed the concept of chemical individuality, in which each of us differs in our health status and susceptibility to various illnesses because of our individual genetic makeup. Indeed, in 1902, he wrote: … the factors which confer upon us our predisposition and immunities from disease are inherent in our very chemical structure, and even in the molecular groupings which went to the making of the chromosomes from which we sprang. The goal of individualized genomic medicine is to use knowledge of an individual’s genetic variants relevant to maintaining health or treating illness as a routine part of medical care. Now, more than a hundred years after Garrod’s visionary pronouncement, in the era of human genomics, we have the means to assess an individual’s genotype at every locus by genome sequencing (whole genome sequencing [WGS]). WGS is a comprehensive test capable of detecting nearly all DNA variation in a genome. WGS can identify the 7000 diseases described in the Online Mendelian Inheritance in Man database (www.omim.org) that have a known molecular basis. These include those we have discussed extensively in this textbook, such as cystic fibrosis, Duchenne muscular dystrophy, familial hypercholesterolemia, and hemophilia. Patients may present with unusual constellations of features, or with common conditions such as autism spectrum disorder, cardiomyopathy, congenital heart disease, epilepsy, cancer, schizophrenia, or dementia, although this list is not comprehensive. WGS is broader in scope than other commonly used genetic tests (see Box 19.5), and data can be analyzed in both hypothesis-driven and hypothesis-generating ways. For these reasons, WGS will most certainly eclipse exome sequencing, large next-generation sequencing gene panel tests, and chromosomal microarray analysis in the future. Genome sequencing is a 3-stage process. First, a medical geneticist or other health care professional obtains the required information on the patient’s phenotype and family history. Second, a clinical laboratory geneticist analyzes the genome data. Third, a physician compares the genetic findings to the clinical manifestation to assess the diagnostic fit or associated risk. The overall goal of interpreting a genetic variant is to explain it in the context of all or part of the clinical manifestation. The main aim of genome sequencing as a clinical diagnostic test is to identify these variations. Some laboratories in North America will also search for secondary findings, which are disease-causing variants in genes associated with medically actionable conditions that are unrelated to the initial intent for testing. The procedure of sequencing is safe; however, possible negative consequences are tied to how results are interpreted and disclosed. First, genome sequencing may be misinterpreted as a diagnostic that can answer all clinical questions. The clarity in clinical data and family history is still essential for interpreting findings. A positive result does not necessarily explain all of the patient’s characteristics, and a negative test does not indicate that there was no genetic component nor invalidate an obvious clinical diagnosis. Second, because of ongoing understanding and the characterization of new information, the classification of a genetic variant may change over time. The majority of ancient peoples other than Europeans are under-represented in the big-scale reference databases of genomic variation that guide interpretation; thus, misdiagnosis is a risk for these people who needs to be taken into consideration. Third, genetic test results might provide information about the person, their family members, or their connections to one another that was not previously considered. These facts underscore the need for thorough pre- and post-test counseling and qualified genetics professionals. The majority of the data come from prospective clinical trials testing in clinically diverse populations with anticipated rare genetic diseases. Primary outcome measures are frequently diagnostic yield or time to diagnosis. Clinical usefulness and cost-effectiveness are desirable secondary outcomes. Genome sequencing has a greater diagnostic yield than exome sequencing and BOX 19.5 OVERVIEW OF SELECTED CLINICAL GENETIC TEST MODALITIES For phenotypes with known genetic heterogeneity, the following tests are commonly employed in clinical practice: Chromosomal microarray analysis: a genome-wide test that typically detects only copy number variations (i.e., chromosome imbalances). Next-generation sequencing gene panel test: a targeted test focusing on a predefined list of genes, which typically detects only exonic sequence-level variants, deletions or duplications in those genes. Exome sequencing: a genome-wide test that typically detects only exonic sequence-level variants, or a subset of exon-level deletions or duplications (CNVs). Genome sequencing (also whole genome sequencing or WGS): this approach offers myriad advantages as a single comprehensive test. Current short-read genome sequencing can reliably detect sequence, structural and copy number variations, both within and outside of exons, as well as clinically relevant short tandem repeats, pseudogenes and mitochondrial DNA variation. ▶

422 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE disease by 65 to 85% in this group. In contrast, attempts to screen for carriers of sickle cell disease in the US Black community have been...

Ch19 · Pt13 CHAPTER 19 — Application of Genomics to Medicine and Individualized Health Care 423 chromosomal microarray analysis, while being increasingly price competitive. Different phenotype categories are associated with different diagnostic yields. For individuals with severe-to-profound intellectual disability, such yields can be less than 10%, but they can be as much as 50% or greater for other indications. Genome sequencing with a rapid turnaround time is most commonly utilized in neonatal and pediatric intensive care settings. In children and adults with suspect genetic conditions that have high genetic heterogeneity, genome sequencing is expected to become a first-tier test (i.e., a broad genetic differential diagnosis with many candidate genes or loci) instead of the second-tier technique it has typically been used for so far. This will cut down on the time it takes to conduct several genetic tests. Genomic testing may reveal pharmacogenetic profiles, reproductive carrier status information, and genetic risk profiles for later-onset diseases. The use of genome sequencing as a preventative health tool in seemingly healthy people is uncertain at this time, but it has tremendous potential for the future. Ongoing research is needed to examine the clinical usefulness, cost-effectiveness, and possible unintended future consequences of genome sequencing in our healthcare system. In certain circumstances, the added yield of genome sequencing over exome sequencing is modest; nevertheless, this gap will increase with improvements in data analysis and bigger data sets to compare against. The anticipation of additional clinically relevant information arising from as-yet-unexplored areas of the genome is also driving investments in genome sequencing technology. Ensuring equitable access to care informed by the DNA code, irrespective of postal code, is a challenge in many countries and needs to be a priority for policy-makers. PRECISION CHILD HEALTH Precision Child Health (PCH) refers to a concept and movement to transform pediatric health care through the integration of data from all domains of a child’s determinants of health (genes, biology, environment). This must go along with patient-reported perceptions of their health and objectively measured physiology in order to predict, prevent, diagnose, and treat disease in a targeted, individualized way. Application of bioinformatics, computing tools, and advanced statistical approaches applied to these integrated data resources will support the evaluation and discovery of rapid, accurate, and cost-effective diagnostics, individualized therapies, and enhanced drug safety and efficacy. Insights unlocked by PCH will fuel the next generation of data-guided quality improvement initiatives and preventative care. This strategy has the potential to radically transform our approach to healthcare delivery by unlocking efficiencies, decreasing preventable harm, and enhancing our approaches to patient-centered care that will eventually be extrapolated to all of medicine. GENERAL REFERENCES Feero WG, Guttmacher AE, Collins FS: Genomic medicine—an updated primer, N Engl J Med 362:2001–2011, 2010. Ginsburg G, Willard HF, editors: Genomic and personalized medicine, ed 2, vols 1 & 2, New York, 2012, Elsevier. Kitzmiller JP, Groen DK, Phelps MA, et al: Pharmacogenomic testing: relevance in medical practice, Cleve Clin J Med 78:243–257, 2011. Schrodi SJ, Mukherjee S, Shan Y, et al: Genetic-based prediction of disease traits: prediction is very difficult, especially about the future, Frontiers Genet 5:1–18, 2014. REFERENCES FOR SPECIFIC TOPICS Amstutz U, Carleton BC: Pharmacogenetic testing: time for clinical guidelines, Pharmacol Ther 89:924–927, 2011. Bardolia C, Matos A, Michaus V, et al: Utilizing pharmacogenomics to reduce adverse drug events, Am J Biomed Sci Res, 2020. https:// biomedgrid.com/pdf/AJBSR. MS. ID.001638.pdf Bennett MJ: Newborn screening for metabolic diseases: saving children’s lives and improving outcomes, Clin Biochem 47(9):693–694, 2014. Deignan JL, Astbury C, Cutting GR, et al: CFTR variant testing: a technical standard of the American College of Medical Genetics and Genomics (ACMG), Gen Med Off J Am Coll Med Genet 22(8):1288– 1295, 2020. https://doi.org/10.1038/s 41436-020-0822-5 Dorschner MO, Amendola LM, Turner EH, et al: Actionable, pathogenic incidental findings in 1,000 participants’ exomes, Am J Hum Genet 93:631–640, 2013. Ferrell PB, Mc Leod HL: Carbamazepine, HLA-B*1502 and risk of Stevens-Johnson syndrome and toxic epidermal necrolysis: US FDA recommendations, Pharmacogenomics 9:1543–1546, 2008. Green RC, Roberts JS, Cupples LA, et al: Disclosure of APOE genotype for risk of Alzheimer’s disease, N Engl J Med 361:245–254, 2009. Ingelman-Sundberg M, Rodriguez-Antona C: Pharmacogenetics of drug-metabolizing enzymes: implications for a safer and more effective drug therapy, Philos Trans R Soc Lond B Biol Sci 360:1563– 1570, 2005. https://doi.org/10.1098/rstb.2005.1685 Johnston JJ, Dirksen RT, Girard T, et al: Variant curation expert panel recommendations for RYR1 pathogenicity classifications in malignant hyperthermia susceptibility, Gen Med Off J Am Coll Med Genet 23(7):1288–1295, 2021. https://doi. org/10.1038/s 41436-021-01125-w Karczewski KJ, Daneshjou R, Altman RB: Pharmacogenomics, PLo S Comput Biol 8(12):e 1002817, 2012. Kohane IS, Hsing M, Kong SW: Taxonomizing, sizing, and overcoming the incidentalome, Genet Med 14:399–404, 2012. Mallal S, Phillips E, Carosi G, et al: HLA-B*5701 screening for hypersensitivity to abacavir, N Engl J Med 358:568–579, 2008. Mayo Clinic Laboratories: Test ID: CARBR. https://www.mayocliniclabs.com/test-catalog/Overview/610048#Clinical-and-Interpretive. Mc Carthy JJ, Mc Leod HL, Ginsburg GS: Genomic medicine: a decade of successes, challenges and opportunities, Sci Transl Med 5: 189sr 4, 2013. Mounzer K, Hsu R, Fusco JS, et al: HLA-B*57:01 screening and hypersensitivity reaction to abacavir between 1999 and 2016 in the OPERA® observational database: a cohort study, AIDS Res Ther 16:1, 2019. https://doi.org/10.1186/s 12981-019-0217-3 Pharm GKB: Annotation of CPIC guideline for desflurane and CACNA1S, RYR1. https://www.pharmgkb.org/chemical/ PA164749136/guideline Annotation/PA166180457 Pharm GKB: Dosing guidelines. https://www.pharmgkb.org/guide lines Relling MV, Klein TE: CPIC: clinical pharmacogenetics implementation consortium of the pharmacogenomics research network, Clin Pharmacol Ther 89(3):464–467, 2011. Topol EJ: Individualized medicine from prewomb to tomb, Cell 157:241–253, 2014. Urban TJ, Goldstein DB: Pharmacogenetics at 50: genomic personalization comes of age, Sci Transl Med 6:220ps 1, 2014. Zanger UM, Schwab M: Cytochrome P450 enzymes in drug metabolism: regulation of gene expression, enzyme activities, and impact of genetic variation, Pharm Ther 138:103–141, 2013. https://doi.org/ 10.1016/j.pharmthera.2012.12.007 Zhu Y, Swanson KM, Rojas RL, et al: Systematic review of the evidence on the cost-effectiveness of pharmacogenomics-guided treatment for cardiovascular diseases, Genet Med 22:475–486, 2020. https://doi.org/10.1038/s 41436-019-0667-y ▶

Ch19 · Pt14 424 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE PROBLEMS 1. In a population sample of 1 million Europeans, idiopathic cerebral vein thrombosis (i CVT) occurred in 18, consistent with an expected rate of 1 to 2 per 100,000. All the individuals were tested for factor V Leiden (FVL). Assuming an allele frequency of 2.5% for FVL, how many homozygotes and how many heterozygotes for FVL would you expect in this sample of 1 million people, assuming Hardy-Weinberg equilibrium? Among the individuals affected with i CVT, two were heterozygotes for FVL and one was homozygous for FVL. a. Set up a 3 × 2 table for the association of the homozygous FVL genotype, the heterozygous FVL genotype, and the wild-type genotype for i CVT. b. What is the relative risk for i CVT in a FVL heterozygote versus in the wild-type genotype? c. What is the risk in a FVL homozygote versus in wild type? d. What is the sensitivity of testing positive for either one or two FVL alleles for i CVT? e. What is the positive predictive value of being homozygous for FVL? Heterozygous? 2. In a population sample of 100,000 European women taking oral contraceptives, deep venous thrombosis (DVT) of the lower extremities occurred in 100, consistent with an expected rate of 1 per 1000. Assuming an allele frequency of 2.5% for factor V Leiden (FVL), how many homozygotes and how many heterozygotes for FVL would you expect in this sample of 100,000 women, assuming Hardy- Weinberg equilibrium? Among the affected individuals, 58 were heterozygotes for FVL and three were homozygous for FVL. Set up a 3 × 2 table for the association of the homozygous FVL genotype, the heterozygous FVL genotype, and the wild-type genotype for DVT of the lower extremity. What is the relative risk for DVT in a FVL heterozygote using oral contraceptives versus in women with the wild-type genotype taking oral contraceptives? What is the risk in a FVL homozygote versus in wild type? What is the sensitivity of testing positive for either one or two FVL alleles for DVT while taking oral contraceptives? Finally, what is the positive predictive value for DVT of being homozygous for FVL while taking oral contraceptives? Heterozygous? 3. What steps should be taken when a newborn phenylketonuria (PKU) screening test comes back positive? 4. Newborn screening for sickle cell disease can be performed by hemoglobin electrophoresis or high-performance liquid chromatography (HPLC), which separates hemoglobin A and S, thereby identifying individuals who are heterozygotes as well as those who are homozygotes for the sickle cell variant. What potential benefits might accrue from such testing? What harms? 5. Toxic epidermal necrolysis (TEN) and the Stevens- Johnson syndrome (SJS) are two related, life-threatening skin reactions that occur in ~1 per 100,000 individuals in China, most commonly as a result of exposure to the antiepileptic drug carbamazepine. These conditions carry a significant mortality rate of 30 to 35% (TEN) and 5 to 15% (SJS). It was observed that individuals who suffered this severe immunologic reaction carried a particular major histocompatibility complex class 1 allele, HLA-B*1502, as do 8.6% of the Chinese population. In a retrospective cohort study of 145 patients who received carbamazepine therapy, 44 developed either TEN or SJS. Of these, all 44 carried the HLA-B*1502 allele, whereas only three of those who received the drug without incident were HLA-B*1502 positive. What is the sensitivity, specificity, and positive predictive value of this allele for TEN or SJS in individuals receiving carbamazepine? 6. A 21-month-old boy with ventricle atrioventricular septal defect and complete pulmonary atresia and history of thrombotic event was put on standard warfarin dosing, with a goal INR (international normalized ratio) of 2.5 to 3. After a second warfarin dose, the INR level rose to 8. Parents took the child to the emergency room where he was observed overnight. Upon review of his pharmacogenomics data, it was found that the patient was warfarin sensitive, as he was a poor metabolizer for the metabolizing enzyme CYP2C9. Warfarin is broken down in the liver by CYP2C9 enzyme to its inactive metabolites. Absent or low CYP2C9 enzyme activity reduces the clearance rates of warfarin. Warfarin exhibits its anticoagulant effect by inhibiting the Vitamin K epoxide reductase (VKORC1) enzyme. VKORC1 converts vitamin K into its active form, which is needed to produce clotting factors. Certain genetic variants in the VKORC1 gene may decrease the level of active vitamin K, thus causing fewer clotting factors to be available. Conclusion: Much lower starting dose would have been sufficient. Question: Review the most common variants in the CYP2C9 gene among different populations. 7. A 10-year-old male with eosinophilic esophagitis (Eo E) was prescribed lansoprazole: 15 mg orally twice daily. Eo E is a chronic immune system disease in which eosinophils build up in the lining of the esophageal tract. Damaged esophageal tissue can lead to difficulty swallowing or cause food impaction and poor appetite. Currently, proton pump inhibitors with/without budesonide slurry are the standard of care for this condition. After 3 months, this child did not show any improvement of symptoms. Pharmacogenomics testing was requested. The metabolizing enzyme CYP2C19 is mainly responsible for the breakdown of proton pump inhibitors such as lansoprazole, omeprazole and pantoprazole. This patient was found to an ultra-rapid CYP2C19 metabolizer. He breaks down lansoprazole much faster than the world population average, leading to a much-decreased plasma concentration, with therapeutic failure. He was switched to rabeprazole which is less influenced by the CYP2C19 metabolizer status. Conclusion: After another 4 months, patient underwent an endoscopy which revealed evidence of healing of the esophageal tract. Question: Which other health condition can be influenced by genetic changes in the CYP2C19 gene? ▶

424 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE PROBLEMS 1. In a population sample of 1 million Europeans, idiopathic cerebral vein thrombosis (i CVT) occurred in 18, consistent with an e...

Chapter 20: Ethical and Social Issues in Genetics and Genomics

Ch20 · Pt1 chapter 20 Ethical and Social Issues in Genetics and Genomics Bartha Maria Knoppers ▶

Ma’n H. Zawati Human genetics and genomics are having a major impact in all areas of medicine and across all age groups and in emerging fields such as epigenetics, cellular genomics, and pathogen geno...

Ch20 · Pt2 426 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE disorder. For some hereditary disorders, prenatal diagnosis remains controversial, particularly when the diagnosis leads to a decision to terminate the pregnancy for a disease that causes various kinds of physical or intellectual disabilities but is not fatal in infancy. Prenatal diagnosis is equally controversial for adult-onset disorders, particularly ones that may be managed or treated. The debate is still ongoing in the community of persons who have a physical or intellectual disability and deaf patients and their families (to name only a few examples) about whether prenatal diagnosis and abortion for these disorders are ethically justified. Other areas of ethical debate include seeking prenatal diagnosis to avoid recurrence of a disorder associated with a mild or cosmetic defect or for putative genetic enhancement, such as genetic variants affecting muscle physiology and therefore athletic prowess. The dilemma lies in attempting to balance, on the one hand, respect for the autonomy of reproductive decision making about the kind of family they wish to have versus, on the other hand, an assessment of how aborting a fetus affected with a disability might be viewed by the broader community of persons with a disability. The dilemma also arises when a couple makes a request for prenatal diagnosis in a pregnancy that is at risk for what most people would not consider a disease or disability at all. Particularly difficult is prenatal diagnosis for selection of sex for reasons other than reducing the risk for sex-limited or X-linked disease. Some genetics professionals are concerned that couples are using assisted reproductive technologies, such as in vitro fertilization and blastomere biopsy, or prenatal sex determination by ultrasonography and abortion, to balance the sexes of the children in their family or to avoid having children of one or the other sex for sociocultural and economic reasons prevalent in their societies. There are already clear signs of a falling ratio of female to male infants from 0.95 to less than 0.85 in certain areas of the world where male children are more highly prized. Some countries explicitly prohibit sex selection by law (absent a link to the presence of a genetic condition). Many of these dilemmas have so far been more theoretical than real. For example, surveys of couples with deafness or achondroplasia show that the couples are concerned about having children who are not deaf or do not have achondroplasia. The vast majority would not actually use prenatal diagnosis and abortion to avoid having children who do not share their conditions. Moreover, as we will see, somatic gene editing will increase the range of interventions and treatments for children and the range of parental and pediatric choices. Yet, in the future, particular alleles and genes that contribute to complex traits, such as intelligence, personality, stature, and other physical characteristics, will likely be identified. Will such nonmedical criteria be viewed as a justifiable basis for prenatal diagnosis? Some might argue that parents are already expending tremendous effort and resources on improving the environmental factors that contribute to healthy, successful children. They might therefore ask why they should not try to improve the genetic factors as well. Others consider prenatal selection for particular desirable genes a dehumanizing step that treats children simply as commodities fashioned for their parents’ benefit. Does a health professional have, on the one hand, a responsibility or, on the other hand, any right to intervene in the decision of a couple concerning the “seriousness” of a disorder? There is little consensus among geneticists as to where or even whether one can draw the line in deciding what constitutes a trait serious enough to warrant prenatal testing. Preimplantation testing of embryos not only moves these dilemmas earlier in time but to date has faced less public scrutiny. Genetic Testing for Predisposition to Disease Another area of medical genetics and genomics in which ethical dilemmas frequently arise is genetic testing of asymptomatic individuals for diseases that may have an onset in life later than the age at which the molecular testing is to be performed. The ethical principles of respect for individual autonomy and beneficence are central to TABLE 20.1 Major Ethical and Policy Issues in Medical Genetics Genetic Testing Prenatal diagnosis, especially for nondisease traits or sex Testing asymptomatic adults for genotypes that predispose to late-onset disease Testing asymptomatic children for genotypes that predispose to adult-onset diseases Secondary and incidental findings and the right “not to know” about clearly deleterious variants that will cause diseases that could be ameliorated or prevented if the risk were known Privacy of Genetic Information Duty to warn and permission to warn family members Misuse of Genetic Information Insurance/employment discrimination based on an employee’s genotype Discrimination in life and health insurance underwriting based on a person’s genotype Genetic Screening Expansion of screening programs Privacy ▶

426 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE disorder. For some hereditary disorders, prenatal diagnosis remains controversial, particularly when the diagnosis leads to a decision to te...

Ch20 · Pt3 CHAPTER 20 — Ethical and Social Issues in Genetics and Genomics 427 testing in this context. At one end of the spectrum is testing for late-onset, highly penetrant neurologic disorders, such as Huntington disease (see Chapter 13) (Case 24). For such diseases, individuals carrying a variant allele may be asymptomatic but will almost certainly develop a devastating illness later in life for which there is currently little or no treatment. For these asymptomatic individuals, is knowledge of the test result more beneficial than harmful, or vice versa? There is no simple answer. Studies demonstrate that some individuals at risk for Huntington disease choose not to undergo testing and would rather not know their risk, whereas others choose to undergo testing. Those who choose testing and test positive have been shown to sometimes have a transient period of depression, but with few suffering severe depression, and many report positive benefits in terms of the knowledge provided to make life decisions about marriage and choice of career. Those who choose testing and are found not to carry the trinucleotide expansion allele report positive benefits of relief, but they can also experience negative emotional responses due to guilt for no longer being at risk for a disease that either affects or threatens to affect many of their close relatives. In any case, the decision to undergo testing is a highly personal one that must be made only after thorough review of the issues with a genetics professional. The balance for or against testing of unaffected, at- risk individuals shifts when testing indicates a predisposition to a disease for which intervention and early treatment are available. For example, in autosomal dominant hereditary breast cancer, individuals carrying various pathogenic variants in BRCA1 or BRCA2 have a 50% to 90% chance of developing breast or ovarian cancer (see Chapter 16) (Case 7). Identification of heterozygous carriers would be of benefit because individuals at risk could choose to undergo more frequent surveillance or have preventive surgery, such as mastectomy, oophorectomy, or both, recognizing that these measures can reduce but not completely eliminate the increased risk for cancer. What if surveillance and preventive measures were more definitive, as they are in familial adenomatous polyposis, for which prophylactic colectomy is a proven preventive measure (see Chapter 16)? Moreover, upon testing for any predisposing gene variant(s), individuals incur the risk for serious psychological distress, stigmatization in their social lives, and possible discrimination in insurance and employment (see later). How are respect for a patient’s autonomy, the physician’s duty not to cause harm, and the physician’s desire to prevent illness to be balanced in these different situations? Geneticists would all agree that the decision to be tested or not to be tested is not one made in a vacuum. The patient must make an informed decision using all available information concerning the risk for and severity of the disease, the effectiveness of preventive and therapeutic measures, and the potential harm that could arise from testing. Genetic Testing of Asymptomatic Children Ethical issues in the testing of asymptomatic individuals take on a further degree of complexity when such testing involves minor children (generally, <18 years), particularly children too young to even give assent. There are several reasons why parents may wish to have their children tested for a disease predisposition. Testing asymptomatic children for alleles that predispose to disease can be beneficial, even life saving, if interventions that decrease morbidity or increase longevity are available. One example is testing the asymptomatic sibling of a child with medium-chain acyl-Co A dehydrogenase deficiency (see Chapter 19) (Case 31). However, a minority have argued that even in situations where there are currently no clear medical interventions that might benefit the child, it is the parents’ duty to inform and prepare their children for the future possibility of development of a serious illness. The parents may also seek this information for their own family planning or to avoid what some parents consider the corrosive effects of keeping important information about their children from them. Testing children, however, carries the same risks for serious psychological damage, stigmatization, and certain kinds of insurance discrimination as does testing adults (see later). Children’s autonomy—their ability to make decisions for themselves about their own genetic constitution— must also now be balanced with the desire of parents to obtain and use such information. A different but related issue arises in testing children for the carrier state of a disease that poses no threat to their health but places them at risk for having affected children. Once again, the debate centers on the balance between respect for children’s autonomy in regard to their own procreation when they could choose as adults and the desire on the part of well-meaning parents to educate and prepare children for the difficult decisions and risks that lie ahead once they reach childbearing age. Most bioethicists believe (and the American College of Medical Genetics and Genomics [ACMG] agrees) that predictive pediatric genetic testing should generally be deferred until a child is sufficiently mature, unless an intervention in childhood would reduce mortality or morbidity. Testing for adult-onset conditions should likewise typically be deferred until the child reaches a sufficient level of maturity. The European Society of Human Genetics echoes this view, suggesting that genetic testing decisions that affect children should be approached with a certain degree of caution. Minors, as soon as their maturity and degree of understanding permit, should have their perspectives taken into account and should be permitted to decide personally whether to undergo asymptomatic genetic testing when they are sufficiently well informed and capable of understanding the test and its consequences. The Canadian College of Medical Geneticists (CCMG) takes a slightly different approach, noting that while asymptomatic pediatric testing might ▶

CHAPTER 20 — Ethical and Social Issues in Genetics and Genomics 427 testing in this context. At one end of the spectrum is testing for late-onset, highly penetrant neurologic disorders, such as Hunti...

Ch20 · Pt4 428 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE sometimes be in the best interests of the child, but testing for adult-onset conditions should generally be deferred until such children are competent to consent to such testing on their own behalf. In exceptional circumstances, parents may insist that the asymptomatic testing of a child be carried out. The CCMG notes that physicians are not obliged to carry out such testing if it is not in the best interests of the child concerned. Gene Therapy Genetic testing inevitably raises the possibility of gene therapy. Such therapy is subject to much public scrutiny as a potential option for patients living with serious, and even incurable, diseases. This technology promises to replace genes that cause medical problems with genes that do not, or even “turn off” genes in the body that cause illness. Beyond the safety and consent issues, gene therapy raises other ethical, legal, and social concerns. As a new and expensive therapy, we need to think about how and who determines whether a condition is serious enough for its use, what uses are for therapy versus enhancement, and how do we make sure that everyone has access to it? However, the main debate is the difference between somatic and germline gene therapy. Somatic therapies target genes in specific cells, such as lungs or skin cells, and cannot be passed on to a person’s children. On the other hand, germline therapies make changes in reproductive cells (e.g., egg or sperm cells), correcting genes that are subsequently inherited by future generations. While germline therapy could prevent future generations in a family from having a particular genetic condition and could eliminate certain serious diseases altogether from society (e.g., Huntington disease), this therapy could have unexpected effects on the future child to be born. Finally, gene therapy could be used to select or enhance different human characteristics (e.g., height, intelligence, athletic skills), which may result in society being less accepting of people who are different or have a certain disability. While somatic therapies are beginning to be used on patients (e.g., spinal muscular atrophy, sickle cell), no country has decided to allow germline therapy. But the debate continues and will require considering the views of communities, patients, and families, as well as the ethical and legal issues and the possible long-term effects for society. Incidental and Secondary Findings From Exome and Whole Genome Sequencing Another area of controversy has arisen in patients who have given consent for exome or whole genome sequencing (ES/WGS) to find a genetic basis for their undiagnosed diseases (see Chapter 19). Laboratories searching the exomes or genomes of such patients usually develop a primary candidate gene list based on the phenotype of the patient. The laboratory considers deleterious variants in these genes as their primary findings (i.e., the results that are actively being sought as the primary target of the testing). In the process of analyzing an exome or genome, however, pathogenic variants may be discovered incidentally in genes known to be associated with diseases unrelated to the phenotype for which the sequencing test was originally conducted. If the pathogenic variants uncovered as incidental findings cause serious diseases that can be ameliorated or prevented, then is there benefit of drawing up a list of genes that every laboratory doing ES/WGS would deliberately analyze in every patient, even though they are not relevant to the primary goal of finding the genetic cause for a patient’s unexplained disease? Pathogenic and likely pathogenic variants in this list of genes would be secondary findings that would be sought regardless of whether the patient wishes to know these results, because the patient’s providers deem the benefit of knowing is so compelling for the patient’s health that it outweighs the requirement of patient autonomy, to be able to choose what kind of information the patient wants to know. The ACMG has drawn up a list of secondary findings that a laboratory should seek. The current list (SF v 3.0) includes 73 genes, most of which are involved in serious hereditary cancer and cardiovascular syndromes that are (1) life threatening, (2) not readily diagnosable before the onset of symptoms, and (3) preventable or treatable. The secondary finding gene list is subject to ongoing refinement and will presumably grow over time. Furthermore, whether a given gene variant should always be a secondary finding that must be sought is also undergoing reevaluation. The current ACMG recommendation is that patients should be provided with appropriate counseling and then given the opportunity prior to testing to agree or to refuse to have such secondary findings looked for and reported. Other jurisdictions take a wide variety of approaches to the return of individual findings. The European Union’s General Data Protection Regulation (GDPR), for example, maintains a right of access to genetic data that works to facilitate the return of individual results and secondary findings. In France, the Code de la Santé Publique requires subjects to consent that findings associated with severe genetic abnormalities be returned. German policy documents are somewhat more sweeping, requiring the return of any medically relevant findings. Other countries, Israel and Italy, for example, require that any returned information be accompanied by access to genetic counseling. In June 2021, the Global Alliance for Genomics and Health released an international policy document on the return of clinically actionable findings in genomics research. Among other things, the policy emphasizes the importance of adhering to clear research protocols, upfront resourcing of any commitments to return findings, and linking return to current clinical practices and standards of care. Newborn Screening Although newborn screening programs with implicit parental consent following notification (see Chapter 19) are one of the great triumphs of modern genetics in ▶

428 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE sometimes be in the best interests of the child, but testing for adult-onset conditions should generally be deferred until such children ar...

Ch20 · Pt5 CHAPTER 20 — Ethical and Social Issues in Genetics and Genomics 429 improving public health and considered to be in the best interests of the child, questions about newborn screening programs still arise in some countries. First, should parents be asked to provide active consent or can they simply be offered the opportunity to opt out of the program? Second, who has access to samples and data, and how can we make sure that samples are not used for purposes other than the screening tests for which they were collected and for which consent was given (or at least, not withheld)? In the United States, these questions came to a head in the area of newborn screening in the state of Texas in 2019 when a group of parents of children sued the state because blood spots obtained through an opt-out process for newborn screening had been diverted to the Department of Defense and private companies and used for purposes other than newborn screening, without parental consent. Texas agreed to destroy their collection of more than 5 million blood spots. In doing so, the state lost samples that could have been used for legitimate purposes, such as developing new newborn screening tests and for quality control of current testing efforts. Increasingly, distinctions are being made between the right of the at-risk, asymptomatic newborn to be found via screening, followed by testing and treatment and the need for parental permissions. It should be noted that children have their own right to the “highest attainable standard of health” and their interests are “primary” according to the 1989 Convention on the Rights of the Child. Thus, many countries continue to notify parents about newborn screening but do not require an explicit, written consent. The same does not hold, however, as concerns further storage or use for research progress where parental permission is usually required. Polygenic Risk Scores PRSs provide individuals with a risk level for developing a range of disorders and diseases to which there is a genetic contribution. Through algorithms, PRSs give the relative risk level for a particular disease but are unable to assign an absolute lifetime risk measurement. PRS testing is capable of identifying risk scores for a wide range of conditions, from breast cancer to type 2 diabetes, schizophrenia to atrial fibrillation. Fundamentally, PRS testing depends on the comparison of individual genotyping results with genome-wide association study data. Insofar as many large-scale genomic research projects are not demographically representative of populations apart from those with European ancestry, it is likely that the risk categories are not generalizable and that benefits of PRS testing will not be equitably distributed. PRS also raises issues of communication and understanding of their meaning by health professionals and patients alike. PRIVACY OF GENETIC INFORMATION Legal protections for genetic information are not uniform across the globe or even within different jurisdictions in the same country. In the United States, the primary set of regulations governing the privacy of health information, including genetic information, is the Privacy Rule of the Health Insurance Portability and Accountability Act (HIPAA). The HIPAA rule sets criminal and civil penalties for disclosing such information without authorization to others, including other providers, except under a defined set of special circumstances. When it was enacted in 2018, Europe’s GDPR upended the privacy rules to which genetic information are subject, both within and outside of Europe. In particular, the GDPR created a context-specific and risk-based approach to data protection, permitting a limited degree of flexibility for the processing of personal data in the scientific research context. Different data processing standards apply in the GDPR depending on whether individual data are identifiable, pseudonymized (i.e., coded), or anonymized. Anonymized data are not considered to be personal data, since data of this kind cannot be directly associated with an identifiable person. The GDPR explicitly recognizes that genetic data fall within the special category of sensitive personal data, though it is unclear at present how genetic data might be distinguished within the text of the GDPR from the biologic materials from which it is obtained. There is additional disagreement about how sensitive genetic data can best be protected and yet still shared for research and care. It is not clear, for example, whether genetic data can be fully anonymized within the meaning of the GDPR. Neither are international transfers of such data clearly addressed or facilitated. Privacy Issues for Family Members in a Family History Patients are free to provide their physicians with a complete family medical history or communicate with their physicians about conditions that run in the family. Privacy legislation does not prevent individuals from gathering medical information about their family members or from deciding to share this information with their health care providers, but it is best to openly address this need for shared familial data with one’s patient. This information becomes part of the individual’s medical record and is treated as “protected health information” about the individual but often is not protected health information for the family members included in the medical history. In other words, often only patients, and not their family members, may exercise their privacy rights to their own family history information in the same fashion as any other information in their personal medical records, including the ability to elect to control disclosure to others. Duty to Warn and Permission to Warn Family Members A patient’s desire to have medical information kept confidential is one facet of the concept of patient autonomy, in which patients have the right to make their own ▶

CHAPTER 20 — Ethical and Social Issues in Genetics and Genomics 429 improving public health and considered to be in the best interests of the child, questions about newborn screening programs still ar...

Ch20 · Pt6 430 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE decisions about how their individual medical information is used and communicated to others. Medical confidentiality is also an ethical and legal norm. Genetics, however, more than any other branch of medical practice, is concerned with both the patient and the family. A serious ethical and legal dilemma can arise in the practice of genetic medicine when patients’ insistence that their medical information be kept strictly private restrains the geneticist from letting other family members know about their risk for a condition, even when such information could be beneficial to their own health and the health of their children (see Box 20.1). In this situation, is the genetics practitioner obligated to respect patient autonomy by keeping information confidential, or is the practitioner permitted or, more forcefully, does the practitioner have a duty to inform other family members and/or their providers? Is there a duty to warn? If so, is informing patients that they should share the information with relatives sufficient to discharge the practitioner’s duty? Judges have ruled in a number of court cases in the United States on whether or not a health care practitioner is permitted or is even required to override a patient’s wishes for confidentiality. The precedent-setting case was not one involving genetics. In the 1976 State Supreme Court case in California, Tarasoff v. the Regents of the University of California, judges ruled that a psychiatrist who failed to warn law enforcement that his client had declared an intention to kill a young woman was liable in her death. The judges declared that this situation is no different from one in which physicians have a duty to protect the contacts of a patient with a contagious disease by warning them that the patient has the disease, even against the express wishes of the patient. In the realm of genetics, a duty to warn was mandated in a case in New Jersey, Safer v. Estate of Pack (1996), in which a panel of three judges concluded that a physician had a duty to warn the daughter of a man with familial adenomatous polyposis of her risk for colon cancer. The Court found that “there is no essential difference between the type of genetic threat at issue here and the menace of infection, contagion, or a threat of physical harm.” They added that the duty to warn relatives is not automatically fulfilled by telling the patient that the disease is hereditary and that relatives need to be informed. Along similar lines, the 2001 Molloy v. Meier case in the Minnesota Supreme Court found that physicians may owe a duty to warn to third parties who are not patients of the physician, in particular to expecting mothers with respect to genetic risks to their future children. The Molloy case BOX 20.1 CASE STUDIES DUTY TO WARN: PATIENT AUTONOMY AND PRIVACY VERSUS PREVENTING HARM TO FAMILY MEMBERS A woman first presents with an autosomal dominant disorder at the age of 40 years, undergoes testing, and is found to carry a particular variant in a gene known to be causative of this disorder. She is planning to discuss the results with her teenage daughter but insists that her younger adult half-siblings (from her father’s second marriage after her mother’s and father’s divorce) not be told that they might be at risk for this disorder and that testing is available. How does a practitioner reconcile the obligation to respect the patient’s right to privacy with a desire not to cause her relatives harm by failing to inform them of their risk? There are many questions to answer in determining whether “a serious threat to another person’s health or safety” exists to justify unauthorized disclosure of risk to a relative. Clinical Questions What is the penetrance of the disorder, and is it age dependent? How serious is the disorder? Can it be debilitating or life threatening? How variable is the expressivity? Are there interventions that can reduce the risk for disease or prevent it altogether? Is this a condition that will be identified by routine medical care, once it is symptomatic, in time for institution of preventive or therapeutic measures? The risk to half-siblings of the patient is either 50% or negligible, depending on which parent passed the variant allele to the patient. What does the family history reveal, if anything, about the parent in common between the patient and her half-siblings? Is the patient’s mother still alive and available for testing? Counseling Questions Was the patient informed at the time of testing that the results might have implications for other family members? Did she understand in advance that she might be asked to warn her relatives? What are the reasons for withholding the information? Are there unresolved issues, such as resentment, feelings of abandonment, or emotional estrangement, that are sources of psychological pain that could be addressed for her own benefit as well as to help the patient clarify her decision making? Are the other family members already aware of the possibility of this hereditary disease, and have they made an informed choice not to seek testing themselves? Would the practitioner’s warning be seen as an unwarranted intrusion of psychologically damaging information, or would their risk come as a complete surprise? Legal and Practical Questions Does the practitioner have the information and resources required to contact all the half-siblings without the cooperation of the patient? Could the practitioner have reached an understanding, or even a formal agreement, with the patient in advance of testing that she would help in informing her siblings? Would asking for such an agreement be seen as coercive and lead to the patient’s depriving herself of the testing she needs for herself and her children? What constitutes adequate discharge of the practitioner’s duty to warn? Is it sufficient to provide a form letter for the patient to show to relatives that discloses the absolute minimal amount of information needed to inform them of a potential risk? ▶

430 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE decisions about how their individual medical information is used and communicated to others. Medical confidentiality is also an ethical and...

Ch20 · Pt7 CHAPTER 20 — Ethical and Social Issues in Genetics and Genomics 431 bears a degree of resemblance to the Watters v. White case decided by the Quebec Court of Appeal in 2012. The central dispute in that case surrounded the failure of two physicians to provide a warning to relatives of their patients about the possibility their children would inherit a serious genetic disorder. After a decision at trial finding the physicians had not met their obligation to warn, the Court of Appeal overturned, finding, among other things, that the duty to warn operates only in the presence of a clear risk to third parties. Yet, in the genetics context, probabilistic risks are often not clear. In the United Kingdom, the 2020 ABC v. St George’s Healthcare NHS Trust case addressed a similar set of facts. In that case, the UK High Court found that a claimant alleging breach of a duty to warn of genetic risk could recover against a health care provider if, on balance, the need to warn relatives of the risk of disease appears to outweigh the wishes of the affected individual. This decision suggests that health care providers might in some circumstances be obliged to warn relatives of risk of a serious genetic disorder even against a patient’s wishes. Several modern commenters diverge slightly from this perspective, arguing that while physicians may have an ethical duty to provide warnings to third parties, this ethical duty is usually most appropriately discharged by assisting and encouraging their patients to provide warnings to their family members. ARTIFICIAL INTELLIGENCE AND INCREASED DIGITIZATION Hardly any field is as affected by the increasingly significant influence of artificial intelligence (AI) and digitization as genomics. AI systems in genomics research, for example, are capable of sifting through massive volumes of data, assessing linkages, determining patterns, and making novel findings that promise to translate into treatments and prevention that improve human health. The digitization of health information makes it easier than ever to share health genomic data. Cloud computing, for example, provides a secure and efficient mechanism for making genomic data widely available for research, similarly working to accelerate discoveries in the field. Applications of AI and digitization in genomics are many. But the use of AI techniques and increased reliance on digitization also presents a range of challenges, notably that many of these technologies draw on datasets that may work to entrench biases that preexist in the health care system. AI systems that have been trained on data derived from personal health might be especially susceptible to this issue. It is well documented that care access and health outcomes are not equitably distributed across populations. To the extent that AI models learn to make important decisions on the basis of data drawn from contexts in which bias is preexisting, these systems may learn to replicate factors that caused unequal outcomes. AI and digitization also raise substantial concerns about data privacy. As both trends depend on the processing of data drawn from large collections, it may be that certain permutations might lead to the identification of individuals or to data breaches where appropriate safeguards are not maintained. Guidelines from international health organizations, individual national health policy groups, and professional medical organizations are not unanimous on this issue, but some countries have legally mandated communication to at-risk family members. Furthermore, in the United States, the inconsistent case law from state courts must also be considered with respect to legislative and regulatory mandates. Contrary to widespread belief in the United States, the HIPAA Privacy Rule permits a physician to disclose protected health information about a patient to another health care provider who is treating a family member of the physician’s patient without the individual’s authorization, unless the patient has explicitly chosen to impose additional restrictions on the use or disclosure of such protected health information. For example, an individual who has obtained a genetic test may request that the health care provider not disclose the test results. If the health care provider agrees to the restriction, HIPAA prevents disclosing such information without authorization to providers treating other family members who are seeking to identify their own genetic health risks. However, the health care provider should discuss such restrictions with the patient in advance of doing the test and is not obligated to agree to the requested restriction. In France, Article 15 of a newly adopted statute on bioethics has express provisions on familial recontact, including that physicians must inform patients subject to genetic testing that there may be certain risks associated with failing to share genetic information with their relatives. Physicians prescribing tests for a serious genetic abnormality are obliged to enter an agreement with their patient for the possible communication of results identifying the tested abnormality. The tested patient is encouraged to contact potentially affected relatives for whom they are able to obtain contact information. Such communication may be delegated to the physician. Where a diagnosed individual refuses to communicate the diagnosis of a severe genetic abnormality, for which preventative measures are justified, the bioethics statute further requires that a physician communicate the diagnosis in question to the Conseil national pour l'accès aux origines personnelles. The Conseil then informs potentially affected relatives of the diagnosed person. Although the genetics practitioner is most knowledgeable about the clinical aspects of the disease, the relevance of the family history, and the family risk assessment, the many legal and ethical controversies surrounding a possible duty to warn suggest that consultation with legal and bioethics experts is advisable should a conflict arise over the release of a patient’s medical information. ▶

CHAPTER 20 — Ethical and Social Issues in Genetics and Genomics 431 bears a degree of resemblance to the Watters v. White case decided by the Quebec Court of Appeal in 2012. The central dispute in tha...

Ch20 · Pt8 432 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE USE OF GENETIC INFORMATION BY EMPLOYERS AND INSURERS The fourth major ethical principle is justice—the requirement that everyone be able to benefit equally from progress in medical genetics. Justice is a major concern in the area of the use of genetic information in employment and health insurance. Whether healthy individuals could be denied employment or health insurance because they carry a genetic predisposition to disease was not a settled issue in the United States until passage of the landmark Genetic Information Nondiscrimination Act (GINA) of 2008. Under this act, private employers with 15 or more employees are prohibited from deliberately seeking or using genetic information, including family history, to make an employment decision because genetic information was not considered to be relevant to an individual’s current ability to work. Similarly, GINA prohibits most group health insurers from denying insurance or adjusting group premiums based on the genetic information of members of the group. Significantly, GINA does not apply to life, disability, and long-term care insurance. Insurers that sell such products insist that they must have access to all pertinent genetic information about individuals that they themselves have when making a decision to purchase one of these policies. Life insurance companies calculate their premiums on the basis of actuarial tables of age-specific survival averaged over the population; premiums will not cover losses if individuals with private knowledge that they are at higher risk for disease conceal this information and buy extra life or long-term disability insurance, a practice referred to as adverse selection. If adverse selection were widespread, the premiums for the entire population would have to increase so that in essence, the entire population would be subsidizing the increased coverage for a minority. Adverse selection is likely to be a real phenomenon in some circumstances; in one study of asymptomatic individuals tested for the APOE ε4 allele, those who chose to know that they tested positive were found to be nearly six times more likely to purchase extra long- term care insurance than those who did not choose to know their APOE genotype. Knowledge that one carried an APOE ε4 allele did not, however, affect life, health, or disability insurance purchases. There must be, however, a clear distinction between what are already phenotypic manifestations of a disease, such as hypertension, hypercholesterolemia, and diabetes mellitus, and what are predisposing alleles, such as BRCA1 mutations (see Chapter 16) and APOE ε4 alleles (see Chapters 9 and 13), that may never result in overt disease in the individual who carries such an allele. At present, there is little evidence that life insurance companies have actually engaged in discriminatory underwriting practices on the basis of genetic testing. Nevertheless, the fear of such discrimination, and the negative impact that discrimination would have on people obtaining clinical testing for their own health benefit as well as on their willingness to participate in genetic research, has led to proposals to ban the use of genetic information in life insurance. In the United Kingdom, for example, life insurance companies have voluntarily agreed to an extended moratorium on the use of genetic information in most life underwriting, except when large policies are involved, or, in the specific case of Huntington disease, for which disclosure of a positive test result by the patient is required. Countries outside of the United States take a variety of statutory approaches on discrimination as concerns life and disability insurance. In 2017, Canada, a country with universal health care, adopted the Genetic Non-Discrimination Act. The Act prohibits employers and insurance providers from requiring genetic testing. Insurers in particular are prohibited from requiring the provision of genetic test results as a provision of coverage. Certain jurisdictions, including France, adopt a broad- based human rights approach to prohibiting genetic discrimination. Others have adopted moratoria in certain applications of genetic testing in an effort to curb potential discrimination. The United Kingdom and Australia have taken this approach. Still other jurisdictions, such as Japan, rely on existing legal frameworks to prevent genetic discrimination. Importantly, countries with national health care systems may not face substantial problems with genetic discrimination in health insurance though it may affect life and disability insurance as well as employment, hence attracting regulation. For most countries, there is widespread agreement that genetic discrimination should not be permitted in insurance and employment. EUGENIC AND DYSGENIC EFFECTS OF MEDICAL GENETICS The Issue of Eugenics The term eugenics, introduced by Darwin’s cousin Francis Galton in 1883, refers to the improvement of a population by selection of only its “best” specimens for breeding. Plant and animal breeders have followed this practice since ancient times. In the late 19th century, Galton and others began to promote the idea of using selective breeding to improve the human species, thereby initiating the eugenics movement, which was widely advocated for the next half-century. The ideal qualities that the eugenics movement sought to promote through the encouragement of certain kinds of human breeding were more often than not defined by social, ethnic, and economic prejudices and fed by antiimmigrant and racist sentiments in society. What we now would consider a lack of education was described then as familial “feeble-mindedness”; what we now would call rural poverty was considered by eugenicists to be hereditary “shiftlessness.” The scientific difficulties in determining whether traits or characteristics are heritable and to what extent heredity contributes to a trait were badly overestimated because most human traits, even those with some genetic component, are ▶

432 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE USE OF GENETIC INFORMATION BY EMPLOYERS AND INSURERS The fourth major ethical principle is justice—the requirement that everyone be able to...

Ch20 · Pt9 CHAPTER 20 — Ethical and Social Issues in Genetics and Genomics 433 complex in their inheritance pattern and are influenced strongly by environmental factors. Thus, by the middle of the last century, many scientists began to appreciate the theoretical and ethical difficulties associated with eugenics programs. Eugenics is commonly thought to have been largely discredited when it was resurrected and used in Nazi Germany as a justification for mass murder. However, it should be pointed out that in North America and Europe, involuntary sterilization of institutionalized individuals deemed to be mentally incompetent or disabled was carried out under laws passed in the early part of the 20th century in support of eugenics and was continued for many years after the Nazi regime was destroyed. Genetic Counseling and Eugenics Genetic counseling, with the aim of helping patients and their families manage the pain and suffering caused by genetic disease, should not be confounded with the eugenic goal of reducing the incidence of genetic disease or the frequency of alleles considered deleterious in the population. Helping patients and families come to free and informed decisions, particularly concerning reproduction, without coercion, forms the basis for the concept of nondirective counseling (see Chapter 17). Nondirectiveness asserts that individual autonomy is paramount and must not to be subordinated to reducing the burden of genetic disease on society or to a theoretical goal of “improving the gene pool.” Some, however, have argued that true nondirective counseling is a myth, often acclaimed but not easy to accomplish because of the personal attitudes and values the counselor brings to the counseling session. Moreover, patients often explicitly request more directed guidance. Nonetheless, despite the difficulties in attaining the ideal of nondirective counseling, the ethical principles of respect for autonomy, beneficence, nonmaleficence, and justice remain at the heart of all genetic counseling practice, particularly in the realm of individual reproductive decision making. The Issue of Dysgenics The opposite of eugenics is dysgenics, a deterioration in the health and well-being of a population by practices that allow the accumulation of deleterious alleles. In this regard, the long-term impact of activities in medical genetics that can affect gene frequencies and the incidence of genetic disease may be difficult to predict. In the case of some single-gene defects, medical treatment can have a dysgenic effect by reducing selection against a particular genotype, thereby allowing the frequency of harmful genes and consequently of disease to increase. The effect of relaxed selection is likely to be more striking for autosomal dominant and X-linked disorders than for autosomal recessive disorders, in which the majority of variant alleles is found in silent heterozygous carriers. For example, if successful treatment of Duchenne muscular dystrophy were to be achieved, the incidence of the disease would rise sharply because the DMD genes of the affected males would then be transmitted to all their daughters. The effect of this transmission would be to greatly increase the frequency of carriers in the population. In contrast, if all persons affected with cystic fibrosis could survive and reproduce at a normal rate, the incidence of the disease would rise from 1 in 2000 to only ~1 in 1550 over 200 years. Common genetic disorders with complex inheritance, discussed in Chapter 9, could theoretically also become more common if selection were removed, although it is likely that as with autosomal recessive diseases, most of the many susceptibility alleles are distributed among unaffected individuals. Consequently, reproduction by affected persons would have little effect on susceptibility allele frequencies. As prenatal diagnosis (see Chapter 18) becomes widespread, increasing numbers of pregnancies in which the fetus has inherited a genetic defect may be terminated. The effect on the overall incidence of disease is quite variable. In a disorder such as Huntington disease, prenatal diagnosis and pregnancy termination would have a large effect on the incidence of the responsible gene. For most other severe X-linked or autosomal dominant disorders, some reduction might occur, but the disease will continue to recur owing to new mutations. In the case of autosomal recessive conditions, the effect on the frequency of the mutant allele, and consequently of the disease, of aborting all affected pregnancies would be small because most of these alleles are carried silently by heterozygotes. One theoretical concern is the extent to which pregnancy termination for genetic reasons is followed by reproductive compensation—that is, by the birth of additional, unaffected children, many of whom are carriers of the deleterious gene. Some families with X-linked disorders have chosen to terminate pregnancies in which the fetus was male, but of course daughters in such families, although unaffected, may be carriers. Thus reproductive compensation has the potential long- term consequence of increasing the frequency of the genetic disorder that led to the loss of an affected child. GENETICS AND GENOMICS IN MEDICINE The 20th century will be remembered as the era that began with the rediscovery of Mendel’s laws of inheritance and their application to human biology and medicine, continued with the discovery of the role of DNA in heredity, and culminated in the completion of the Human Genome Project. At the beginning of the 21st century, the human species has, for the first time: An increasingly complete representative sequence of its own DNA A comprehensive, albeit likely incomplete, inventory of its genes A vigorous ongoing effort to identify and characterize variants in DNA sequence and copy number ▶

CHAPTER 20 — Ethical and Social Issues in Genetics and Genomics 433 complex in their inheritance pattern and are influenced strongly by environmental factors. Thus, by the middle of the last century,...

Ch20 · Pt10 434 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE A rapidly expanding knowledge base that includes diverse populations and in which various diseases and disease predispositions will be attributable to such variation Powerful new sequencing technologies that allow sequencing of an exome or genome at a tiny fraction of the cost of the first human genome sequence With such knowledge comes powerful capabilities as well as great responsibilities. Ultimately, genetics and genomics in medicine is not about knowledge for its own sake, but for the sake of sustaining wellness, improving health, relieving suffering, and enhancing human dignity. The challenge confronting us all, both future health professionals and members of society at large, is to make sure that the advances in human genetics and genomics knowledge and technology are used responsibly, fairly, and humanely. ACKNOWLEDGMENTS The authors (Bartha Maria Knoppers and Ma’n H. Zawati) would like to acknowledge the contributions of the original authors of this chapter. They would also like to acknowledge the assistance of Michael Lang and Minh Thu Nguyen, both Academic Associates at the Centre of Genomics and Policy in Mc Gill University’s Faculty of Medicine and Health Sciences. Ma’n H. Zawati acknowledges the generous support of the Fonds de recherche du Québec-Santé Junior 1 Research Scholar program. GENERAL REFERENCES Beauchamp TL, Childress JF: Principles of biomedical ethics, ed 5, New York, 2001, Oxford University Press. Gostin LO, Wiley LF: Public health law and ethics, ed 3, Oakland, 2018, University of California Press. Kevles D: In the name of eugenics: genetics and the uses of human heredity, Cambridge, 1995, Harvard University Press. Milunsky A, Milunsky JM, editors: Genetic disorders and the fetus— Diagnosis, prevention, and treatment ed 8, Oxford, 2021, Wiley Blackwell. REFERENCES FOR SPECIFIC TOPICS American Academy of Pediatrics: Policy statement: ethical and policy issues in genetic testing and screening of children, Pediatrics 131:620–622, 2013. Biesecker LG: Incidental variants are critical for genomics, Am J Hum Genet 92:648–651, 2013. Borry P: Genetic testing in asymptomatic minors, Eur J Hum Gen 17:711–719, 2009. Canadian Pediatric Society: Guidelines for genetic testing of healthy children, Paediatr Child Health 8(1):42–45, 2003. Elger B, Michaud K, Mangin P: When information can save lives: the duty to warn relatives about sudden cardiac death and environmental risks, Hastings Center Report 40:39–45, 2010. Global Alliance for Genomics and Health: 2021 policy on clinically actionable genomic research results POL 007, v 1.0, 2021. HIPAA regulations on family history. http://www.hhs.gov/ocr/privacy/ hipaa/faq/family_medical_history_information/index.html. Human Cell Atlas: Ethics Toolkit, Pediatric Template Consent Form, v. 1.0 (2022). https://docs.google.com/document/d/11-B68-w O5rl Ouz OKx A9v Ma D7Tonhb 7W8/edit. Joly Y: Looking beyond GINA: policy approaches to address genetic discrimination, Ann Rev Genomics Hum Genet 21:491–507, 2020. Kleiderman E, Ravitsky V, Knoppers BM: The ‘serious’ factor in germline modification. J Med Ethics 45:508–513, 2019. Knoppers BM, Doerr M, Wallace S, et al: Pediatric Consent to Genetic Research: Clauses, Global Alliance for Genomics and Health, 2021. <https://www.ga 4gh.org/wp-content/uploads/Pediatric-Consent-toGenetic-Research_-Clauses-1.pdf>. Knoppers BM, Isasi R, Caulfield T, et al: Human gene editing: revisiting Canadian policy, NPJ Regen Med 2:3, 2017. Knoppers BM, Kekesi-Lafrance K: The genetic family as patient? Am J Bioethics 20(6):77–80, 2020. Knoppers BM, Thorogood A, Zawati MH: Letter: relearning the 3 R’s? Reinterpretation, recontact, and return of genetic variants, Genet Med, 2019. Mac Ewen JE, Boyer JT, Sun KY: Evolving approaches to the ethical management of genomic data, Trends Genet 29:375–382, 2013. Martin AR: Clinical use of current polygenic risk scores may exacerbate health disparities, Nat Genet 51:584–591, 2019. Mc Guire AL, Joffe S, Koenig BA, et al: Point-counterpoint, Ethics and genomic incidental findings, Science 340:1047–1048, 2013. Middleton A: Professional duties are now considered legal duties of care within genomic medicine, Eur J Human Genet 28:1301–1304, 2020. Offit K, Thom P: Ethicolegal aspects of cancer genetics, Cancer Treat Res 155:1–14, 2010. Rothstein M: Reconsidering the duty to warn genetically at-risk relatives, Genet in Med 20:285–290, 2018. Shabani M, Borry P: Rules for processing genetic data for research purposes in view of the new EU General Data Protection Regulation, Eur J Hum Gen 26(2):149–156, 2018. Thorogood A, Zawati MH, Knoppers BM: Oversight, governance, and policy for making decisions about return of individual genomic findings, Sec Find Genom Res Trans Appl Genomics 29–41, 2020. Visscher PM, Gibson G: What if we had whole-genome sequence data for millions of individuals? Genome Med 5:80, 2013. Yurkiewicz IR, Korf BR, Lehmann LS: Prenatal whole-genome sequencing—Is the quest to know a fetus’s future ethical? N Engl J Med 370:195–197, 2014. Zawati MH, Thorogood A: The physician who knew too much: a comment on Watters v White, Health L J 21:1–27, 2014. PROBLEMS 1. A couple with two children is referred for genetic counseling because their younger son, age 12 years, has a movement disorder for which testing for juvenile Huntington disease (Case 24) is being considered. What are the ethical considerations for the family in testing? 2. A research project screened more than 40,000 consecutive, unselected births for the number of X chromosomes and the presence of a Y chromosome and correlated the sex chromosome karyotype with the sex assigned by visual inspection in the newborn nursery. The purpose of the project was to observe infants with sex chromosome abnormalities (see Chapter 6) prospectively for developmental difficulties. What are the ethical considerations in carrying out this project? 3. In the case described in the Box in the section on duty to warn, consider what might be your course of action if you were the genetic counselor and the disease in question were the following: hereditary breast and ovarian cancer due to BRCA1 mutations (see Chapter 16) (Case 7); malignant hyperthermia due to RYR1 (ryanodine receptor) variants (see Chapter 19); early-onset, familial Alzheimer disease due to a PSEN1 (presenilin 1) variant (see Chapter 13) (Case 4); neurofibromatosis due to NF1 variants (see Chapter 7) (Case 34); or type II diabetes mellitus. 4. Draw up a list of a dozen genes and disorders that you believe should be analyzed and reported if they emerge as secondary findings during ES/WGS for undiagnosed diseases. Explain how and why you chose each of these dozen genes and conditions. ▶

434 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE A rapidly expanding knowledge base that includes diverse populations and in which various diseases and disease predispositions will be attri...

🧬 Part 10: Prenatal Diagnosis, Genomic Medicine, and Ethical Issues English

Chapter 18: Preconception and Prenatal Screening and Diagnosis

Chapter 19: Application of Genomics to Medicine and Individualized Health Care

Chapter 20: Ethical and Social Issues in Genetics and Genomics