Part 4 — Thompson & Thompson Genetics and Genomics in Medicine

Chapter 7: Patterns of Single Gene Inheritance

Ch7 · Pt1 chapter 7 Patterns of Single-Gene Inheritance Neal Sondheimer In Chapter 1 we introduced and briefly characterized the three main categories of genetic disorders – single gene, chromosomal, and complex. In this chapter the typical patterns of transmission of single-gene disorders are discussed in detail, building on the mechanisms of gene and genome transmission presented generally in Chapters 2 and 3; the emphasis here is on the various inheritance patterns of genetic disease in families. Later, in Chapter 9, we will examine more complex patterns of inheritance, including multifactorial disorders that result from the interaction between variants at one or more genes, as well as environmental factors. OVERVIEW AND CONCEPTS Genotype and Phenotype For autosomal loci (and X-linked loci in females), the genotype of a person at a locus is determined by the alleles occupying that locus on the two homologous chromosomes (Fig. 7.1). Genotype should not be confused with haplotype, which refers to the set of alleles at two or more neighboring loci on one of the two homologous chromosomes. More broadly, the term genotype can refer to all the allele pairs that collectively make up an individual’s genetic constitution across the entire genome. Phenotype, as described initially in Chapter 3, is the expression of genotype as a morphologic, clinical, cellular, or biochemical trait, which may be clinically observable or may only be detected by blood or tissue testing. The phenotype can be qualitative – such as the presence or absence of a disease – or can be quantitative, such as measured body mass index or a range of blood glucose levels. A phenotype may, of course, be either normal or pathologic in a given individual, but in this book, which emphasizes disorders of medical significance, the focus is on disease phenotypes (i.e., genetic disorders). When a person has a pair of identical alleles at a locus encoded in nuclear DNA, they are said to be homozygous, or a homozygote. When the combination of alleles matches to the human reference genome it is referred to as homozygous wild-type. It is important to understand that the reference sequence is merely one possible combination of alleles and that many allelic variants are not associated with disease. When two different sets of alleles are present at a locus, a person is heterozygous, or a heterozygote. The term compound heterozygote is used to describe a genotype in which two different variants from a reference sequence are present, rather than one wild-type and one variant allele. These terms (homozygous, heterozygous, and compound heterozygous) can be applied either to a person or to a genotype. In the special case in which an XY male has a variant allele for a gene located on the X chromosome, they are referred to as hemizygous. Mitochondrial DNA is still another special case. In contrast to the two copies of each gene per cell, mitochondrial DNA molecules are typically present in hundreds or thousands of copies per cell (see Chapter 2). For this reason, the terms homozygous, heterozygous, and hemizygous are not used to describe genotypes at mitochondrial loci. A single-gene disorder is one that is determined primarily by the alleles at a single locus. The known single-gene diseases are maintained in Online Mendelian Inheritance in Man (OMIM; https://omim.org), an indispensable resource for medical geneticists created by the late Victor A. Mc Kusick. Most of these diseases follow one of the classic inheritance patterns in families (autosomal recessive, autosomal dominant, X linked) and are therefore referred to as mendelian because, like the characteristics of the garden peas Gregor Mendel studied, they occur on average in fixed and predictable proportions among the offspring of specific types of matings. OMIM additionally catalogues mitochondrial disorders, defects due to imprinting, and disorders where the genetic basis is not yet known, as well as genes of known function. Pathogenic sequence variants in a single gene may produce diverse phenotypic effects in multiple organ systems, with a variety of signs and symptoms occurring at different points during the life span. To cite just one example, individuals with a pathogenic variant in the VHL gene can have hemangioblastomas of the brain, spinal cord, and retina; renal cysts; pancreatic cysts; renal cell carcinoma; pheochromocytoma; endolymphatic tumors of the inner ear; as well as tumors of the epididymis in males or of the broad ligament of the uterus in females. All of these disease manifestations stem from the same single variant. Under these ▶

chapter 7 Patterns of Single-Gene Inheritance Neal Sondheimer In Chapter 1 we introduced and briefly characterized the three main categories of genetic disorders – single gene, chromosomal, and comple...

Ch7 · Pt2 110 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE circumstances, the disorder is said to exhibit pleiotropy (from Greek pleion and tropos, “more turns”), and the expression of the gene defect is said to be pleiotropic. Many pleiotropic effects are due to differences in the role of a gene in distinct cell types. With the example of VHL, the impact of pathogenic variants in VHL is cell type specific because the loss of cell cycle regulation gives rise to characteristic problems in specific cell types. Single-gene disorders affect children disproportionately but not exclusively. Serious single-gene disorders affect 1 in 300 neonates and are responsible for an estimated 16% of pediatric hospitalizations. Although less than 10% of single-gene disorders manifest after puberty, and only 1% occur after the end of the reproductive period, mendelian disorders are nonetheless important to consider in adult medicine. There are hundreds of mendelian disorders whose phenotypes include common adult illnesses such as heart disease, stroke, cancer, and diabetes. Although mendelian disorders are by no means the major contributory factor in causing these common diseases in the population at large, they are important in individual patients because of their significance for the health of other family members and because of the availability of genetic testing and detailed management options for many of them. Penetrance and Expressivity For some genetic conditions, a disease-causing genotype is always fully expressed at birth as an abnormal phenotype. Clinical experience, however, teaches that other disorders are not expressed at all or may vary substantially in their signs and symptoms, clinical severity, or age of onset, even among members of a family who all share the same disease-causing genotype. Geneticists use distinct terms to describe such differences in clinical expression. Penetrance is the probability that an allele or alleles will have any phenotypic expression at all. When the frequency of expression of a phenotype is less than 100% – that is, when some of those who have the relevant genotype completely fail to express it – the disorder is said to show reduced or incomplete penetrance. Penetrance is all or nothing. It is the percentage of people at any given age with a predisposing genotype who are affected, regardless of the severity. Penetrance of some disorders is age dependent – that is, it may occur any time, from early in intrauterine development all the way to the postreproductive years. Some disorders are lethal prenatally, whereas others can be recognized prenatally (e.g., by ultrasonography; see Chapter 18) but are consistent with a live born infant; still others may be recognized only at birth (congenital). Other disorders have their onset typically or exclusively in childhood or in adulthood. It is even possible that two individuals in the same family with the same diseasecausing genotype may develop the disease at very different ages. In contrast to penetrance, expressivity refers not to the presence or absence of a phenotype but to the severity of expression of that phenotype among individuals with the same disease-causing genotype. When the severity of disease differs in people who have the same genotype, the phenotype is said to show variable expressivity. Even in the same family, two individuals carrying the same pathogenic variants may have some signs and symptoms in common, whereas their other disease manifestations may be quite different, depending on which tissues or organs happen to be affected. The challenge to the clinician caring for these families is to not miss very subtle signs of a disorder in a family member and, as a result, either mistake mild expressivity for lack of penetrance or infer that the individual does not have the disease-causing genotype. Phenotype Locus 1 Locus 2 A B a b Figure 7.1 The concepts of genotype and phenotype. (Left) The genotype refers to information encoded in the genome. Diagram of one pair of homologous chromosomes and two loci on that chromosome, Locus 1 and Locus 2, in an individual who is heterozygous at both loci. They have alleles A and a at locus 1 and alleles B and b at locus 2. The locus 1 genotype is Aa, while the locus 2 genotype is Bb. The two haplotypes on these homologous chromosomes are A-B and a-b. (Right) The phenotype is the physical, clinical, cellular, or biochemical manifestation of the genotype, as illustrated here by morphometric aspects of an individual’s face. ▶

110 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE circumstances, the disorder is said to exhibit pleiotropy (from Greek pleion and tropos, “more turns”), and the expression of the gene defec...

Ch7 · Pt3 CHAPTER 7 — Patterns of Single-Gene Inheritance 111 PEDIGREES Single-gene disorders are characterized by their patterns of transmission in families. A usual first step is to obtain information about the patient’s family history and to summarize the details in the form of a pedigree – a graphical representation of the family tree – with use of standard symbols (Fig. 7.2). Some of these symbols and drawing styles are strongly established, such as the use of a square symbol for a male and a circle for a female. Others vary among users and evolve to accommodate changing needs (e.g., to differentiate sex from gender or Figure 7.2 Symbols used in pedigree charts. Although there is no uniform system of pedigree notation, the symbols used here are commonly used by professionals in the field of genetic counseling. 1Modifiers below symbol: AMAB (assigned male at birth), AFAB (assigned female at birth), UAAB (unassigned at birth), no notation = unknown or not specified 2LMP = last menstrual period (date) 3Note that this symbol may be inappropriate when multiple genotypes are involved (Practice Resource Focused Revision: Standardized pedigree nomenclature update centered on sex and gender inclusivity: A practice resource of the National Society of Genetic Counselors Robin L. Bennett et al.) J Genet Couns. 2022;00:1–11. Male Spontaneous abortion /miscarriage Stillbirth SB Sex or gender unknown or non-binary 1 Female 2 Number of individuals in specified category Affected with phenotype specified in key Segments indicate components of phenotype as specified in key Currently non-expressing carrier (typically for dominant phenotype) Monozygotic twins Dizygotic twins Twins of unknown zygosity? Abortion /termination of pregnancy (TOP) Carrier for recessive phenotype specified in key 3 Marriage or union Proband Consultand P C Union ended Consanguineous union Sibship Adopted out of family Sibship with known genotypes CFTR: F508del/F508del CFTR: +/+ Deceased Pregnancy with information LMP [date]2 P Adopted into family No offspring Infertility [Cause, if known] ▶

CHAPTER 7 — Patterns of Single-Gene Inheritance 111 PEDIGREES Single-gene disorders are characterized by their patterns of transmission in families. A usual first step is to obtain information about t...

Ch7 · Pt4 112 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE to accommodate assisted reproduction options). How to differentiate phenotype and genotype can be a point for consideration, especially as sequence information becomes more prevalent. Many professionals advocate the need for standardization, particularly as computergenerated pedigree drawings become more widespread, but there is not yet one established authority. Drawings in this text reflect a variety of current styles of presenting such pedigrees. The most important considerations are to be clear and practical and to define the symbols and abbreviations used for the drawing. The extended family depicted in such pedigrees is a kindred (Fig. 7.3). An affected individual through whom a family is first brought to medical attention (i.e., is ascertained) is the proband, propositus, or index case. The person who consults a health professional is referred to as the consultand (or perhaps patient or client) who may or may not themself be affected. Probands and consultands are sometimes differentiated on the pedigree with P or C beside their respective arrows. A family may have more than one proband if they are ascertained through more than one source. Brothers and sisters are called sibs or siblings, and a family of sibs forms a sibship. Relatives are classified as first degree (parents, sibs, and offspring), second degree (grandparents and grandchildren, uncles and aunts, nephews and nieces, and half-sibs), or third degree (e.g., first cousins), and so forth, depending on the number of steps in the pedigree between the two relatives. Couples who have one or more ancestors in common are consanguineous. If the proband is the only affected member in a family, that person is an isolated (or sometimes sporadic) case. When there is a definitive diagnosis based on comparisons to other patients, wellestablished patterns of inheritance in other families with the same disorder can often be used as a basis for counseling, even with an isolated case. Examining a pedigree is an essential first step in determining the inheritance pattern of a genetic disorder in a family. There are, however, situations that may make this difficult to discern in an individual pedigree. For example, in a family with a lethal disorder affecting a fetus early in pregnancy, one may observe only multiple miscarriages or reduced fertility. For phenotypes with delayed onset, a family may include members who have not yet reached the age at which the disease reveals itself. Nonpenetrance or variable expressivity may make it difficult to obtain accurate information about the existence of relatives carrying a pathogenic genotype. Family relationships may be inaccurately described. Finally, in smaller families, the proband may happen to be the only affected family member, making determination of any inheritance pattern very difficult. PATTERNS OF INHERITANCE The patterns of inheritance shown by single-gene disorders in families depend chiefly on two factors: Whether the chromosomal location of the gene locus is on an autosome (chromosomes 1–22), on a sex chromosome (X and Y chromosomes), or in the mitochondrial genome Whether the phenotype is dominant (expressed when only one chromosome carries the pathogenic allele) or recessive (expressed only when both chromosomes of a pair carry pathogenic alleles at a locus) The different patterns of transmission of the autosomes, sex chromosomes, and mitochondria during meiosis result in distinctive inheritance patterns of pathogenic alleles on these different types of chromosome (see Chapter 2). Because only one of the two copies of each autosome passes into a single gamete during meiosis, males and females heterozygous for a I II III IV V 1 8 7 6 5 4 3 2 1 2° 4° 4° 1° 1° 1° *2° and *4° 1 2 3 4 6 5 4 3 2 1 1 2 5 6 7 8 9 2° 2° 2° 2° 2° 2° 1° 1° 1° 3° 1° 1° 1° 3° 3 4 Figure 7.3 Relationships within a kindred. Generations are designated with roman numerals; individuals within each generation are specified by arabic numerals above the symbols. The proband, III-5(arrow), represents an isolated case of a genetic disorder. She has four siblings: III-3, III-4, III-7, and III-8. Her partner/spouse is III-6, and they have three children (their F1 progeny). The proband has nine first-degree (1°) relatives (her parents, siblings, and offspring), nine second-degree (2°) relatives (grandparents, uncles and aunts, nieces and nephews, and grandchildren), two third-degree (3°) relatives (first cousins), and four fourth-degree (4°) relatives (first cousins once removed). IV-3, IV-5, and IV-6 are second cousins of IV-1 and IV-2. IV-7 and IV-8, whose parents are consanguineous, are doubly related to the proband: second-degree relatives through their father and fourth-degree relatives through their mother. ▶

112 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE to accommodate assisted reproduction options). How to differentiate phenotype and genotype can be a point for consideration, especially as s...

Ch7 · Pt5 CHAPTER 7 — Patterns of Single-Gene Inheritance 113 pathogenic allele on an autosome have a 50% chance of passing that allele on to any offspring, regardless of the child’s sex. Pathogenic alleles on the X chromosome, however, are not distributed equally to sons and daughters. Males pass their Y chromosome to their sons and their X to their daughters; they therefore cannot pass an allele on the X chromosome to their sons and always pass the allele to their daughters (unless it is at one of the pseudoautosomal loci; see Chapter 6). Because mitochondria are inherited from the mother only, regardless of the sex of the offspring, variants in the mitochondrial genome are not inherited according to a mendelian pattern. Autosomal, X-linked, and mitochondrial inheritance will be discussed in the rest of the chapter that follows. Dominant and Recessive Traits Autosomal Loci As classically defined, a phenotype is recessive if it is expressed only in homozygotes or compound heterozygotes, all of whom lack a wild-type allele, and never in heterozygotes, who do have a wild-type allele. In contrast, a dominant inheritance pattern occurs when a phenotype is expressed in heterozygotes as well as in homozygotes (or compound heterozygotes). For the vast majority of inherited dominant diseases, homozygotes or compound heterozygotes for pathogenic alleles at autosomal loci are more severely affected than are heterozygotes, an inheritance pattern known as incompletely dominant (or semidominant). Very few diseases are known in which homozygotes (or compound heterozygotes) show the same phenotype as heterozygotes; such a disorder is referred to as a pure dominant disease. Finally, if phenotypic expression of both alleles at a locus occurs in a compound heterozygote, inheritance is termed codominant. ABO Blood Group. One medically important trait that demonstrates codominant expression is the ABO blood group system important in blood transfusion and tissue transplantation. The A, B, and O alleles at the ABO locus form a three-allele system in which two alleles (A and B) govern expression of either the A or B carbohydrate antigen on the surface of red cells as a codominant trait; a third allele (O) results in expression of neither the A nor the B antigen and is recessive. The difference between the A and B antigen is which of two different sugar molecules makes up the terminal sugar on a cell surface glycoprotein called H. Whether the A or B form of the glycoprotein is made is specified by an enzyme encoded by the ABO gene that adds one or the other sugar molecule to the H antigen, depending on which version of the enzyme is encoded by alleles at the ABO locus. There are, therefore, four phenotypes possible: O, A, B, and AB (Table 7.1). Type A individuals have antigen A on their red blood cells, type B individuals have antigen B, type AB individuals have both antigens, and type O individuals have neither. A feature of the ABO groups not shared by other blood group systems is the reciprocal relationship, in an individual, between the antigens present on the red blood cells and the antibodies in the serum (see Table 7.1). When the red blood cells lack antigen A, the serum contains anti-A antibodies; when the cells lack antigen B, the serum contains anti-B. Formation of anti-A and anti-B antibodies in the absence of prior blood transfusion is believed to be a response to the natural occurrence of A-like and B-like antigens in the environment (e.g., in bacteria). X-Linked Loci For X-linked disorders, a condition expressed only in hemizygotes and never in heterozygotes has traditionally been referred to as an X-linked recessive, whereas a phenotype that is always expressed in heterozygotes as well as in hemizygotes has been called X-linked dominant. Because of epigenetic regulation of X-linked gene expression in carrier females, due to X chromosome inactivation (introduced in Chapters 3 and 6), it can be difficult to determine phenotypically whether a disease with an X-linked inheritance pattern is dominant or recessive. Some geneticists have, therefore, chosen not to use these terms when describing the inheritance of X-linked disease. Strictly speaking, the terms dominant and recessive refer to the inheritance pattern of a phenotype rather than to the alleles responsible for that phenotype. Similarly, a gene is not dominant or recessive; it is the phenotype produced by a particular pathogenic allele in that gene that shows dominant or recessive inheritance. AUTOSOMAL PATTERNS OF MENDELIAN INHERITANCE Autosomal Recessive Inheritance Autosomal recessive disease occurs only in individuals with pathogenic variants on both inherited alleles and no wildtype allele. Such homozygotes or compound heterozygotes TABLE 7.1 ABO Genotypes and Serum Reactivity Genotype Phenotype in RBCs Reaction With Anti-A Reaction With Anti-B Antibodies in Serum OO O − − Anti-A, anti-B AA or AO A + − Anti-B BB or BO B − + Anti-A AB AB + + Neither − Represents no reaction; + represents reaction. RBC, Red blood cell. ▶

CHAPTER 7 — Patterns of Single-Gene Inheritance 113 pathogenic allele on an autosome have a 50% chance of passing that allele on to any offspring, regardless of the child’s sex. Pathogenic alleles on...

Ch7 · Pt6 114 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE must have inherited a pathogenic allele from each parent, each of whom is (barring rare exceptions that we will consider later) a heterozygote for that allele. When a disorder shows recessive inheritance, the pathogenic variant responsible generally reduces or eliminates the function of the gene product: a so-called loss-of-function mutation. For example, many recessive diseases are caused by variants that impair or eliminate the function of an enzyme. In a heterozygote, a remaining normal gene copy is able to compensate for the pathogenic allele and prevent the disease from occurring. However, when no wild-type allele is present, as in homozygotes or compound heterozygotes, disease occurs. Disease mechanisms and examples of recessive conditions are discussed in detail in Chapters 12 and 13. Autosomal recessive disorders may appear whenever two parents are at least carriers for the condition, here with the genotype R/r (Table 7.2). Carriers are unaffected heterozygotes. In the common case where we consider two carrier parents, the risk of transmission of disease is 25%, since each parent passes an allele at random to their offspring. Autosomal recessive disorders may also occur when one parent is a carrier and the other parent has the disease (genotype r/r). In this case the risk of transmission is 50% since the affected parent must transmit a pathogenic allele and the carrier parent transmits the pathogenic allele 50% of the time. Autosomal recessive disorders will always appear in offspring when both parents are affected by the identical condition, since neither parent has a wildtype allele to transmit. Often in autosomal recessive disorders, the proband may be the only affected family member, but if any others are affected, they are usually in the same sibship and not elsewhere in the kindred (Fig. 7.4). Sex-Influenced Autosomal Recessive Disorders Because males and females both have the same complement of autosomes, autosomal recessive disorders generally show the same frequency and severity in males and females. There are, however, exceptions. Some autosomal recessive diseases demonstrate a sex-influenced phenotype – that is, the disorder is expressed in both sexes but with different frequencies or severity. For example, hereditary hemochromatosis is an autosomal recessive phenotype that is 5 to 10 times more common in males than in females (Case 20). Affected individuals have enhanced absorption of dietary iron that can lead I II III IV Figure 7.4 Pedigree showing autosomal recessive inheritance. Filled symbols represent individuals affected with the trait. Those with dots represent obligate carriers of the variant allele for the recessive trait but are unaffected. Many others in the pedigree also have significant likelihood to be carriers. TABLE 7.2 Autosomal Recessive Inheritance Two carrier parents Parent 2 Genotype R/r Gametes Risk for Disease R r Parent 1 genotype R/r Gametes R R/R R/r ¼ Unaffected (R/R) ½ Unaffected carriers (R/r) ¼ Affected (r/r) r R/r r/r Parent 2 genotype r/r gametes One Carrier and One Affected Parent r r Parent 1 genotype R/r Gametes R R/r R/r ½ Unaffected carriers (R/r) ½ Affected (r/r) r r/r r/r Parent 2 genotype r/r gametes Two Affected Parents r r Parent 1 genotype r/r Gametes r r/r r/r All affected (r/r) r r/r r/r The wild-type allele is denoted by uppercase R, a pathogenic allele by lowercase r. ▶

114 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE must have inherited a pathogenic allele from each parent, each of whom is (barring rare exceptions that we will consider later) a heterozygo...

Ch7 · Pt7 CHAPTER 7 — Patterns of Single-Gene Inheritance 115 to iron overload and serious damage to the heart, liver, and pancreas. The lower incidence of the clinical disorder in homozygous females is believed to be due to their lower dietary iron intake, lower alcohol usage, and increased iron loss through menstruation. Gene Frequency and Carrier Frequency Pathogenic alleles responsible for a recessive disorder are generally rare, so most people will not have even one such copy. Because an autosomal recessive disorder must be inherited from both parents, the risk that any carrier will have an affected child depends partly on the chance that their partner is also a carrier of a pathogenic allele for the condition. Thus, knowledge of the carrier frequency of a disease in the population is clinically important for genetic counseling. As an example, the most common autosomal recessive disorder in individuals of European ancestry is cystic fibrosis (CF) (Case 12), caused by pathogenic variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (see Chapter 13). Among this population, ~1 in 2500 individuals has two pathogenic CFTR alleles and has the disease, from which we can infer that 1 in 24 individuals is a carrier. (How one calculates heterozygote frequencies in autosomal recessive conditions will be addressed in Chapter 10.) Pathogenic variants may be transmitted from generation to generation without appearing in a homozygous or compound heterozygous state and causing overt disease. The presence of such hidden recessive genes is not revealed unless the carrier has children with someone who also carries a pathogenic allele at the CFTR locus and both deleterious alleles are inherited. Estimates of the number of deleterious alleles in each of our genomes range from 50 to 200, based on examining an individual’s complete exome or genome sequence for clearly deleterious variants in the coding regions of the genome (see Chapter 4). This estimate is imprecise, however. It may be an underestimate because it does not include variants whose deleterious effect is not obvious from a simple examination of the DNA sequence. Alternatively, it may be an overestimate because it includes variants in many genes that are not known to cause disease. Consanguinity Because most pathogenic variants are generally uncommon in the population, people with rare autosomal recessive disorders are often compound heterozygotes rather than true homozygotes. One well-recognized exception to this rule occurs when an affected individual inherits the exact same pathogenic allele from both parents because the parents are consanguineous (i.e., they are related and carry the identical allele inherited from a common ancestor). Consanguinity in the parents of a patient with a genetic disorder is strong evidence (although not proof) for the autosomal recessive inheritance of that condition. For example, the disorder in the pedigree in Fig. 7.5 is likely to be an autosomal recessive trait, even though other information in the pedigree may seem insufficient to establish this inheritance pattern. Consanguinity is more frequently found in the background of individuals with very rare conditions than in those with more common recessive conditions. This is because it is less likely that two individuals at random in the population will both be carriers of a very rare disorder by chance alone than it is that they would both be carriers because they inherited the same pathogenic allele from a single common ancestor. For example, in xeroderma pigmentosum (Case 48), a very rare autosomal recessive condition of DNA repair (see Chapter 16), more than 20% of cases occur among the offspring of first cousins. In contrast, in more common recessive conditions, most children are born to ostensibly unrelated persons, each of whom happens by chance to be a carrier. Thus, most affected persons with a relatively common disorder, such as phenylketonuria, do not have consanguineous parents because pathogenic variants are common in the general population. How consanguinity is measured is described in Chapter 10. The genetic risk to the offspring of related people is not as great as is sometimes imagined. For first cousins, the absolute risk to offspring, including not only known autosomal recessive diseases but also stillbirth, neonatal death, and congenital malformation, is 3% to 5%, approximately double the overall background risk of 2% to 3% for offspring born to an unrelated couple (see Chapter 17). Because consanguinity can be seen in any population, it is always important to ascertain in every family. CHARACTERISTICS OF AUTOSOMAL RECESSIVE INHERITANCE An autosomal recessive phenotype, if not isolated, is typically seen only in the sibship of the proband, not in parents, offspring, or other relatives. For most autosomal recessive traits, males and females are equally likely to be affected. Parents of an affected child are asymptomatic carriers of pathogenic alleles (obligate carriers). The parents of the affected person may in some cases be consanguineous. This is especially likely if the gene responsible for the condition is rare in the population. I II III IV Figure 7.5 Pedigree in which parental consanguinity suggests autosomal recessive inheritance. Arrow indicates the proband. ▶

CHAPTER 7 — Patterns of Single-Gene Inheritance 115 to iron overload and serious damage to the heart, liver, and pancreas. The lower incidence of the clinical disorder in homozygous females is believe...

Ch7 · Pt8 116 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE The recurrence risk for each sib of the proband is 1 in 4 (25%). Unaffected sibs of proband have a ⅔ chance of being carriers. Autosomal Dominant Inheritance More than half of all known mendelian disorders are inherited as autosomal dominant traits. The incidence of some autosomal dominant disorders can be high. For example, adult polycystic kidney disease (Case 37) occurs in 1 in 1000 individuals in the United States. Other autosomal dominant disorders show a high frequency only in certain populations from specific geographic areas (e.g., the frequency of familial hypercholesterolemia [Case 16] affects 1 in 100 for Afrikaner populations in South Africa; myotonic dystrophy affects 1 in 550 in the Charlevoix and Saguenay–Lac Saint Jean regions of northeastern Quebec). The burden of autosomal dominant disorders is further increased because of their hereditary nature; when they are transmitted through families they raise medical and even social problems, not only for individuals but also for whole kindreds, often through many generations. The risk and severity of dominantly inherited disease in the offspring depend on whether one or both parents are affected and whether the trait is a pure dominant or is incompletely dominant. There are a number of ways that one pathogenic allele can cause a dominantly inherited trait to occur in a heterozygote despite the presence of a normal allele. Disease mechanisms in various dominant conditions are discussed in Chapter 12. Denoting D as the pathogenic variant and d as the wild-type allele, the parents of children with an autosomal dominant disease can be two heterozygotes (D/d) or, more frequently, a heterozygote (D/d) and a homozygote for a normal allele (d/d). TABLE 7.3 Autosomal Dominant Inheritance One Affected Parent and One Unaffected Parent Parent 2 Genotype d/d Gametes Risk for Disease d d Parent 1 genotype D/d Gametes D D/d D/d ½ Affected (D/d) ½ Unaffected (d/d) d d/d d/d Two Affected Parents Parent 2 genotype D/d gametes Risk for Disease D d Parent 1 genotype D/d Gametes D D/D D/d Strictly dominant ¾ Affected (D/D and D/d) ¼ Unaffected (d/d) Incompletely dominant ¼ Severely affected (D/D) ½ Affected (D/d) ¼ Unaffected (d/d) d D/d d/d The pathogenic allele causing dominantly inherited disease is denoted by uppercase D; the normal or wild-type allele is denoted by lowercase d. As seen in Table 7.3, each child born to a couple where one parent has the D/d genotype and the other the d/d genotype has a 50% chance of receiving the affected parent’s allele D and a 50% chance of receiving the normal allele d. In the population as a whole, then, the offspring of D/d by d/d parents are ~50% D/d and 50% d/d. Of course, each pregnancy is an independent event, not governed by the outcome of previous pregnancies. Thus, within a family, the distribution of affected and unaffected children may be quite different from the theoretic expected ratio of 1:1, especially if the sibship is small. Typical autosomal dominant inheritance can be seen in the pedigree of a family with a dominantly inherited form of hereditary deafness (Fig. 7.6A). In practice, homozygotes for dominant phenotypes are not often seen, but the offspring of two affected individuals with the genotype D/d could have a D/D genotype 25% of the time. The potential to observe individuals with the D/D genotype may also be limited if the phenotype causes early lethality (see the description of incompletely dominant inheritance later). Pure Dominant Inheritance As mentioned earlier, very few human disorders demonstrate a purely dominant pattern of inheritance. Even Huntington disease (Case 24), which is frequently considered to be a pure dominant because the nature and severity of symptoms in heterozygotes and homozygotes is similar, appears to have a somewhat accelerated time course from onset to death in homozygous individuals, compared with that of heterozygotes. Incompletely Dominant Inheritance As introduced in Chapter 4, achondroplasia (Case 2) is an incompletely dominant skeletal disorder (shortlimbed dwarfism and large head) caused by certain variants in the fibroblast growth factor receptor 3 gene (FGFR3). Most individuals with achondroplasia have ▶

116 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE The recurrence risk for each sib of the proband is 1 in 4 (25%). Unaffected sibs of proband have a ⅔ chance of being carriers. Autosomal Dom...

Ch7 · Pt9 CHAPTER 7 — Patterns of Single-Gene Inheritance 117 I II III IV I II III 2 3 I II FGFR3 arg 248cys A B C Figure 7.6 (A) Pedigree showing typical inheritance of a form of adult-onset progressive sensorineural hearing loss (DFNA1) inherited as an autosomal dominant trait. (B) Pedigree showing inheritance of achondroplasia, an incompletely dominant (or semidominant) trait. (C) Pedigree showing a sporadic case of thanatophoric dwarfism, a genetic lethal, in the proband (arrow). normal intelligence and lead normal lives within their physical capabilities. A pedigree with two parents heterozygous for the most common pathogenic variant that causes achondroplasia is shown in Fig. 7.6B. The deceased child, individual III-3, was a homozygote for the condition and had a disorder far more severe than in either parent, resulting in death soon after birth. Sex-Limited Phenotype in Autosomal Dominant Disease As discussed earlier for the autosomal recessive condition, hemochromatosis, autosomal dominant phenotypes may also demonstrate a sex ratio that differs significantly from 1:1. Extreme divergence of the sex ratio is seen in sex-limited phenotypes, in which the defect is transmitted as an autosomal trait but expressed in only one sex. An example is male-limited precocious puberty, an autosomal dominant disorder in which affected boys develop secondary sexual characteristics and undergo an adolescent growth spurt at ~4 years of age. In some families, the cause has been traced to variants in the LCGR gene, which encodes the receptor for luteinizing hormone; these pathogenic variants constitutively activate the receptor’s signaling action, even in the absence of its hormone. The defect shows no effect in heterozygous females. The pedigree in Fig. 7.7 shows that, although the disease can be transmitted by unaffected (nonpenetrant carrier) females, it can also be transmitted directly from father to son, showing that it is autosomal, not X linked. For disorders in which affected males do not reproduce, however, it is not always easy to distinguish sexlimited autosomal inheritance from X-linked inheritance, because the critical evidence—absence of male-to-male transmission, cannot be provided. In that case, other lines of evidence, particularly gene mapping to learn whether the responsible gene maps to the X chromosome or to an autosome (see Chapter 11), can determine the pattern of inheritance and the consequent recurrence risk (Box 7.1). I II III Figure 7.7 Part of a large pedigree of male-limited precocious puberty. This autosomal dominant disorder can be transmitted by affected males or by unaffected carrier females. Male-to-male transmission shows that inheritance is autosomal, not X linked. Transmission of the trait through carrier females shows that inheritance cannot be Y linked. Arrow indicates proband. ▶

CHAPTER 7 — Patterns of Single-Gene Inheritance 117 I II III IV I II III 2 3 I II FGFR3 arg 248cys A B C Figure 7.6 (A) Pedigree showing typical inheritance of a form of adult-onset progressive sensor...

Ch7 · Pt10 118 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE A B Figure 7.8 Split-hand deformity, an autosomal dominant trait involving the hands and feet, in a 3-month-old boy. (A) Upper part of body. (B) Lower part of body. (From Kelikian H: Congenital deformities of the hand and forearm, Philadelphia, 1974, WB Saunders.) BOX 7.1 CHARACTERISTICS OF AUTOSOMAL DOMINANT INHERITANCE The phenotype usually appears in every generation, each affected person having an affected parent. Exceptions or apparent exceptions to this rule in clinical genetics are (1) cases originating from new mutations and (2) cases in which the disorder is not expressed (nonpenetrant) or is expressed only subtly in a person who has inherited the responsible pathogenic allele. Any child of an affected parent has a 50% risk for inheriting the trait. This is true for most families, in which the other parent is phenotypically normal. Because statistically each family member is the result of an “independent event,” wide deviation from the expected 1:1 ratio may occur by chance in a single family. Phenotypically normal family members do not transmit the phenotype to their children. Failure of penetrance or subtle expression of a condition may lead to apparent exceptions to this rule. Males and females are equally likely to transmit the phenotype, to children of either sex. In particular, maleto-male transmission can occur, and males can have unaffected daughters. A significant proportion of isolated cases are sporadic due to new mutation. The less the fitness, the greater is the proportion of cases due to new mutation. ▶

Effect of Incomplete Penetrance, Variable Expressivity, and New Mutations on Autosomal Dominant Inheritance Patterns Some of the difficulties raised by incomplete penetrance in fully understanding the...

Ch7 · Pt11 CHAPTER 7 — Patterns of Single-Gene Inheritance 119 Finally, in classic autosomal dominant inheritance, every affected person in a pedigree has an affected parent, who also has an affected parent, and so on, as far back as the disorder can be traced (see Fig. 7.6A). In fact, however, many dominant conditions of medical importance occur because of a spontaneous, de novo mutation in a gamete inherited from a noncarrier parent (see Fig. 7.6C). An individual with an autosomal dominant disorder caused by a new mutation will look like an isolated case, and his or her parents, aunts and uncles, and cousins will all be unaffected noncarriers. This person will still be at risk for passing the altered allele down to his or her own children, however. Once a new mutation has arisen, the variant allele will be transmitted to future generations following standard principles of inheritance; as we discuss in the next section, its survival in the population depends on the fitness of persons carrying it. Relationship Between New Mutation and Fitness in Autosomal Dominant Disorders In many disorders, whether a condition demonstrates an obvious pattern of transmission in families depends on whether individuals affected by the disorder can reproduce. Geneticists coined the term fitness as a measure of the impact of a condition on reproduction. Fitness is defined as the ratio of the number of offspring of individuals affected with the condition who survive to reproductive age, compared to the number of offspring of individuals who do not carry the pathogenic allele. Fitness ranges from 0 (affected individuals never have children who survive to reproductive age) to 1 (affected individuals have the same number of offspring as unaffected controls). Although we will explore the impact of mutation, selection, and fitness on allele frequencies in greater detail in Chapter 10, here we discuss examples that illustrate the major concepts and range of impact of fitness on autosomal dominant conditions. At one extreme are disorders that have a fitness of 0; patients with such disorders never reproduce, and the disorder is referred to as genetic lethal. One example is the severe short-limb dwarfism syndrome known as thanatophoric dysplasia that occurs in heterozygotes for certain pathogenic alterations in the FGFR3 gene (see Fig. 7.6C). Thanatophoric dysplasia is lethal in the neonatal period, and therefore all probands with the disorder must be due to new mutations because these variants cannot be transmitted to the next generation. At the other extreme are disorders that have virtually normal reproductive fitness because of a late age of onset or a mild phenotype that does not interfere with reproduction. If the fitness is normal, the disorder will only rarely be the result of new mutation; a patient is much more likely to have inherited the pathogenic variant, and the pedigree is likely to show multiple affected individuals with clear-cut autosomal dominant inheritance. Lateonset progressive hearing loss is a good example of such an autosomal dominant condition, with a fitness of ~1 (see Fig. 7.6A). Thus, there is an inverse relation between the fitness of a given autosomal dominant disorder and the proportion of individuals with the disorder who inherited the defective gene, versus those who received it due to a new mutation. The measurement of mutation frequency and the relation of mutation frequency to fitness will be discussed further in Chapter 10. It is important to note that fitness is not simply a measure of physical or intellectual disability. Some individuals with an autosomal dominant disorder may appear phenotypically normal but have a fitness of 0; at the other extreme, individuals may have normal or nearnormal fitness, despite being affected by an autosomal dominant condition with an obvious and severe phenotype, such as familial Alzheimer disease (Case 4). X-LINKED INHERITANCE In contrast to genes on the autosomes, genes on the X and Y chromosomes are distributed unequally to males and females in families. The patrilineal inheritance of the Y chromosome is straightforward. However, there are very few strictly Y-linked genes, almost all of which are involved in primary sex determination or the development of secondary male characteristics, as discussed in Chapter 6, and they will not be considered here. Approximately 800 protein-coding and 300 noncoding RNA genes have been identified on the X chromosome to date, of which over 300 genes are presently known to be associated with X-linked disease phenotypes. Phenotypes determined by genes on the X have a characteristic sex distribution and a pattern of inheritance that is usually easy to identify and easy to distinguish from the patterns of autosomal inheritance we just explored. Because males have one X chromosome but females have two, there are only two possible genotypes in males and four in females with respect to pathogenic alleles at an X-linked locus. A male with a pathogenic allele at an X-linked locus is hemizygous for that allele, whereas females may be a homozygote for the wild-type allele, a homozygote for a pathogenic allele, a compound heterozygote for two different pathogenic alleles, or a heterozygous carrier of a pathogenic allele. For example, if XH is the wild-type allele for an X-linked disease gene and Xh, is the disease allele, the genotypes expected in males and females are as in Table 7.4. III II I P Figure 7.9 Pedigree of split-hand deformity demonstrating of non-penetrance in the mother of the proband (arrow) and his sister, the consultand. Reduced penetrance must be taken into account in genetic counseling. ▶

CHAPTER 7 — Patterns of Single-Gene Inheritance 119 Finally, in classic autosomal dominant inheritance, every affected person in a pedigree has an affected parent, who also has an affected parent, and...

Ch7 · Pt12 120 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE X Inactivation, Dosage Compensation, and the Expression of X-Linked Genes As introduced in Chapters 3 and 6, X inactivation is a normal physiologic process in which most of the genes on one of the two X chromosomes in normal females, but not the genes on the single X chromosome in males, are inactivated in somatic cells, thus equalizing the expression of most X-linked genes between the two sexes. The clinical relevance of X inactivation in X-linked diseases is profound. It leads to females having two cell populations, which express alleles of X-linked genes from one or the other of the two X chromosomes (see Fig. 3.14 and further discussion in Chapter 6). These two cell populations are thus genetically identical but functionally distinct, and both cell populations in human females can be readily detected for some disorders. For example, in Duchenne muscular dystrophy (Case 14), female carriers exhibit typical mosaic expression of their dystrophin immunostaining (Fig. 7.10). Depending on the pattern of random inactivation of the two X chromosomes, two female heterozygotes for an X-linked disease may have very different clinical presentations because they differ in the proportion of cells that have the pathogenic allele on the active X in a relevant tissue (as seen in manifesting heterozygotes, as described later). Recessive and Dominant Inheritance of X-Linked Disorders As mentioned earlier in this chapter, the use of the terms dominant and recessive is different for X-linked conditions than for autosomal disorders. So-called X-linked dominant and recessive patterns of inheritance are typically distinguished on the basis of the phenotype in heterozygous females. Some X-linked phenotypes are consistently apparent clinically in carriers, at least to some degree; these are referred to as dominant. Other X-linked phenotypes are typically not observed in heterozygous females and are considered to be recessive. The difficulty in classifying an X-linked disorder as dominant or recessive arises because females who are heterozygous for the same pathogenic allele in a family may or may not demonstrate the disease, depending on the pattern of random X inactivation and the proportion of the cells in pertinent tissues that have the pathogenic allele on the active or inactive X. Nearly a third of X-linked disorders are penetrant in some (but not all) female heterozygotes and cannot be classified as either dominant or recessive. Even for disorders that can be so classified, they show incomplete penetrance that varies as a function of X inactivation patterns, not inheritance patterns. Because clinical expression of an X-linked condition does not depend strictly on the particular gene involved, or even the particular A B C Figure 7.10 Immunostaining for dystrophin in muscle specimens. (A) A normal female (×480). (B) A male with Duchenne muscular dystrophy (DMD) (×480). (C) A carrier female (×240). Staining creates the bright signals seen here encircling individual muscle fibers. Muscle from DMD patients lacks dystrophin staining. Muscle from DMD carriers exhibits both positive and negative patches of dystrophin immunostaining, representing fibers with either the normal or pathogenic allele on the active X. Images courtesy K. Arahata, National Institute of Neuroscience, Tokyo. TABLE 7.4 Genotypes and Phenotypes in X-Linked Disease Genotypes Phenotypes Males Hemizygous XH Unaffected Hemizygous Xh Affected Females Homozygous XH/XH Unaffected Heterozygous XH/Xh Carrier (may or may not be affected) Homozygous (or compound heterozygous) Xh/Xh Affected ▶

120 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE X Inactivation, Dosage Compensation, and the Expression of X-Linked Genes As introduced in Chapters 3 and 6, X inactivation is a normal phys...

Ch7 · Pt13 CHAPTER 7 — Patterns of Single-Gene Inheritance 121 pathogenic variant in the same family, some geneticists have recommended dispensing altogether with the terms recessive and dominant for X-linked disorders. Be that as it may, the terms are widely applied to X-linked disorders, and we will continue to use them, recognizing that they describe extremes of a continuum of penetrance and expressivity in female carriers of X-linked diseases. X-Linked Recessive Inheritance The inheritance of X-linked recessive phenotypes follows a well-defined and easily recognized pattern (Fig. 7.11 and Box 7.2). An X-linked recessive trait is expressed phenotypically in all males who receive the variant allele, and, consequently, X-linked recessive disorders are generally restricted to males. Hemophilia A is a classic X-linked recessive disorder in which the blood fails to clot normally because of a deficiency of factor VIII, a protein in the clotting cascade (Case 21). The hereditary nature of hemophilia and even its pattern of transmission have been recognized since ancient times, and the condition became known as the “royal hemophilia” because of its occurrence among descendants of Britain’s Queen Victoria, who was a carrier. As in the earlier discussion, suppose Xh represents a pathogenic allele of factor VIII causing hemophilia A, and XH represents the normal allele. The sons of a male with hemophilia and a noncarrier female receive their I II III IV 3 3 5 2 Figure 7.11 Pedigree pattern demonstrating an X-linked recessive disorder such as hemophilia A, transmitted from an affected male through females to an affected grandson and great-grandson. TABLE 7.5 X-Linked Recessive Inheritance Affected Male and Noncarrier Female Female Genotype XH/XH Gametes Risk for Disease XH XH Male Genotype Xh/Y Gametes Xh XH/Xh XH/Xh All females carriers (XH/Xh) All males unaffected (XH/Y) Y XH/Y XH/Y Unaffected Male and Carrier Female Female Genotype XH/Xh Gametes Risk for Disease XH Xh Male Genotype XH/Y Gametes XH XH/XH XH/Xh ¼ Noncarrier female (XH/XH) ¼ Carrier female (XH/Xh) ¼ Normal male (XH/Y) ¼ Affected male (Xh/Y) Y XH/Y Xh/Y The wild-type allele at the X-linked hemophilia locus is denoted as XH, and the pathogenic allele is denoted as Xh. BOX 7.2 CHARACTERISTICS OF X-LINKED RECESSIVE INHERITANCE The incidence of the trait is much higher in males than in females. Heterozygous females are usually unaffected, but some may express the condition with variable severity as determined by the pattern of X inactivation. The gene responsible for the condition is transmitted from an affected man through all his daughters. Any of his daughters’ sons has a 50% chance of inheriting it. The pathogenic allele is never transmitted directly from father to son, but it is transmitted by an affected male to all his daughters. The pathogenic allele may be transmitted for many generations through a series of carrier females; if so, the affected males in a kindred are related through females. A significant proportion of isolated cases are due to new mutation. ▶

father’s Y chromosome and a maternal X and are unaffected, but the daughters receive the paternal X chromosome with its hemophilia allele and are obligate carriers. Children of obligate carrier female...

Ch7 · Pt14 122 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE More commonly, a female carrier of an X-linked allele who has phenotypic expression of the disease is referred to as a manifesting heterozygote. Whether a female carrier will be a manifesting heterozygote depends on a number of features of X inactivation. First, as we saw in Chapter 3, the choice of which X chromosome is to become inactive is random, but it occurs when there is a relatively small number of cells in the developing female embryo. By chance alone, therefore, the fraction of cells in various tissues of carrier females in which the normal or pathogenic allele happens to remain active may deviate substantially from the expected 50%, resulting in unbalanced or skewed X inactivation (see Fig. 6.12A). A female carrier may have signs and symptoms of an X-linked disorder if the skewed inactivation is unfavorable (i.e., a large majority of the active X chromosomes in pertinent tissues happen to contain the deleterious allele active). Favorably unbalanced or skewed inactivation also occurs, in which the pathogenic allele is preferentially on the inactive X in some or all tissues of an unaffected heterozygous female. Such skewed inactivation may simply be due to chance alone, as we just saw (albeit in the opposite direction). However, in certain X-linked conditions, there is reduced cell survival (or a proliferative disadvantage) for those cells that originally had the pathogenic allele on the active X early in development. This results in a pattern of highly skewed inactivation that favors cells with the normal allele on the active X in relevant tissues. For example, highly skewed X inactivation is the rule in female carriers of certain X-linked immunodeficiencies, in whom only those early progenitor cells that happen to carry the normal allele on their active X chromosome can populate certain lineages in the immune system. X-Linked Dominant Inheritance As discussed earlier, an X-linked phenotype can be described as dominant if it is regularly expressed in heterozygotes. X-linked dominant inheritance (Table 7.6) can readily be distinguished from autosomal dominant inheritance by the lack of male-to-male transmission, which is TABLE 7.6 X-Linked Dominant Inheritance Unaffected Male and Affected Female Female Genotype XD/Xd Gametes Risk for Disease XD Xd Male Genotype Xd/Y Gametes Xd XD/Xd Xd/Xd ¼ Affected females (XD/Xd) ¼ Unaffected females (Xd/Xd) ¼ Affected males (XD/Y) ¼ Unaffected males (Xd/Y) Y XD/Y Xd/Y Affected Male and Affected Female Female Genotype Xd/Xd Gametes Risk for Disease Xd Xd Male Genotype XD/Y Gametes XD XD/Xd XD/Xd All females affected (XD/Xd) All males unaffected (Xd/Y) Y Xd/Y Xd/Y The wild-type allele at the hypophosphatemic rickets locus is denoted as Xd, and the pathogenic allele is denoted as XD. impossible for X-linked inheritance because males transmit the Y chromosome, not the X, to their sons. Thus the distinguishing feature of a fully penetrant X-linked dominant pedigree (Fig. 7.12) is that all the daughters and none of the sons of affected males are affected; if any daughter is unaffected or any son is affected, the inheritance must be autosomal, not X linked. The pattern of inheritance through females is no different from the autosomal dominant pattern; because females have a pair of X chromosomes just as they have pairs of autosomes, each child of an affected female has a 50% chance of inheriting the trait, regardless of sex. Across multiple families with an X-linked dominant disease, the expression is usually milder in heterozygous females because the pathogenic allele is located on the inactive X chromosome in a proportion of their cells. Thus, most X-linked dominant disorders are incompletely dominant, as is the case with most autosomal dominant disorders (Box 7.3). BOX 7.3 CHARACTERISTICS OF X-LINKED DOMINANT INHERITANCE Affected males with unaffected partners have no affected sons and no unaffected daughters. Both male and female offspring of female carriers have a 50% risk for inheriting the phenotype. The pedigree pattern is similar to that seen with autosomal dominant inheritance. Affected females are approximately twice as common as affected males, but affected females typically have milder (although variable) expression of the phenotype. One example of an X-linked dominant disorder is X-linked hypophosphatemic rickets (also known as vitamin D–resistant rickets), in which the ability of the kidney tubules to reabsorb filtered phosphate is impaired. This disorder fits the criterion of an X-linked dominant disorder in that both sexes are affected, although the serum phosphate level is less depressed and the rickets less severe in heterozygous females than in affected males. ▶

X-Linked Dominant Disorders With Male Lethality Although most X-linked conditions are typically apparent only in males, a few rare X-linked defects are expressed exclusively, or almost exclusively, in...

Ch7 · Pt15 CHAPTER 7 — Patterns of Single-Gene Inheritance 123 I II III IV Figure 7.12 Pedigree pattern demonstrating X-linked dominant inheritance. I II III Figure 7.13 Pedigree pattern demonstrating X-linked dominant inheritance of a disorder that is lethal in males during the prenatal period. the same families are completely unaffected (Fig. 7.14). The disorder is due to loss-of-function variants in the protocadherin gene 19, an X-linked gene that encodes a cell surface molecule expressed on neurons in the central nervous system. The explanation for this unusual pattern of inheritance is not clear. It is hypothesized that the epilepsy occurs in females because mosaicism for expression of protocadherin 19, resulting from random X inactivation in the brain, disrupts communication between groups of neurons with and without the cell surface protein. Neurons in males uniformly lack the cell surface molecule, but their brains are apparently spared cell-cell miscommunication by a different, compensating protocadherin. Relationship Between New Mutation and Fitness in X-Linked Disorders Just as with autosomal dominant disorders, new mutations account for a significant fraction of isolated cases of many X-linked diseases. Males carrying variants causing X-linked disorders are exposed to selection that is complete for some disorders, partial for others, and absent for still others, depending on the fitness of the genotype. Males carrying pathogenic alleles for X-linked disorders such as Duchenne muscular dystrophy (Case 14)—a disease of muscle that affects young boys, do not reproduce. Fitness of affected males is currently 0, although the situation I II III 1 2 3 4 5 6 7 8 9 10 11 1 2 1 2 3 4 5 6 7 8 9 10 Figure 7.14 Pedigree pattern of familial female epilepsy and cognitive impairment, demonstrating its X-linked dominant inheritance with sparing of males hemizygous for a premature termination variant in the PCDH19 gene. These X-linked dominant conditions are lethal in males before birth (Fig. 7.13). Typical pedigrees of these conditions show transmission by affected females, who produce affected daughters, normal daughters, and normal sons in equal proportions (1:1:1); affected liveborn males are not seen. Rett syndrome (Case 40) is a striking disorder that occurs nearly exclusively in females and meets all criteria for being an X-linked dominant disorder that is usually lethal in hemizygous males. The syndrome is characterized by normal prenatal and neonatal growth and development, followed by the rapid onset of neurologic symptoms in affected girls. The disease mechanism is thought to reflect abnormalities in the regulation of a set of genes in the developing brain; the cause of male lethality is unknown but presumably reflects a requirement during early development for at least one functional copy of the MECP2 gene on the X chromosome. X-Linked Dominant Disorders With Male Sparing Other disorders are manifest only in carrier females because hemizygous males are largely spared the consequences of the variant they carry. One such disorder is female-limited, X-linked epilepsy and cognitive impairment. Affected females are asymptomatic at birth and appear to be developing normally but begin to have seizures, generally in the second year of life, after which development begins to regress. Most affected females go on to be developmentally delayed, which can vary from mild to severe. In contrast, male hemizygotes in ▶

CHAPTER 7 — Patterns of Single-Gene Inheritance 123 I II III IV Figure 7.12 Pedigree pattern demonstrating X-linked dominant inheritance. I II III Figure 7.13 Pedigree pattern demonstrating X-linked d...

Ch7 · Pt16 124 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE may change as a result of advances in research aimed at therapy for affected boys (see Chapter 14). In contrast, individuals with hemophilia (Case 21) also have reduced fitness, but the condition is not a genetic lethal. Affected males have, on average, ~70% as many offspring as unaffected males do, and fitness of affected males is therefore ~0.70. This fitness may also increase with improvements in the treatment of this disease. When fitness is reduced, the pathogenic alleles that these males carry are lost from the population. In contrast to autosomal dominant conditions, however, pathogenic alleles for X-linked diseases with reduced fitness may be partially or completely protected from selection when present in females. Thus, even in X-linked disorders with a fitness of 0, less than half of new cases will be due to new mutations. The overall incidence of the disease, then, will be determined both by the transmittal of a pathogenic allele from a carrier mother and by the rate of de novo mutations at the responsible locus. The balance between new mutation and selection will be discussed more fully from the population genetics perspective in Chapter 10. PSEUDOAUTOSOMAL INHERITANCE As we first saw in Chapter 2, meiotic recombination between X-linked loci only occurs between the two homologous X chromosomes and is, therefore, restricted to females. X-linked loci do not participate in meiotic recombination in males, who have a Y chromosome and only one X chromosome. There are, however, a small number of contiguous loci, located at the tips of the p and q arms of the sex chromosomes, that are homologous between X and Y and recombine in male meiosis. As a consequence, during spermatogenesis, a pathogenic allele at one of these loci on the X chromosome can be transferred onto the Y chromosome and passed on to male offspring, thereby demonstrating the male-to-male transmission characteristic of autosomal inheritance. Because these unusual loci on the X and Y mimic autosomal inheritance but are not located on an autosome, they are referred to as pseudoautosomal loci; the segments of the X and Y chromosomes where they are located are referred to as the pseudoautosomal regions. One example of a disease caused by a pathogenic variant at a pseudoautosomal locus is dyschondrosteosis, a dominantly inherited skeletal dysplasia with disproportionate short stature and deformity of the forearms. Although a greater prevalence in females than in males initially suggested an X-linked dominant disorder, the presence of male-to-male transmission clearly ruled out strict X-linked inheritance (Fig. 7.15). Variants in the SHOX or SHOXY gene, located in the pseudoautosomal region on Xp and Yp, respectively, are responsible for this condition. MOSAICISM Although we are used to thinking of ourselves as being composed of cells that all carry the same complement of genes and chromosomes, this is in reality an oversimplified view. Mosaicism is the presence in an individual or a tissue of at least two cell lineages that differ genetically but are derived from a single zygote. Mutations that occur after conception in a single cell in either prenatal or postnatal life can give rise to clones of cells genetically different from the original zygote because, given the nature of DNA replication, the altered allele will persist in all the clonal descendants of that cell (Fig. 7.16). Mosaicism for numeric or structural abnormalities of chromosomes is a clinically important phenomenon (see Chapters 5 and 17), and somatic mutation is recognized as the major contributor to most types of cancer (see Chapter 16). Mosaicism can affect any cells or tissue within a developing embryo or at any point from after conception to adulthood. It can be a diagnostic dilemma to determine just how widespread the mosaic pattern is. For example, the population of cells that carry a mutation in a mosaic pregnancy might be (a) found only in extraembryonic tissue and not in the embryo proper (confined placental mosaicism, see Chapter 18), (b) present in some tissues of the embryo but not in the gametes (pure somatic mosaicism), (c) restricted to the gamete lineage only and nowhere else (pure germline mosaicism), or (d) present in both somatic lineages and the germline. This all depends on whether the mutation occurred before or after the separation of the inner cell mass, the germline cells, and the somatic cells during embryogenesis (see Chapter 18). Because there are ~30 mitotic divisions in the cells of the germline before meiosis in the female and several hundred in the male (see Chapter 2), there is ample opportunity for mutation to occur in germline I II III IV Figure 7.15 Pedigree showing inheritance of dyschondrosteosis due to variants in SHOX, a pseudoautosomal gene on the X and Y chromosomes. The arrow shows a male who inherited the trait on his Y chromosome from his father. His father, however, inherited the trait on his X chromosome from his mother. From Shears DJ, Vassal HJ, Goodman FR, et al: Mutation and deletion of the pseudoautosomal gene SHOX cause Leri-Weill dyschondrosteosis, Nat Genet 19:70–73, 1998. ▶

124 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE may change as a result of advances in research aimed at therapy for affected boys (see Chapter 14). In contrast, individuals with hemophilia...

Ch7 · Pt17 CHAPTER 7 — Patterns of Single-Gene Inheritance 125 cells after the separation from somatic cells, resulting in pure gonadal mosaicism. Determining whether mosaicism for a mutation is present only in the germline or only in somatic tissues may be difficult. Failure to find evidence in a subset of cells from a readily accessible somatic tissue (e.g., peripheral white blood cells, skin, or buccal cells) does not ensure that the mutation is not present elsewhere in the body, including the germline. Segmental Mosaicism A mutation affecting morphogenesis and occurring during embryonic development might be manifested as a segmental or patchy abnormality, depending on the stage at which the mutation occurred and the lineage of the somatic cell in which it originated. For example, NF1 (Case 34) is sometimes segmental, affecting only one part of the body. Segmental NF1 is caused by somatic mosaicism for the outcome of a mutation that occurred after conception. Although the parents of such an individual would be unaffected and considered not at risk for transmitting the mutated allele, a patient with segmental NF1 could be at risk for having an affected child, whose phenotype would be typical for NF1; that is, not segmental. Whether the individual is at risk for transmitting the defect will depend on whether the mutation occurred before separation of germline cells from the somatic cell line. Germline Mosaicism In pedigrees with germline mosaicism, unaffected individuals with no evidence of a given disease-causing mutation in their genome (as evidenced by the failure to find an altered allele the mutation in DNA extracted from their peripheral white blood cells) may still be at risk for having more than one child who inherited the mutation from them (Fig. 7.17). The existence of germline mosaicism means that geneticists and genetic counselors must be aware that normal examination results and normal gene test results of the parents of a child with an autosomal dominant or X-linked phenotype do not necessarily mean there is no risk of recurrence. The impact of this possibility on risk assessment will be discussed further in Chapter 17. Mutation Mutated cells Figure 7.16 Schematic representation of a mutation occurring after conception, during mitotic cell divisions. Such a mutation can lead to a proportion of cells carrying the mutation – that is, to either somatic or germline mosaicism, depending on the stage of embryonic or postnatal development where the mutation occurred. No pathogenic variant FBN1 variant Arg 1137Pro No pathogenic variant DMD variant Ala 68Asp A B Figure 7.17 Pedigrees demonstrating two affected half-siblings with the autosomal dominant disorder Marfan syndrome (family A) and the X-linked condition Becker muscular dystrophy (family B). In family A, the affected children have the same single nucleotide variant inherited from their father, who is unaffected and does not carry the variant in DNA from examined somatic tissues. He must have been a mosaic for the FBN1 variant in his germline. In family B, the affected children have the same single nucleotide variant inherited from their mother who is unaffected and does not carry the variant in DNA from examined somatic tissues. She must have been a mosaic for the DMD variant in her germline. ▶

CHAPTER 7 — Patterns of Single-Gene Inheritance 125 cells after the separation from somatic cells, resulting in pure gonadal mosaicism. Determining whether mosaicism for a mutation is present only in...

Ch7 · Pt18 126 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE PARENT-OF-ORIGIN EFFECTS ON INHERITANCE PATTERNS Unusual Inheritance Patterns due to Genomic Imprinting According to Mendel’s laws of heredity, a pathogenic allele of an autosomal gene is equally likely to be transmitted from a parent of either sex to an offspring of either sex; similarly, a female is equally likely to transmit a variant X-linked allele to a child of either sex. Originally, little attention was paid to whether the sex of the parent had any effect on the expression of the genes each parent transmits. As discussed in Chapter 6, we now know that in some genetic disorders, such as Prader-Willi syndrome (Case 38) and Angelman syndrome, expression of the disease phenotype depends on whether the pathogenic allele has been inherited from the father or from the mother. This phenomenon is known as genomic imprinting. The hallmark of genomic imprinting is that the sex of the parent who transmits the pathogenic allele determines whether there is expression of the disorder in a child (see Chapter 8). This is very different from sex-limited inheritance (described earlier in this chapter), in which expression of the disease depends on the sex of the child who inherits the pathogenic allele. Imprinting can cause unusual inheritance patterns: a disorder can appear to be inherited in a dominant manner when transmitted from one parent, but not from the other. For example, the hereditary paragangliomas (PGLs) are a group of autosomal dominant disorders in which multiple tumors develop in sympathetic and parasympathetic ganglia of the autonomic nervous system. Individuals with paraganglioma can also develop a catecholamine-producing tumor known as a pheochromocytoma, either in the adrenal medulla or in sympathetic ganglia along the vertebral column. A pedigree of one type of PGL family, caused by a pathogenic variant in the SDHD gene, is shown in Fig. 7.18. The striking observation is that, although both males and females can be affected, this is typically only if they inherited the variant from their father. A male heterozygote who has inherited his variant from his mother will remain unaffected throughout life but is still at a 50% risk for transmitting the variant to each of his children, who are then at high risk for developing the disease. DYNAMIC MUTATIONS: UNSTABLE REPEAT EXPANSIONS In all types of inheritance presented thus far in this chapter, the responsible variant allele, once it occurs, is stable when transmitted from one generation to the next; that is, all affected members of a family share the identical inherited variant. In contrast, an entirely different class of genetic disease is has been recognized, diseases due to dynamic mutations that change from generation to generation (see Chapter 4). These conditions are characterized by an unstable expansion within the affected gene of a segment of DNA that consists of tandem repeating units of three or more nucleotides. Many such repeat units consist of three nucleotides, such as CAG or CCG; the repeat being CAGCAGCAGCAG... or CCGCCGCCGCCG, for example. In general, gene loci associated with these diseases are polymorphic; that is, alleles in the normal population have a variable number of repeat units, as we saw in Chapter 4. As the gene is passed from generation to generation, the number of repeats can increase and undergo expansion, far beyond the normal range, leading to abnormalities in gene expression and function. I II III IV 1 2 3 4 5 6 7 8 9 10 17 16 15 14 13 12 11 18 + + + + + + + + + + 1 2 3 4 5 6 7 8 9 10 1 2 1 2 3 4 + + + + + + + + + + Figure 7.18 Pedigree of a family with paraganglioma syndrome 1 caused by a variant in the SDHD gene. Individuals II-1, II-2, II-4, III2, III-3, III-9, III-10, IV-6, IV-7, IV-11, and IV-14 each inherited the variant from their mothers but are unaffected. However, when the males in this group pass on the variant, those children can be affected. In addition to the imprinting, the family demonstrates the effect of reduced and age-dependent penetrance in the children (III-6, IV-10, IV-17) of heterozygous fathers. The + and − symbols refer to the presence or absence of the SDHD variant in this family. ▶

126 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE PARENT-OF-ORIGIN EFFECTS ON INHERITANCE PATTERNS Unusual Inheritance Patterns due to Genomic Imprinting According to Mendel’s laws of heredi...

Ch7 · Pt19 CHAPTER 7 — Patterns of Single-Gene Inheritance 127 The discovery of this unusual group of conditions has dispelled the orthodox notions of germline stability and provided a biologic basis for peculiarities of familial transmission (discussed in the next section) that previously had no known mechanistic explanation. More than 70 diseases are known to result from unstable repeat expansions of this type. All of these conditions are primarily neurologic. Here, we will review the inheritance patterns of two different diseases that illustrate the effects that different dynamic mutations can have on patterns of inheritance. A more complete description of the pathogenic mechanisms of unstable repeat disorders is given in Chapter 13. Polyglutamine Disorders Several different neurologic diseases share the property that the protein encoded by the implicated gene has a variable-length string of consecutive glutamine residues, which can be encoded by the trinucleotide CAG. These so-called polyglutamine disorders result when an expansion of the CAG repeat leads to a protein with more glutamines than is compatible with normal function. Huntington disease (HD) is a well-known disorder that illustrates many of the common genetic features of such disorders (Case 24). The neuropathology is dominated by degeneration of the striatum and the cortex. Individuals first present clinically in midlife, manifesting a characteristic phenotype of motor abnormalities (chorea, dystonia), personality changes, a gradual loss of cognition, and ultimately death. For a long time, HD was thought to be a typical autosomal dominant condition with age-dependent penetrance. The disease is transmitted from generation to generation with a 50% risk to each offspring. Heterozygous and homozygous individuals carrying the abnormal allele have very similar phenotypes, although homozygotes may have a more rapid course of their disease. There are, however, obvious peculiarities that cannot be explained by simple autosomal dominant inheritance. First, the disease appears to develop at an earlier and earlier age in successive generations, a phenomenon referred to as anticipation. Second, anticipation seems to occur only when the pathogenic allele is transmitted by an affected father and not by an affected mother, a situation known as parental transmission bias. The peculiarities of inheritance of HD are now readily explained by the discovery that the pathogenic allele is composed of an abnormally long CAG expansion in the coding region of the HTT gene. Normal individuals carry alleles with between 9 and 35 CAG repeats in their HTT gene, with the average being 18 or 19. Individuals affected with HD, however, have 36 or more repeats, with the average being around 46. Repeat numbers of 36 to 50 usually result in disease later in life, which explains the age-dependent penetrance that is a hallmark of this condition. A borderline repeat number of 36 to 39, although usually associated with HD, can be found in a few individuals who show no signs of the disease even at a fairly advanced age. The age of onset varies with how many CAG repeats are present (Fig. 7.19). 20 40 60 80 Age at onset (years) Normal range ≤ 35 Reduced penetrance range 36–39 Fully penetrant range ≥ 40 20 30 40 50 60 70 80 90 100 110 120 Number of CAG repeats in HTT Figure 7.19 Graph correlating approximate age of onset of Huntington disease with the number of CAG repeats found in the HTT gene. The solid line is the average age of onset, and the shaded area shows the range of age of onset for any given number of repeats. (Data courtesy Dr. M. Macdonald, Massachusetts General Hospital, Boston.) ▶

CHAPTER 7 — Patterns of Single-Gene Inheritance 127 The discovery of this unusual group of conditions has dispelled the orthodox notions of germline stability and provided a biologic basis for peculia...

Ch7 · Pt20 128 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE How, then, does an individual come to have an expanded CAG repeat in his or her HTT gene? First, the person may have inherited it from a parent who has an allele expanded beyond the normal range but has not yet developed the disease. Second, the person may have inherited an expanded repeat from a parent with repeat length of 36 to 39 which may or may not cause disease in the parent’s lifetime but may have expanded on transmission, resulting in earlier-onset disease in later generations (i.e., anticipation). For example, in the pedigree shown in Fig. 7.20, individual I-1, now deceased, was diagnosed with HD at the age of 64 years; he was heterozygous for an expanded allele with 37 CAG repeats and a normal, stable allele with 25 repeats. Four of his children inherited the unstable allele, with CAG repeat lengths ranging from 42 to more than 100 repeats. Finally, unaffected individuals may carry alleles with repeat lengths at the upper limit of the normal range (29–35 CAG repeats) that can expand further during meiosis. CAG repeat alleles at the upper limits of normal that do not cause disease but are capable of expanding into the disease-causing range are known as intermediate alleles (previously premutations). Expansion in HTT alleles shows a paternal transmission bias and occurs most frequently during male gametogenesis; thus, the severe early-onset juvenile form of the disease, seen with the largest expansions (70–121 repeats), is always paternally inherited. Fragile X Syndrome The fragile X syndrome (Case 17) is the most common heritable form of moderate intellectual disability. The name fragile X refers to a cytogenetic marker on the X chromosome at Xq 27.3, a so-called fragile site induced in cultured cells in which the chromatin fails to condense properly during mitosis. The syndrome is inherited as an X-linked disorder with penetrance in females in the 50 to 60% range. The fragile X syndrome has a frequency of 1 in 3000 to 4000 male births; it is so common that it requires consideration in the differential diagnosis of intellectual disability or autism in both males and females. Like HD, fragile X syndrome is caused by an unstable repeat expansion. However, in this case, a massive expansion of a different triplet repeat, CGG, occurs in the 5′ untranslated region of a gene called FMR1. The normal number of repeats is up to 55, whereas more than 200 (and even up to several thousand) repeats are found in individuals with the “full” fragile X syndrome allele. The syndrome is due to a lack of expression of the FMR1 gene and failure to produce the encoded protein. The expanded repeat leads to excessive methylation of cytosines in the promoter of FMR1. As discussed in Chapter 3, DNA methylation at Cp G islands prevents normal promoter function and leads to gene silencing. Triplet repeat numbers between 55 and 200 constitute an intermediate premutation stage of the fragile X syndrome. Expansions in this range are unstable when they are transmitted from mother to child and have an increasing tendency to undergo full expansion to more than 200 copies of the repeat during gametogenesis in the female but almost never in the male. The risk for expansion increases dramatically with increasing premutation size (Fig. 7.21). The overall premutation frequency in females in the population is estimated to be greater than 1 in 200. 100 80 60 40 20 Frequency of expansion to full mutation (%) Number of repeats in a premutation allele 60–69 80–89 >100 56–59 14% 20% 58% 72% 94% 100% 70–79 90–99 Figure 7.21 Frequency of expansion of a premutation triplet repeat in FMR1 to a full mutation in oogenesis as a function of the length of the premutation allele carried by a heterozygous female. The risk for fragile X syndrome to her sons is approximately half this frequency because there is a 50% chance a son will inherit the expanded allele. The risk for fragile X syndrome to her daughters is approximately one-fourth this frequency because there is a 50% chance a daughter would inherit the full mutation, and penetrance of the full mutation in a female is ~50%. (From Nolin SL: Familial transmission of the FMR1 CGG repeat, Am J Hum Genet 59:1252-1261, 1996. The University of Chicago Press.) 25 2 1 1 2 3 4 5 37 42 70 55 103 I II Figure 7.20 Pedigree of family with Huntington disease. Shown beneath the pedigree is a Southern blot analysis for CAG repeat expansions in the HTT gene. In addition to a normal allele containing 25 CAG repeats, individual I-1 and his children, II-1, II-2, II-4, and II-5, are all heterozygous for expanded alleles, each containing a different number of CAG repeats. The repeat number is indicated below each individual. II-2, II-4, and II-5 are all affected; individual II-1 is unaffected at the age of 50 years but will develop the disease later in life. (Data courtesy Dr. Ben Roa, Baylor College of Medicine, Houston, Texas.) ▶

128 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE How, then, does an individual come to have an expanded CAG repeat in his or her HTT gene? First, the person may have inherited it from a par...

Ch7 · Pt21 CHAPTER 7 — Patterns of Single-Gene Inheritance 129 Similarities and Differences in Huntington Disease and Fragile X Pedigrees A comparison of HD with the fragile X syndrome reveals some similarities but also many differences, illustrating many of the features of disorders due to dynamic mutations: Intermediate/Premutation expansions causing an increased risk for passing on full expansion alleles are the rule in both of these disorders. Anticipation is common in both. However, the number of repeats in intermediate alleles for HD is 29 to 35, far smaller than the 55 to 200 repeats in fragile X syndrome premutations. Premutation carriers for fragile X syndrome are at risk for adult-onset ataxia (in males) and ovarian failure (in females). Intermediate allele carriers in HD are, by definition, disease-free. The expansion of premutation alleles occurs primarily in the female germline in fragile X syndrome; in contrast, the largest expansions causing juvenileonset HD occur in the male germline. MATERNAL INHERITANCE OF DISORDERS CAUSED BY VARIANTS IN THE MITOCHONDRIAL GENOME All the patterns of inheritance described thus far are explained by variants in the nuclear genome, in either autosomal or X-linked genes. However, some inherited diseases that do not show patterns typical of mendelian inheritance are caused by pathogenic variants in the mitochondrial genome (mtDNA), which manifest strictly maternal inheritance. Disorders caused by pathogenic variants in mtDNA have several unusual features that result from the unique characteristics of mitochondrial biology and function. As introduced in Chapter 2, not all the RNA and protein synthesized in a cell are encoded in the DNA of the nucleus; a small but important fraction is encoded by genes in mtDNA. The mitochondrial genome consists of 37 genes that encode 13 subunits of enzymes involved in oxidative phosphorylation, as well as ribosomal RNAs and transfer RNAs required for translating the transcripts of the mitochondria-encoded polypeptides. Because mitochondria are essential to the normal functioning of nearly all cells, disruption of energy production by pathogenic variants in mtDNA often results in severe disease, affecting many different tissues. Thus, pleiotropy is the rule, not the exception, in mitochondrial disorders. More than 100 different pathogenic variants have been identified in mtDNA that can cause a range of human diseases—often involving the central nervous and musculoskeletal systems, such as myoclonic epilepsy with ragged-red fibers (Case 33). In this section we will focus on the distinctive pattern of inheritance related to three unusual features of mtDNA: maternal inheritance, replicative segregation, and homoplasmy and heteroplasmy. The underlying mechanisms of mitochondrial disorders are discussed in more detail in Chapter 13. Maternal Inheritance of mtDNA The first defining characteristic of the genetics of mtDNA is its maternal inheritance. Sperm mitochondria are generally not present in the zygote so that only the maternal mtDNA is transmitted to the next generation. Thus, the children of a female who has an mtDNA variant may inherit it, whereas none of the offspring of a male carrying the same variant will. Pedigrees of such disorders are quite distinctive, as shown by the strictly maternal inheritance of an mtDNA variant causing Leber hereditary optic neuropathy (Fig. 7.22). Although maternal inheritance is the general expectation, at least one instance of paternal inheritance of mtDNA has occurred in a patient with a mitochondrial myopathy. Consequently, in individuals with apparently sporadic mtDNA mutations, the rare occurrence of paternal mtDNA inheritance must be considered (Box 7.4). Replicative Segregation A second feature of the mitochondrial genome is the stochastic nature of segregation during mitosis and meiosis. The number of mtDNA copies per cell is not fixed and is substantially higher than the number of nuclear DNA copies, with cells having as many as hundreds of thousands of mtDNA copies. In addition there is no fixed phase of the cell cycle for the replication of mtDNA. At cell division, the copies of mtDNA in each of the I II III Figure 7.22 Pedigree of Leber hereditary optic neuropathy, a form of adult-onset blindness caused by a defect in mitochondrial DNA. Inheritance is only through the maternal lineage, in agreement with the known maternal inheritance of mitochondrial DNA. Note that no affected male transmits the disease. ▶

CHAPTER 7 — Patterns of Single-Gene Inheritance 129 Similarities and Differences in Huntington Disease and Fragile X Pedigrees A comparison of HD with the fragile X syndrome reveals some similarities...

Ch7 · Pt22 130 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE mitochondria in each cell sort randomly to the daughter cells, in stark contrast to the highly predictable and programmed segregation of the 46 nuclear chromosomes. This process is known as replicative segregation and can result in significant variability in manifestations of mitochondrial disorders among different tissues and/or individuals. Homoplasmy and Heteroplasmy The presence of a high copy number creates an additional distinctive feature in the genetics of mtDNA. The terms heterozygous and homozygous, used to describe the presence of one or two allelic variants of a nuclear gene, are inexact for mtDNA. Instead, the term for the uniform presence of an identical mitochondrial sequence is homoplasmic. When a variant sequence of mtDNA is also present, within a cell, tissue, or organism, the term used to describe this is heteroplasmic. When a sequence variant first occurs in the mtDNA, it is present in only one of the mtDNA molecules in a mitochondrion. As mtDNA replicates, the mitochondria undergo fission and fusion, and the variant and wild-type DNA are distributed randomly into daughter organelles, which – simply by chance – may contain different proportions of the two allelic variants. The cell, which now contains mitochondria containing different mixtures of mtDNAs, in turn distributes those mitochondria randomly to its daughter cells. Daughter cells may thus have different levels of heteroplasmy (Fig. 7.23). A key feature of the heteroplasmic state is that the ratio of the two allelic variants is not fixed over time and may change with further replication and cell division. Because the phenotypic expression of a pathogenic variant in mtDNA depends on a quantitative value—the relative proportions of normal- and pathogenic-allele- bearing mtDNA in the cells making up different tissues—reduced penetrance and variable expression are typical features of mitochondrial disorders (Case 33). Most pathogenic variants in mtDNA are only present and transmitted in a state of heteroplasmy, since they would reduce reproductive fitness to 0 if they were homoplasmic. The exceptions (including Leber hereditary optic neuropathy as described earlier) cause disorders that are either incompletely penetrant or are not reproductively lethal. Maternal inheritance in the presence of heteroplasmy in the mother is associated with additional features of mtDNA genetics that are of medical significance. First, the number of mtDNA molecules within developing oocytes is reduced before being subsequently amplified to the massive number (up to 106 copies) seen in mature oocytes. This restriction and subsequent amplification of mtDNA during oogenesis is termed the mitochondrial genetic bottleneck. Consequently, variability in the proportion of variant mtDNA molecules seen in the offspring of a mother with heteroplasmy arises, at least in part, from the sampling of a reduced subset of the mtDNAs after the mitochondrial bottleneck that occurs in oogenesis. The heteroplasmy of the resulting oocytes is a distribution of values based on the heteroplasmy of the mother herself. As might be expected, mothers with a high heteroplasmy for a pathogenic variant are more likely to have clinically affected offspring than are mothers with a lower proportion. Mothers may also have offspring who, by chance, are homoplasmic for the absence of a pathogenic variant. Mutant mitochondria Normal mitochondria Clonal mtDNA proliferation Random Segregation N Disease phenotype Normal phenotype Threshold for phenotypic expression N N N N N Figure 7.23 Replicative segregation of a heteroplasmic mitochondrial variant. Random partitioning of variant and wild-type mtDNA through multiple rounds of mitosis produces a collection of daughter cells with wide variation in heteroplasmy. Cell and tissue dysfunction results when the fraction of mitochondria that are carrying a variant exceeds a threshold level. N, Nucleus. ▶

130 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE mitochondria in each cell sort randomly to the daughter cells, in stark contrast to the highly predictable and programmed segregation of the...

Ch7 · Pt23 CHAPTER 7 — Patterns of Single-Gene Inheritance 131 CORRELATING GENOTYPE AND PHENOTYPE An important component of medical genetics is identifying and characterizing the genotypes responsible for particular disease phenotypes. In doing so, it is important not to adhere to an overly simplistic view that each disease phenotype is caused uniquely by one particular variant in a specific gene or that pathogenic variants in a particular gene always cause the same phenotype. In fact, there is often substantial heterogeneity in the complex relationship(s) among disease phenotypes, the genes that are altered in those diseases, and the nature of the variants found in those genes. Three main types of heterogeneity are distinguished, as will be illustrated in detail in Chapters 12 and 13. Here, we introduce them and outline their distinguishing features. Allelic heterogeneity, in which different variants in a gene may produce the same phenotype Locus heterogeneity, in which variants in different genes may cause the same phenotype Clinical or phenotypic heterogeneity, also referred to as a phenotypic diversity at a locus, in which different variants in a gene may result in different phenotypes Allelic Heterogeneity There may be more than one pathogenic variant at a locus. Allelic heterogeneity may be responsible for differences in the severity or degree of pleiotropy demonstrated for a particular condition. As one example, more than 2000 different variants have been found worldwide in the cystic fibrosis transmembrane conductance regulator gene (CFTR) among patients with CF (Case 12). Sometimes these different variants result in clinically indistinguishable disorders. In other cases, different variants at the same locus produce a similar phenotype but along a continuum of severity. In autosomal recessive disorders, in particular, the fact that many individuals are compound heterozygotes for two different alleles further adds to phenotypic variability of a disorder. For example, homozygotes or compound heterozygotes for many CFTR variants have classic CF with pancreatic insufficiency, severe progressive lung disease, and congenital absence of the vas deferens in males, whereas others with combinations of other variants may have lung disease but normal pancreatic function; still others will have only the abnormality of the male reproductive tract. Allelic heterogeneity may also manifest in the pattern of inheritance demonstrated for a particular condition. For example, in retinitis pigmentosa, a common cause of hereditary visual impairment due to photoreceptor degeneration, some variants in the ORP1 gene, encoding an oxygen-regulated photoreceptor protein, cause an autosomal recessive form of the disease, whereas others in the same gene result in an autosomal dominant form. Locus Heterogeneity Locus heterogeneity describes the situation in which clinically similar and even indistinguishable disorders may arise from variants in different loci in different individuals. For some phenotypes, pedigree analysis alone has been sufficient to demonstrate locus heterogeneity. Taking retinitis pigmentosa again as an example, it was recognized many years ago that the disease occurs in both autosomal and X-linked forms. Now, pedigree analysis combined with gene mapping has demonstrated that this single clinical entity can be caused by variants in at least 96 different genes, of which 89 are autosomal, 6 are X linked, and one is Y linked! Clinical Heterogeneity Different variants in the same gene may produce very dissimilar phenotypes in different families: a phenomenon known as clinical or phenotypic heterogeneity. This situation occurs with variants in the LMNA gene, which encodes a nuclear membrane protein. Different LMNA variants have been associated with at least a half dozen phenotypically distinct disorders, including a form of muscular dystrophy, one form of hereditary dilated cardiomyopathy, one form of the Charcot-Marie-Tooth peripheral neuropathy, a disorder of adipose tissue called lipodystrophy, and the premature aging syndrome known as Hutchinson-Gilford progeria. IMPORTANCE OF THE FAMILY HISTORY IN MEDICAL PRACTICE Among medical specialties, medical genetics is distinctive in that it focuses not only on the patient but on the entire family. A comprehensive family history is an important first step in the analysis of any disorder, regardless of whether the disorder is known to be genetic. As the late Barton Childs stated succinctly: “to fail to BOX 7.4 CHARACTERISTICS OF MITOCHONDRIAL INHERITANCE All children of a female homoplasmic for a pathogenic variant will inherit the variant; the children of a male carrying a similar variant will not. Females heteroplasmic for a pathogenic variant will pass the variant on to many of their children. The heteroplasmy in her offspring is based on a distribution and cannot be easily predicted in advance. The fraction of pathogenic variant, and therefore the risk and severity of disease, can vary considerably depending on the quantitative level of maternal heteroplasmy, as well as on random chance due to the oocyte bottleneck. The heteroplasmy in different tissues of an individual can vary tremendously, thereby causing a spectrum of disease among the members of a family. Pleiotropy and variable expressivity in different affected family members are also frequent. ▶

CHAPTER 7 — Patterns of Single-Gene Inheritance 131 CORRELATING GENOTYPE AND PHENOTYPE An important component of medical genetics is identifying and characterizing the genotypes responsible for partic...

Ch7 · Pt24 132 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE take a good family history is bad medicine.” Despite the sophisticated cytogenetic, molecular, and genome testing now available to geneticists, an accurate family history (including the family pedigree) still remains a fundamental tool for all physicians and genetic counselors. They use it for determining the pattern of inheritance of a disorder in the family, forming a differential diagnosis, determining what genetic testing might be needed, and designing an individualized management and treatment plan for their patients. Furthermore, recognizing a familial component to a medical disorder allows the risk in other family members to be estimated so that proper management, prevention, and counseling can be offered to the patient and the family, as we will discuss in many of the chapters to follow. ACKNOWLEDGMENT We thank Carolyn Applegate, Jodie Vento and Cheryl Shuman for contributing to this chapter. GENERAL REFERENCES Bennett RL, French KS, Resta RG, et al: Standardized human pedigree nomenclature: update and assessment of the recommendations of the National Society of Genetic Counselors, J Genet Counsel 17:424–433, 2008. Online Mendelian Inheritance in Man (OMIM), Baltimore, 2022, Johns Hopkins University, http://omim.org/ Rimoin DL, Pyeritz RE, Korf BR, editors: Emery and Rimoin’s essential medical genetics, Oxford, 2013, Academic Press. Scriver CR, Beaudet AL, Sly WS, et al: The metabolic and molecular bases of inherited disease, ed 8, New York, 2000, Mc Graw Hill. Updated online version available at https://ommbid.mhmedical.com/ PROBLEMS 1. Cathy and Calvin are pregnant for the second time. Their first child, Donald, has cystic fibrosis (CF). Cathy has two brothers, Charles and Colin, and a sister, Cindy. Colin and Cindy are unmarried. Charles is married to an unrelated woman, Carolyn, and has a 2-year-old daughter, Debbie. Cathy’s parents are Bob and Betty. Betty’s sister Barbara is the mother of Cathy’s husband, Calvin. There is no family history of CF except for Donald. a. Sketch the pedigree, using standard symbols. b. Which people in this pedigree are obligate heterozygotes? Which are likely heterozygotes? 2. George and Grace, who have normal hearing, have eight children; two of their five daughters and two of their three sons have congenital hearing loss. Another couple, Harry and Helen, both with normal hearing, also have eight children; two of their six daughters and one of their two sons are hearing impaired. A third couple, Gilbert and Gisele, each with congenital hearing loss, have four children, who are all affected by hearing loss. Gilbert and Gisele’s daughter Hedy marries Horace, a hearing impaired son of George and Grace, and Hedy and Horace in turn have four hearing impaired children. Hedy and Horace’s eldest son Isaac marries Ingrid, a daughter of Harry and Helen; although both Isaac and Ingrid are hearing impaired, their six sons all have normal hearing. Sketch the pedigree and answer the following questions. (Hint: How many different types of congenital hearing loss are segregating in this pedigree?) a. State the probable genotypes of Isaac and Ingrid’s children. b. Why are all the children of Gilbert and Gisele and of Hedy and Horace hearing impaired? 3. Consider the following situations: a. Retinitis pigmentosa occurs in X-linked and autosomal forms. b. Two parents each have a typical case of familial hypercholesterolemia: hypercholesterolemia, arcus corneae, tendinous xanthomas, and deficiency of low- density lipoprotein (LDL) receptors, and family history of the disorder. Their child has very high plasma cholesterol level at birth and within a few years develops xanthomas and generalized atherosclerosis. c. A couple with normal vision, from an isolated community, have a child with autosomal recessive gyrate atrophy of the retina. The child grows up, marries another member (with normal vision) of the same community, and has a child with the same eye disorder. d. A child has severe neurofibromatosis 1 (NF1). Her father is phenotypically normal; her mother seems clinically normal but has several large café au lait spots and areas of hypopigmentation; slit-lamp examination shows a few Lisch nodules (hamartomatous growths on the iris). e. Parents of normal stature have a child with achondroplasia. f. An adult male with myotonic dystrophy has cataracts, frontal balding, and hypogonadism, in addition to myotonia. g. A man with vitamin D -resistant rickets transmits the condition to all his daughters, who have a milder form of the disease than their father; none of his sons is affected. The daughters have approximately equal numbers of unaffected sons, affected sons, unaffected daughters, and affected daughters, the affected sons being more severely affected than their affected sisters. h. A boy has progressive muscular dystrophy with onset in early childhood and is wheelchair-bound by age 12 years. An unrelated man also has progressive muscular dystrophy but is still ambulant at the age of 30 years. Molecular analysis shows that the individuals have a large but different deletion in the dystrophin gene. Which of the concepts listed here are illustrated by situations a. to h.? Variable expressivity Consanguinity X-linked dominant inheritance New mutation Allelic heterogeneity Locus heterogeneity Homozygosity for an autosomal dominant trait Pleiotropy 4. Don and his maternal grandfather Barry both have hemophilia A. Don’s partner Diane is his maternal first cousin. Don and Diane have one son, Edward, and two daughters, continued ▶

132 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE take a good family history is bad medicine.” Despite the sophisticated cytogenetic, molecular, and genome testing now available to geneticis...

Ch7 · Pt25 CHAPTER 7 — Patterns of Single-Gene Inheritance 133 Elise and Emily, all of whom have hemophilia A. They also have an unaffected daughter, Enid. a. Draw the pedigree. b. Why are Elise and Emily affected? c. What is the probability that a son of Elise would have hemophilia? What is the probability that her daughter would have hemophilia? d. What is the probability that a son of Enid would have hemophilia? A daughter? 5. A couple has a child with NF1. Both parents are clinically normal, and neither of their families shows a positive family history. a. What is the probable explanation for NF1 in their child? b. What is the risk for recurrence in other children of this couple? c. If the husband has another child by a different mother, what would the risk for NF1 be? d. What is the risk that any offspring of the affected child will also have NF1? 6. Before starting her family, the consultand (arrow) wants to know the risk that a child of hers and her husband’s would have a birth defect because they are related (see pedigree). The family history reveals no known recessive disease. What is the chance that such a child could be homozygous for a variant for a recessive disorder carried by the woman who is her great-grandmother and her partner’s grandmother? I II III IV V 7. Given the following pedigree, what is/are the most likely inheritance pattern(s); possible but less likely inheritance pattern(s); incompatible inheritance pattern(s)? Patterns are autosomal recessive, autosomal dominant, X-linked recessive, X-linked dominant, and mitochondrial. Justify your choices. I II III ▶

PROBLEMS—CONT’D .

Chapter 8: Principles of Clinical Epigenetics

Ch8 · Pt1 chapter 8 Principles of Clinical Epigenetics Sarah Goodman ▶

Cheryl Cytrynbaum Rosanna Weksberg INTRODUCTION Epigenetics is a nascent and quickly evolving field. As defined in Chapter 3, epigenetics refers to the study of modifications to DNA or DNA packaging...

Ch8 · Pt2 136 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Recent work in epigenetics has highlighted the role of epigenetics in human health outcomes, specifically the relationship between DNA methylation and genetic variation, including genetic background or population- level variation, polygenic risk scores, and single deleterious gene variants. In this context, a risk allele can predispose individuals to a certain outcome or phenotype following an exposure, such as an adverse reaction to a medication; the underlying mechanism that drives this reaction may be epigenetic in nature and function via DNA methylation and histone modification changes. As such, if we were to measure DNA methylation in a group of individuals with and without the risk allele, with and without exposure to the medication, the methylation patterns would reflect not just the allele or the exposure, but both. These so-called epialleles represent important biomarkers of the past exposure and may be valuable in a clinical setting. For example, epialleles could provide biologic validation of an exposure to a specific toxin, such as a past exposure to secondhand cigarette smoke in an individual with a respiratory disease and a negative smoking history. EPIGENETIC MACHINERY The epigenetic machinery within cells consists of a set of enzymes with specific functions that maintain transcriptional programs and 3D DNA structure, collectively known as epigenetic regulators. Later in the chapter we focus on the epigenetics regulators involved in histone post-translational modifications and DNA methylation. The set of epigenetic regulators involved with histone modifications is much larger than the set targeting DNA methylation due to the large number of epigenetic marks that modify histones. As such, histone marks are abbreviated by the histone, the modified amino acid and its position, and the epigenetic mark. For example, H3K9ac denotes acetylation of the ninth amino acid residue (a lysine or K in standard amino acid abbreviation) of the histone H3 protein. Within the two groups of genes (i.e., histone and DNA epigenetic regulators) are so- called writers, erasers, and readers of epigenetic marks. Writers place chemical marks on DNA or histones and often carry the term transferase, which connotes this activity. A few examples include the group DNA methyltransferases, histone-lysine methyltransferases, and histone acetyltransferases. Erasers remove chemical marks and include enzyme groups such as histone deactylases and histone demethylases. TET enzymes are the erasers of DNA methylation, for which demethylases are not known to exist. Rather TET proteins initiate a stepwise enzymatic process of methyl group removal that results in demethylation. Readers are usually nonenzymatic proteins that bind to specific chemical marks. The fourth and broadest group is remodelers. These enzymes work within large protein complexes to alter chromatin state/3D structure, typically at the nucleosome level. This includes changing the conformation of the nucleosome DNA, the position of the nucleosome along the DNA, or exchanging histone variants within a nucleosome. The association between epigenetic regulators and genetic disorders will be discussed later in the chapter. Specifically, we will describe a group of mendelian neurodevelopmental disorders caused by pathogenic variants in genes encoding epigenetic regulators. Histone and DNA modifications function interdependently, with accumulating evidence for temporal and spatial colocalization of certain groups of epigenetic marks, suggesting the likelihood of combinatorial effects. One example of this interdependency of different modifications is that regions of methylated DNA commonly lack di- and trimethylation of histone H3 at lysine 4 (H3K4me 2 and H3K4me 3, respectively). While DNA methylation is associated with transcriptional repression, these histone methylation marks (H3K4me 2 and H3K4me 3) typically occur at transcriptionally active loci. However, there are many known exceptions to these rules. The current hypothesis as to the mutual interdependence of these two marks is that the presence of DNA methylation excludes the histone methyltransferase enzyme from binding and depositing di- and trimethyl groups to H3K4. In fact, many enzymes that act to deposit or remove chemical modification to histone tails have protein domains that are sensitive to DNA methylation. For example, SETDB1 and SETDB2 are two epigenetic writers that function as histone-lysine methyltransferases; both paralogs contain a methyl-Cp G-binding domain (MBD), which enables the encoded proteins to localize to methylated DNA in addition to cooperation with other proteins known as binding partners. Inversely, other histone-modifying enzymes can prevent the localization of DNA methylation machineries and protein complexes that lead to chromatin compaction. While we have an incomplete understanding of the crosstalk between DNA and histone modifications, they do not act as isolated units. As well, the immense number of possible combinations of various modifications and context sensitivity make for an exceptionally complex regulatory mechanism. EPIGENETICS IN DEVELOPMENT Now that we have discussed how and where epigenetic marks, particularly DNA methylation, exist in the human genome, we will focus on the critical role of epigenetics in human development. Comprehending how these mechanisms function in development will provide a context for their contributions to pathophysiology of certain diseases and disorders. Arguably, one of the most important roles of DNA methylation occurs during embryonic and fetal development, wherein it participates in regulating cell differentiation, conferring a stable cell/tissue-specific identity. As such, DNA methylation displays tissue- and cell-specific patterns. In fact, ▶

136 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Recent work in epigenetics has highlighted the role of epigenetics in human health outcomes, specifically the relationship between DNA methy...

Ch8 · Pt3 CHAPTER 8 — Principles of Clinical Epigenetics 137 tissue of origin is one of the largest determinants of DNA methylation variation in healthy individuals, accounting for greater variation than genetic background. Epigenetic states are most dynamic during germ cell specification and early embryogenesis, two time periods distinguished by epigenetic reprogramming (Fig. 8.1). Our knowledge of these processes comes primarily from studies in mice; however, recent genetic and functional data from human studies have shown that epigenetic reprogramming in the gametes and embryo are generally parallel in humans and mouse, although important differences are being identified that require further investigation. During primordial germ cell specification in a fetus at ~5 weeks of gestation there is global erasure of DNA methylation followed by remethylation and imprint acquisition in the differentiating germ cells prior to maturing into oocytes or sperm depending on the sex of the fetus. The resulting highly divergent DNA methylation patterns are associated with distinct differentiated/ transcriptional states. Together, the DNA and histone modifications, as well as molecules that support 3D DNA structure, constitute the epigenome. To that end, after fertilization, the chromatin in the zygote is generally open but not transcribed. This is followed by rapid remodeling leading to zygotic genome activation at the eight-cell stage in human embryos. Prior to implantation the embryo undergoes genome-wide DNA methylation reprogramming. This comprises rapid and enzymatically driven/active demethylation of the paternal genome. By comparison, demethylation of the maternal genome occurs mainly through passive demethylation over several cell divisions. The lowest levels of methylation in the maternal genome occur at the blastocyst stage, at which time the two parental genomes are comparable. Importantly, the imprinted loci are excluded from this stage of reprogramming, and gametic imprinting marks are retained (see Fig. 8.1). DNA methylation at these loci is protected from genome-wide demethylation/ remethylation in the embryo. The mechanism, although not yet completely understood, involves protein complexes encoded by maternal effect genes. These genes are transcribed from the maternal genome before fertilization, generating transcripts/proteins required by the early embryo before zygotic genome activation occurs at the eight-cell stage. The majority of maternal effect genes have been studied in mice, including their phenotypic outcomes when dysregulated by a targeted deletion. Maternal effect genes serve similar functions in humans in that their epigenomic/organizational role is a requirement for normal developmental competence. See Genomic Imprinting later for phenotypic outcomes associated with pathogenic variants in these genes. Figure 8.1 The life cycle of imprints. DNA methylation reprogramming during human development. Methylation of imprinting centers (ICs) (dashed black line) is erased more slowly than that of the rest of the genome (black line) in primordial germ cells (PGCs) and reestablished with different kinetics in male (paternal ICs, dashed blue line; whole genome, blue line) and female (maternal ICs, dashed red line; whole genome, red line) germ cells. After fertilization, the maternally and paternally derived genomes are widely demethylated, while differential methylation between maternal and paternal IC alleles (50% level) is maintained preimplantation and postimplantation. Factors and events involved in each stage, 5-methylcytosine level and approximate timing of imprint erasure, establishment and preimplantation and postimplantation maintenance are indicated. g DMRs, Germline differentially methylated regions; GVs, germinal vesicles; SCMC, subcortical maternal complex. (From Monk D, Mackay DJG, Eggermann T, et al: Genomic imprinting disorders: lessons on how genome, epigenome and environment interact, Nat Rev Genet 20:235–248, 2019. doi:10.1038/s 41576-018-0092-0.) ▶

Ch8 · Pt4 138 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Following implantation, parallel remethylation of the maternal and paternal genomes occurs in a cell-type– dependent and time-dependent manner. Precursor cells (cells that are not yet terminally differentiated) undergo a stepwise differentiation process in which epigenetics plays a critical role. For example, during differentiation, DNA methylation is required to silence pluripotency factors; the promoters of genes associated with pluripotency, such as Oct 4 and Nanog, are hypermethylated and silenced. As well, DNA methylation acts to upregulate markers associated with germ-layer specificity. In embryonic stem cells lacking DNA methylation, differentiation is inhibited. While some epigenetic processes are thought to drive transcriptional programs based on various inputs, spatial and temporal, other epigenetic changes are believed to enforce these changes and create a barrier that prevents dedifferentiation. The results of these tightly orchestrated epigenetic patterns are lineage- specific transcription profiles that confer cellular identity. Moreover, these profiles are maintained across cell divisions, as epigenetic patterns are mitotically heritable. THE ENVIRONMENT INTERACTS WITH THE EPIGENOME There is a strong interest in epigenetic mechanisms within the developmental origins of health and disease (DOHa D) field. The DOHa D paradigm posits that environmental factors during fetal development and infancy contribute to chronic disease susceptibility. The seminal research in this field identified geographic links between low birthweight in the United Kingdom associated with increased fetal mortality, as well as adult cardiovascular disease. These findings identified poor in utero nutrition and impaired fetal growth as contributing factors to adult cardiovascular disease, initially by observing that regions of England and Wales with the highest rates of coronary heart disease also had increased infant mortality rates in the decades prior. Further work across England and then Europe identified poor prenatal nutrition as an environmental risk for both outcomes, providing strong evidence that prenatal environment contributed to later health outcomes. Longitudinal findings from adults exposed in utero to the Dutch Hunger Winter established many long- term health outcomes of prenatal starvation, including increased risk of obesity, abnormal lipid profiles, cardiovascular disease, and neuropsychiatric disorders. These outcomes differ based on the timing of exposure; those exposed only during early gestations had normal birthweights (but increased risk of obesity), while those exposed at later gestations had reduced birthweights. Importantly, these contrasting phenotypes allow us to define critical periods of development (i.e., during development) for a given biologic system where there exists a window of sensitivity during which certain environmental exposures can cause lasting changes. Here long-term metabolism was altered in response to starvation during early gestation despite the paradoxic healthy birthweights. By contrast, reduced kidney function was observed more prevalently in individuals exposed during midgestation. A continuation of work on this natural experiment also identified corresponding DNA methylation changes, suggesting a role for epigenetics in molecular architecture underlying the physiologic response/changes. Notably, insulin-like growth factor 2 (IGF2), a gene that is critical to prenatal growth and cell proliferation, was found to be hypomethylated (i.e., lower methylation levels that are commonly associated with increased gene activity) in individuals exposed in early gestation, as compared to their unexposed siblings. By comparison, this difference in IGF2 methylation was not observed in pairs of individuals exposed in late gestation and their siblings. The Agouti mouse model is also a classic example of how epigenetic mechanisms act as a temporal bridge between in utero exposures and health outcomes in adulthood. The Agouti gene in mice, which controls fur color via melanin production, is regulated by a cell- type–specific promoter found in the second exon of the gene. This promoter results in gene activation during hair follicle cell development. However, the insertion of an intracisternal A-particle (IAP) retrotransposon in the Agouti gene results in constitutive expression of this gene (i.e., it is expressed in all cells not just hair follicle cells as the retrotransposon contains a cryptic promoter) (Fig. 8.2). This allele is referred to as Avy or the viable yellow allele. The phenotype of these mutant mice includes yellow fur, obesity, type II diabetes, and predisposition to tumors. However, mice with the IAP insertion can have a range of pan-cellular Agouti expression, and the associated phenotypes are dependent on the levels of DNA methylation at the IAP (see Fig. 8.2). Furthermore, a diet rich in methyl donors fed to pregnant Agouti mice can alter the expression of the Agouti gene in the offspring, which will in turn impact long- term health. Mothers heterozygous for the Avy allele, when crossed with heterozygous males and fed with a diet high in methyl donors, will more frequently produce healthy brown Avy offspring, who carry high levels DNA methylation acting to repress this gene. By comparison, bisphenol A, an endocrine disruptor, when fed to pregnant mice leads to more Avy offspring with yellow coat colors and lower levels of DNA methylation. These regions of phenotype-associated DNA methylation, which also vary by maternal nutrition, constitute differentially methylated regions (DMRs). The variation in fur color and health outcomes is especially striking when considering that these mice exhibiting a range of fur color and health outcomes are genetically identically individuals. These examples highlight the environmental influence on epigenetic regulation impacting physiologic outcomes, but also how DNA methylation can act as a biosensor of past in utero environmental factors. ▶

138 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Following implantation, parallel remethylation of the maternal and paternal genomes occurs in a cell-type– dependent and time-dependent m...

Ch8 · Pt5 CHAPTER 8 — Principles of Clinical Epigenetics 139 Additional evidence that environmental factors can influence the imprinting process derives from assisted reproductive technologies (ART). Originally developed in the 1970s ART was developed as a treatment for infertility caused by fallopian tube obstruction. Since then, the indications for ART have grown and include couples at increased risk for genetic disorders as well as diverse causes of female and male subfertility or infertility ART has the potential to disrupt two critical periods of developmental epigenetic reprogramming: oocyte maturation and retention of gametic imprints following fertilization. Ovarian follicular stimulation may activate oocytes that are not yet fully epigenetically reprogramed. In addition, several aspects of ART (in vitro fertilization, intracytoplasmic sperm injection, and freezing of embryos) may deregulate preimplantation epigenetic reprogramming. Therefore the reports are not surprising of increased risks of adverse pregnancy outcomes: specifically, low birthweight for gestational age, preterm birth, congenital malformations, and increased rate of imprinting disorders, including Beckwith-Wiedemann, Russell-Silver, Angelman, and Prader-Willi syndromes (see Genomic Imprinting, later). The risk for each of these syndromes in individuals conceived using ART is increased several fold over the general population risk (e.g., for Beckwith-Wiedemann syndrome this would raise the risk from 1/13,000 to ~1/2500, although the absolute risk remains low). In humans, targeted and genome-wide molecular testing in individuals born following ART has identified DNA methylation alterations not only at a specific locus associated with known clinical entities but also variable dysregulation at multiple imprinted loci, a phenomenon known as multilocus imprinting disorder. Studies in humans and model organisms have implicated both ART processes (hormone therapy, in vitro culture medium) and primary subfertility issues (oocyte/sperm quality or pathogenic variants in maternal effect genes) as contributors to aberrant epigenetic programming in this complex developmental time period. We explored earlier the important prenatal environments in relation to epigenetic and phenotypic changes; A 5' 5' 3' 2 ectopic wildtype,a Cp GSites 1-9 A A vy 3' IAP PS1A 1A B Yellow Slightly mottled Mottled Heavily mottled Pseudo Agouti Figure 8.2 Environmentally induced alterations in the epigenome in Av ▶

y mouse. (A) The Avy allele contains a contraoriented intracisternal A-particle insertion within pseudoexon 1A (PS1A) of the Agouti gene. A cryptic promoter (short arrowhead labeled “Avy ectopic”) dr...

Ch8 · Pt6 140 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE however, plasticity does not end at birth. Across the lifespan, DNA methylation patterns continue to change in both predictable and seemingly random ways. These ongoing changes to the epigenome can be illustrated by aging and twin studies, respectively. With regard to the predictable nature of epigenetic patterns across the life span, DNA methylation is the most accurate biologic predictor of chronologic age. For unknown reasons, a small subset of Cp Gs sites acts as a molecular clock. Furthermore, many health behaviors and disease states are associated with an advanced epigenetic clock (i.e., a predicted age older than one’s chronologic age). Significant gaps between the predicted epigenetic age and chronologic age have been associated with increased mortality and morbidity, which may reflect a relationship between DNA methylation and the aging process. However, this phenomenon is not well understood and currently provides little insight into the molecular mechanisms that underlie the aging process, a situation that will likely be clarified by future research. The second example of DNA methylation patterns across the life span is best observed in monozygotic twins, who are born with nonidentical but highly concordant DNA methylation patterns. These relatively small DNA methylation differences observed at birth are likely related to differences experienced in utero despite sharing an embryonic environment. Following birth, the DNA methylation patterns of monozygotic twins become increasingly divergent with age. This well-described pattern of diverging DNA methylation patterns across the life spans of twins likely occurs in response to ongoing environmental differences as well as stochastic molecular events such as errors in epigenetic machinery. Importantly, DNA methylation differences in monozygotic twins at all ages have been associated with many discordant phenotypes, including psychiatric disorders (e.g., schizophrenia and bipolar disorder) and autoimmune diseases (e.g., lupus erythematosus and multiple sclerosis). This work speaks to plasticity that is mediated by DNA methylation beyond the formative years of fetal development and its role as an interface between one’s environment and health outcomes. THE ROLE OF EPIGENETICS IN HUMAN DISEASE It was the elegant nuclear transfer experiments in mouse embryos that originally led to the discovery that the mammalian maternal and paternal genomic contributions to the fertilized egg, provided by the haploid germ cells, have different effects on the developing embryo. Zygotes were created carrying either two nuclei of maternal or paternal origin generating exclusively embryonic or placental tissue, respectively, but no viable embryos. Evidence in humans of the functional difference between the maternal and paternal genomes came from studying human germ cell tumors, specifically hydatidiform moles and ovarian teratomas. Hydatidiform moles are androgenetic in origin (two paternal genomes, no maternal genome), while ovarian teratomas are gynogenetic (two maternal genomes, no paternal genome). The histopathologic phenotype of ovarian teratomas reveals well-differentiated fetal structures of all three germ layers (ectoderm, mesoderm, endoderm), while the hydatidiform mole contains only extraembryonic trophoblast elements, providing evidence that the maternally and paternally transmitted genomes are not functionally equivalent. We now know that the functional differences between the maternal and paternal genomes are attributed to genomic imprinting. CATEGORIES OF EPIGENETIC DISORDERS Genomic Imprinting As discussed in Chapter 6, imprinted genes are expressed from only one parental allele—that is, although two copies of the gene are present in the cell, only one copy is expressed. Which copy is expressed depends on the parent of origin and is determined by DNA methylation marks. The allele that is expressed is unmethylated, and the allele that is silenced is methylated. Although only a small percentage of human genes undergo genomic imprinting, many of these genes are critical regulators of growth and development, and therefore disruption of their normal monoallelic expression results in disorders that often impact both intrauterine and postnatal growth and neurodevelopment. The majority of imprinted genes are found in clusters, called imprinted domains, in specific chromosome regions (i.e., 15q11-13 and 11p15). Each imprinted domain is controlled by one or more independent imprinting control regions that regulate in cis the expression of target imprinted genes within the domain. More than 120 imprinted genes have been identified across the human genome (Fig. 8.3). Epigenetic changes that impact imprinting centers (ICs) and result in transcriptional silencing of a gene that is normally active are referred to as epimutations. The first human disorders recognized to result from genomic imprinting were Prader-Willi syndrome and Angelman syndrome (see Chapter 6). These two neurodevelopmental disorders result from the absence of paternally or maternally expressed genes, respectively, in the chromosome 15q11-13 imprinted region (which contains a cluster of imprinted genes). One of the characteristics of imprinting disorders is molecular heterogeneity in that there are several different mechanisms, including epigenetic and/or genetic, that can disrupt gene expression. This is seen with both Prader-Willi and Angelman syndromes, which can occur due to chromosome deletions, uniparental disomy (two copies of a single chromosome from one parent) (see Chapter 6; Table 6.5), imprinting defects (i.e., epimutation), and pathogenic sequence variants (UBE3A in Angelman syndrome). ▶

140 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE however, plasticity does not end at birth. Across the lifespan, DNA methylation patterns continue to change in both predictable and seemingl...

Ch8 · Pt7 CHAPTER 8 — Principles of Clinical Epigenetics 141 Other examples of paired human imprinting disorders are Beckwith-Wiedemann and Russell-Silver syndromes, which are two clinically opposite growth disorders that result from dysregulation of imprinted genes in the chromosome 11p15 region. Beckwith- Wiedemann syndrome is characterized by overgrowth, whereas Russell-Silver syndrome is characterized by intrauterine growth restriction and postnatal growth deficiency. Beckwith-Wiedemann syndrome is also associated with an increased risk for the development of embryonal tumors. The chromosome 11p15 region contains a cluster of imprinted genes that are organized into two distinct imprinted domains, each with its own imprinting control region: the IC1 domain in the telomeric region and the IC2 domain in the centromeric region. The IC1 domain contains the IGF2 and H19 genes, and the IC2 domain contains the CDKN1C, KCNQ1, and KCNQ10T1 genes. The genes in these regions undergo parent-of-origin imprinting such that typically IC1 is methylated on the paternally derived chromosome resulting in IGF2 expression (promotes cell growth and proliferation) and silencing of H19. On the maternally derived chromosome IC2 is methylated, resulting in silencing of KCNQ10T1 and expression of KCNQ1 and CDKN1C (negative regulator of cell proliferation). Opposite molecular alterations at IC1 and IC2 lead to an imbalance of growth-promoting and/or growth-suppressing genes in this region, either resulting in overgrowth (Beckwith-Wiedemann) or undergrowth (Russell-Silver). Therefore these conditions are mirror images of each other both clinically and molecularly (Fig. 8.4). Sometimes these two conditions can be seen in the same family when the underlying etiology is a chromosome duplication/deletion that is transmitted through a male versus a female due to parent-of-origin– specific imprinting of the chromosome 11p15 region (Fig. 8.5). The molecular mechanisms that cause these conditions are complex, and similar to the chromosome 15q11-13-related disorders include epigenetic and/or genetic alterations: cytogenetic aberrations, uniparental disomy, loss or gain of methylation at ICs (i.e., epimutation), and pathogenic sequence variants (CDKN1C in Beckwith-Wiedemann syndrome [Case 6]). Whereas imprinting disorders generally result from disturbed methylation in cis at one imprinted locus, there are also reports of individuals with multilocus imprinting disorders (MLID) in which there is aberrant methylation of multiple imprinted loci. Individuals with MLID can present with features specific for a single imprinting disorder or overlapping features of multiple imprinting disorders. MLID can be observed in children conceived via ART or caused by pathogenic variants in the patient’s genome (e.g., ZFP57), or pathogenic variants in maternal effect genes such as NLRP5 or PAD16, which encode proteins that impact imprinted loci in trans (see Epigenetics in Development, earlier). -TP73 -RNU5D-1 -DIRAS3 LRRTM1NAP1L5- -RHOBTB3 GPR1-AS ZDBF2 Chr 1 Chr 2 Chr 4 Chr 5 Chr 6 *GRB10 is maternally expressed in placenta and paternally expressed in brain -IGF2R, SLC22A3 HYMAI PLAGL1 PHACTR2 AIM1 -LIN28B, -FAM50B -GRB10* CALCR TFPI2 SGCE PEG10 PPP1R9A CPA4 MEST MESTIT1 COPG2IT1 KLF14 Chr 7 Chr 8 Chr 10 Chr 11 Chr 12 Chr 13 Chr 14 Chr 15 Chr 16 -KCNK9 -INPP5F_V2 NLRP2 AXL C19MC ZIM2 Chr 19 Chr 20 PEG3 -DNMT1 PSIMCT-1 L3MBTL BLCAP NNAT, MIMT1 AN01WT1- -DLGAP2 H19 IGF2 IGF2A5 INS KCNQ1 KCNQ10T1 CDKN1C SLC22A18 PHLDA2 -WIF1 DLK1 RTL1 IRAIN- -RB1 UBE3A NPAP1 NDN MKRN3 MAGEL2 #SNURF-SNRPN MEG3 MIR337 -ZNF597 NAA60 MEG8 GS-ALPHA, GNASXL, EXON1A, NESPAS, MIR269, MIR268 NESP, Figure 8.3 Ideograms of human imprinted genes. Ideograms were generated using http://www.dna-rainbow.org/ideograms/. An ideogram of each human chromosome known to have an imprinted gene based on the imprinted gene catalogue (http://igc.otago.ac.nz) and Gene Imprint portal (http://www.geneimprint.com) is shown. Imprinted genes are listed on each ideogram if they were designated as imprinted in both of the aforementioned human imprinted gene catalogs. Blue genes are paternally expressed, red genes are maternally expressed, black genes have unknown parent-of-origin expression, gray genes have parental expression that is isoform dependent. Bold genes are implicated in growth, underlined genes play roles in neurodevelopment. Genes in italic have no reported function in growth or neurodevelopment. ▶

CHAPTER 8 — Principles of Clinical Epigenetics 141 Other examples of paired human imprinting disorders are Beckwith-Wiedemann and Russell-Silver syndromes, which are two clinically opposite growth d...

Ch8 · Pt8 142 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Figure 8.5 Pedigree of a family in which a chromosome 11p15 duplication is segregating; different phenotypes determined by parent- of-origin–specific imprinting. Individual II-1 has a diagnosis of Beckwith-Wiedemann syndrome, which is determined to be due to a de novo chromosome duplication of chromosome 11p15 encompassing imprinting center 1 (IC1) on her paternally derived chromosome 11. She therefore has two copies of paternally imprinted genes in this region and one copy of maternally imprinted genes, which leads to relative hypermethylation of IC1. When she passes this chromosome duplication on to her children, the parental imprints will be erased and replaced with maternal imprints. Therefore her daughter (III-2) who inherits the chromosome 11p15 duplication will have two copies of maternally imprinted genes in this region and one copy of paternally imprinted genes, which leads to relative hypomethylation of IC1. This is associated with Russell-Silver syndrome. Figure 8.4 Opposite imprinting alterations on 11p15 can cause opposite phenotypes. Schematic representation of imprinting regulation at imprinting center 1 (IC1) in the chromosome 11p15 region. The highlighted box (middle) represents normal expression in which IC1 is methylated on the paternally derived chromosome and unmethylated on the maternally derived chromosome, resulting in expression of insulin-like growth factor 2 (IGF2) only from the paternal allele. (Top) Loss of methylation at IC1 on the paternal allele results in silencing of IGF2; suppression of IGF2 results in reduced growth and causes Russell-Silver syndrome (RSS). (Bottom) Gain of methylation at IC1 on the maternal allele results in activation of IGF2, which promotes growth and causes Beckwith-Wiedemann syndrome (BWS). Loss and gain of methylation at IC2 (not shown here) can also lead to BWS and RSS. ▶

142 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Figure 8.5 Pedigree of a family in which a chromosome 11p15 duplication is segregating; different phenotypes determined by parent- of-orig...

Ch8 · Pt9 CHAPTER 8 — Principles of Clinical Epigenetics 143 Pathogenic variants in maternal effect genes cause variable imprint dysregulation at multiple imprinted loci resulting in a broad range of clinical presentations, including infertility and adverse reproductive outcomes such as hydatidiform moles, recurrent miscarriages, and one or more imprinting disorders. Therefore, when investigating the etiology of MLID where there is a history of infertility and/or adverse pregnancy outcomes, one must consider testing not only the proband but also the proband ’s mother. Another consideration in the differential diagnosis of overlapping features of multiple imprinting disorders in the same individual is genome-wide paternal isodisomy. While genome-wide uniparental paternal disomy is not associated with a viable pregnancy, mosaicism for genome-wide paternal isodisomy has been reported in several individuals with overlapping features of multiple imprinting disorders; specifically, conditions resulting from uniparental disomy of imprinted chromosome regions (6q24, 11p15, 14q32, 15q11, 20q13). Genome- wide paternal uniparental disomy is typically characterized by mosaicism for paternal uniparental and biparental cell lineages. Clinical presentation depends on percentage of mosaic cells and location of the uniparental lineage. Disorders Involving Unstable Repeat Expansions Epigenetic mechanisms have been shown to play a critical role in the etiology of disorders due to unstable repeat expansions (see Chapter 13). This has been well established for fragile X syndrome, where the expansion of the FMR1 CGG repeat to a full mutation triggers a cascade of epigenetic events, including methylation of the FMR1 promotor, which leads to reduced or absent production of the fragile X mental retardation protein (FMRP). In males with normal size FMR1 alleles, the FMR1 promotor is unmethylated resulting in an open chromatin conformation that allows access of transcription factors to the FMR1 promoter, leading to transcription of FMRP. The importance of DNA methylation in mediating the expression of FMRP is illustrated by rare cases of males with full FMR1 expansion and normal cognition, in whom the FMR1 promoter has been shown to remain unmethylated. Many unstable repeat expansion disorders demonstrate anticipation, whereby increased disease severity and decreased age of onset are observed in subsequent generations. The basis of anticipation is the tendency for unstable repeats to undergo expansion when transmitted from parent to child. It has been proposed that epigenetic factors are involved in both disease pathogenesis and repeat instability. For example, congenital myotonic dystrophy (CDM1) is the most severe form of myotonic dystrophy type 1, a neuromuscular disease caused by the expansion of a CTG repeat in the DMPK gene. CDM1 shows strong genetic anticipation, as well as altered patterns of DNA methylation. Specifically, in individuals with CDM1, cis-regulatory elements upstream and downstream of the DMPK gene are often aberrantly methylated, thereby altering chromatin structure and gene expression at this locus—that is, impairment of these regulatory elements can lead to increased repeat instability providing early evidence for epigenetic involvement in genetic anticipation. Disorders of the Epigenetic Machinery Advances in genome sequencing technology have accelerated the discovery of genes involved in mendelian disorders. Over the last decade, an increasing number of mendelian disorders have been recognized to be caused by sequence variants in genes that are important for maintaining normal epigenetic regulation, including writers, erasers, readers, and chromatin remodelers. Although the majority of these syndromes are caused by loss of function of a single allele (haploinsufficiency) suggesting that these proteins function in a dosage- sensitive manner, both autosomal recessive and X-linked recessive patterns of inheritance are also described. In contrast to classical imprinting disorders that impact imprinted genes in cis, for this group of disorders the epigenetic dysregulation occurs in trans, impacting multiple genomic-wide targets. To date, there are over 80 disorders of the epigenetic machinery that have been identified and likely many more yet to be recognized (Fig. 8.6). These disorders are characterized by a wide range of multisystem anomalies, with the two most common phenotypic features observed being intellectual disability and growth dysregulation. Several of these disorders will be discussed later. Disorders of the Epigenetic Machinery: DNA Methylation There are a small number of genes that regulate DNA methylation marks in contrast to those that regulate histones; these include writers, readers, and erasers. Pathogenic variants in each of these genes are associated with specific phenotypes. Heterozygous germline pathogenic loss-of-function variants in the DNA methyltransferase DNMT3A (a writer) cause Tatton-Brown-Rahman syndrome (TBRS), a nonprogressive neurodevelopmental disorder characterized by increased growth, intellectual disability, and dysmorphic facial features. While constitutional pathogenic variants in DNMT3A cause TBRS, somatically acquired pathogenic variants in DNMT3A are associated with over 20% of acute myeloid leukemia (AML) cases. Notably the same pathogenic variants have been reported in association with both AML and TBRS; however, AML rarely occurs in individuals with TBRS, emphasizing the requirement for multistep ▶

CHAPTER 8 — Principles of Clinical Epigenetics 143 Pathogenic variants in maternal effect genes cause variable imprint dysregulation at multiple imprinted loci resulting in a broad range of clinical p...

Ch8 · Pt10 144 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE deregulation in cancer. Although there is an increased cancer (myeloid neoplasms, including AML) risk above the baseline population risk in individuals with TBRS, this does not meet the threshold for clinical surveillance; therefore cancer screening is not recommended for these individuals. There are other epigenetic regulators associated with mendelian disorders for which somatically acquired pathogenic variants are involved in cancers, including NSD1 and EZH2, which encode two histone methyltransferases (writers). Similarly, the phenotypes associated with germline pathogenic variants in these genes (Sotos syndrome and Weaver syndrome, respectively) have an increased cancer risk above the baseline population risk, which does not meet the threshold for clinical surveillance. Of interest, pathogenic variants in DNMT1, the maintenance methyltransferase, are associated with two distinct progressive adult-onset neurologic disorders. These are the only adult-onset conditions associated with pathogenic variants in an epigenetic regulator that we currently recognize and likely result from the ongoing loss of the cell’s capacity to maintain critical DNA methylation marks. The specific phenotype is determined by the position of the pathogenic variants in the gene. Hereditary sensory and autonomic neuropathy type 1 with dementia and hearing loss (HSAN1E), associated with variants in exon 20, is a disorder that presents in early adulthood with sensory neuropathy and hearing loss and progresses to dementia. The second syndrome, associated with variants in exon 21 of DNMT1, is called autosomal dominant cerebellar ataxia, deafness and narcolepsy and is characterized by adult-onset of narcolepsy followed by the onset of sensorineural deafness, cerebellar ataxia, and ultimately dementia. Methyl-Cp G-binding protein 2 (Me CP2), which functions as a reader of DNA methylation marks, has been studied extensively in part because pathogenic variants in this gene cause a well-recognized neurodevelopmental disorder, Rett syndrome (Case 40), which is characterized by acquired microcephaly, progressive intellectual disability, and loss of motor skills beginning in the first year of life. The majority (90%) of classic Rett syndrome cases are caused by loss-of-function variants in Me CP2, located at Xq 28. Those with classic Rett Recessive Recessive Growth Intellectual disability Dominant X-linked abnormalities Autosomal A and Dominant Remodeler, Writer, and Eraser epigenetic enzyme domains that also carry a Reader domain Figure 8.6 Mendelian disorders of the epigenetic machinery. Over 70 genes with defined epigenetic domains (reader, writer, eraser, remodeler, middle icons) have been linked to mendelian phenotypes. The majority of genes cause disease in the heterozygous state (filled circle). Enzyme domains (writer, eraser, remodeler) are mutually exclusive in any given factor but many coexist with a reader domain (gray shading). Intellectual disability is seen in the vast majority (blue), as are growth abnormalities (orange). A = genes on autosomes; X = genes on the X chromosome. (Modified from Fahrner JA, Bjornsson HT: Mendelian disorders of the epigenetic machinery: postnatal malleability and therapeutic prospects, Human Molecul Genet 28(2):R254–R264, 2019.) ▶

144 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE deregulation in cancer. Although there is an increased cancer (myeloid neoplasms, including AML) risk above the baseline population risk in...

Ch8 · Pt11 CHAPTER 8 — Principles of Clinical Epigenetics 145 syndrome are generally girls who are heterozygous for the loss-of-function variants. When boys with a pathogenic Me CP2 variant or deletion survive until birth they exhibit a severe infantile encephalopathy with seizures. As described earlier, the TET family of enzymes acts as erasers of DNA methylation marks. Whereas disorders involving writers and readers of DNA methylation have been known for some time, only recently was the first neurodevelopmental disorder impacting the DNA methylation eraser system described. TET3 deficiency, or Beck-Fahrner syndrome (BEFAHRS), is caused by either mono- and biallelic pathogenic variants in TET3, which encodes methylcytosine dioxygenase and is characterized by highly variable and nonspecific clinical features, including intellectual disability, features of autism, hypotonia, and dysmorphic facial features. This syndrome can be transmitted in an autosomal recessive or autosomal dominant manner. Disorders of the Epigenetic Machinery: Histones The number of genes involved in regulating histone modifications is much larger than for DNA methylation and include writers, erasers, readers, and chromatin remodelers. This group of disorders often presents with overlapping phenotypes, which can make them difficult to differentiate clinically. This phenotypic overlap can be attributed to the fact that the downstream targets of the various epigenetic regulators, which include different genes, are all involved in the regulation of a common pathway to brain and organ development. This means that distinguishing individual disorders is clinically very challenging. For example, Sotos and Weaver syndromes are two overgrowth conditions with overlapping features caused by different genes that function as epigenetic writers, NSD1 and EZH2, respectively. In spite of the fact that these two genes have different downstream targets, these conditions can be difficult to differentiate clinically especially in the first year or two of life. An accurate clinical diagnosis is important for anticipating the natural history as well as clarifying recurrence risk. Sotos syndrome is associated with significant intellectual and behavioral problems, whereas Weaver syndrome can have relatively mild or no intellectual deficits. There are also differences with respect to the types of cancers and their respective risks in these two conditions, which is important for anticipatory medical care. With respect to recurrence risks, most cases of Sotos syndrome have a de novo etiology, whereas Weaver syndrome is often familial, with a milder presentation in a parent only recognized after an affected child is born. Phenotypic overlap can also be seen when pathogenic variants occur in genes that function as part of multiprotein complexes. In this instance the phenotypic overlap results from the fact that regulation of common downstream targets is disrupted. This can be seen in Kabuki syndrome, a neurodevelopmental disorder characterized by growth deficiency, which can be caused by loss of function variants in one of two genes with opposite functions, KMT2D and KDM6A. KMT2D encodes a histone methyltransferase (writer) and KDM6A encodes a histone demethylase (eraser), two proteins that form a complex and have complementary roles in regulating chromatin state and transcriptional activity at a specific set of target genes. KMT2D adds a methylation mark associated with open chromatin (H3K4me 3), whereas KDM6A removes a mark associated with closed chromatin (H3K27me 3). Both genes facilitate the opening of chromatin and promote gene expression. Disruption of either gene/protein function will disrupt the balance of open versus closed chromatin at overlapping target genes resulting in the same clinical outcome (i.e., Kabuki syndrome). Identification of the specific genetic etiology is important because pathogenic variants in KMT2D are inherited in an autosomal dominant manner and usually occur de novo, whereas KDM6A is an X-linked recessive gene that can have a high risk of recurrence if inherited from a phenotypically normal carrier mother. There are also distinct clinical conditions with overlapping phenotypes that are caused by pathogenic variants in different genes within the same multiprotein complex. This can be seen with Coffin-Siris syndrome (CSS) and Nicolaides-Baraitser syndrome (NCBRS), two neurodevelopmental disorders that are caused by pathogenic variants in the ARID1B, SMARCB1, and SMARCA4 genes (CSS) and SMARCA2 gene (NCBRS). These genes are all part of the BAF chromatin remodeling complex. Although these two conditions have overlapping clinical features, attributable to the common downstream targets of the multiprotein complex that includes the respective causative genes, they also have important differences in natural history that require gene-based diagnosis for optimal management. DIAGNOSTIC TESTING FOR EPIGENETIC DISORDERS In light of the fact that the molecular mechanisms that cause imprinting disorders are heterogeneous, one must often utilize more than a single testing methodology to identify the underlying etiology. The hallmark of imprinting disorders is abnormal DNA methylation patterns. The most effective first line of investigation for imprinting disorders is methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA). See Chapter 5 for a description of the methodology. The benefit of using MS-MLPA is that it can assess both methylation levels and copy number variants across the relevant chromosome region; that is, it can distinguish between methylation abnormalities due to a deletion, uniparental disomy, or imprinting defect. In the case where a methylation abnormality is detected, additional testing may be required to determine the specific underlying molecular etiology. This could involve ▶

CHAPTER 8 — Principles of Clinical Epigenetics 145 syndrome are generally girls who are heterozygous for the loss-of-function variants. When boys with a pathogenic Me CP2 variant or deletion survive...

Ch8 · Pt12 146 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE chromosome microarray analysis to look for a chromosome rearrangement (e.g., deletion not detectable by the targeted probes utilized in MS-MLPA) or additional molecular testing with parental samples to test for uniparental disomy. If MS-MLPA testing is negative, given the underlying molecular heterogeneity in imprinting disorders, additional testing should be considered: specifically, sequence analysis of relevant imprinted genes (i.e., CDKN1C for Beckwith-Wiedemann syndrome or UBE3A for Angelman syndrome) and cytogenetic analysis for chromosome rearrangements that can impact imprinting without a change in methylation detectable by MS-MLPA. Identification of the etiology is critical to determining recurrence risk. For instance, a de novo methylation abnormality, without a concomitant genetic alteration, would confer a very low recurrence risk, whereas a methylation abnormality due to a deletion at an imprinting control region could confer a 50% risk of recurrence if inherited, depending on parent of origin. Another consideration in diagnostic testing for imprinting disorders is the fact that the underlying molecular changes may be present in a mosaic state; that is, some cells will have imprinting aberrations and some cells will have appropriate allelic methylation. This is commonly seen with chromosome 11p15 molecular alterations that cause Beckwith-Wiedemann syndrome, and accounts for some of the 20% of patients with a clinical diagnosis who have negative results following comprehensive molecular testing. Therefore one must be aware that a negative test result does not exclude a diagnosis because of the significant rate of somatic mosaicism. Mendelian disorders of the epigenetic machinery have traditionally been diagnosed via genome sequencing, including targeted single gene or panel testing (if a specific diagnosis is suspected) or genome-wide sequencing. However, sequencing technologies have several limitations, including coverage of noncoding regions, detection of complex sequence variants, and identification of variants of uncertain significance. A promising approach to improving the diagnostic yield of genetic disorders resulting from pathogenic sequence variants in epigenetic regulators involves analysis of genome- wide DNA methylation patterns. In the last few years, unique patterns of DNA methylation alterations, called DNA methylation signatures, have been defined for over 50 different genes. These signatures are developed by comparing peripheral blood–derived DNA for groups of cases with pathogenic variants in a specific gene to controls. The utility of these gene-specific signatures as functional biomarkers for diagnostic testing is increasingly being recognized as a novel tool. These signatures can be used to classify sequence variants of uncertain significance, as either pathogenic or benign, by comparing a DNA methylation profile generated for a specific case to the genome-wide DNA methylation signature for the gene in question and to controls. Given that the DNA methylation signatures to date have been derived in DNA from whole blood and that DNA methylation marks have cell-type specificity, the current signatures are limited to testing in blood-derived DNA samples. In the future as DNA methylation signatures are identified in other cell types, this technology can be more broadly applied. DNA METHYLATION AND CANCER DIAGNOSTICS Cancer, although conventionally considered a genetic disorder, often involves genome-wide epigenetic dysregulation, including alterations to DNA methylation, histone modifications, chromatin remodeling, and micro RNA. Given that one important function of eukaryotic DNA methylation is to maintain genomic stability by regulating the expression of oncogenes and tumor suppressor genes, it is not surprising that epigenetic dysregulation often contributes to tumor development and progression. Some of this epigenetic dysregulation is driven by somatically acquired pathogenic variants in specific chromatin modifier genes. Such variants are frequently observed in malignant cells and can result in aberrant genome-wide methylation changes leading to inappropriately expressed or repressed genes. Pathogenic variants in certain epigenetic regulators are often a hallmark of specific tumor types. DNA methylation patterns are becoming increasingly recognized as valuable diagnostic and prognostic tools in the cancer realm, particularly with respect to their utility in defining specific tumor types. This is especially important in tumors that escape definition by other molecular and pathologic methods. One particularly difficult challenge that can be addressed by DNA methylation is a cancer of unknown origin (i.e., metastatic disease for which the primary tumor is unknown) because the DNA methylation state of cell type at the time of tumor initiation remains identifiable during tumor development and progression. Therefore the DNA methylation profile of a tumor provides not only data about the current state of the cancer epigenome but also defines the tumor’s cell type of origin. As a result, DNA methylation-based clinical diagnosis and prognosis across many different types of primary cancers are now utilized to predict the primary site of metastatic cancers of unknown primaries. In addition to aiding in the diagnosis of tumor origin, DNA methylation-based diagnostics have been shown to play a valuable role in classifying different tumor subtypes, which can be critical in optimizing treatment/ management. One example is the ability to classify tissue samples into one of over 80 central nervous system tumors, and (even more valuable) the ability to identify subgroups within a particular tumor type. This can be seen in the case of medulloblastomas, where DNA methylation signatures have enabled the subclassification so ▶

146 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE chromosome microarray analysis to look for a chromosome rearrangement (e.g., deletion not detectable by the targeted probes utilized in MS-...

Ch8 · Pt13 CHAPTER 8 — Principles of Clinical Epigenetics 147 that four different subtypes are now recognized; these subtypes are associated with dramatic prognostic and therapeutic differences. We expect that ongoing advances in DNA methylation-based diagnostics in cancer will be integrated to improve the broader landscape of cancer diagnostics and treatment for patients. TREATMENT FOR NEURODEVELOPMENTAL DISORDERS CAUSED BY PATHOGENIC VARIANTS IN EPIGENETIC REGULATORS Historically postnatal treatment of neurodevelopmental disorders was not considered feasible because prior to our understanding of neuroplasticity the brain was considered to be a fully developed organ early in life. Early evidence for possible postnatal treatment of neurodevelopmental disorders came from studies of a mouse model of Rett syndrome, in which the restoration of Mecp 2 function led to reversal of advanced neurologic symptoms in adult mice. Given that a large group of neurodevelopmental disorders result from epigenetic dysregulation and that epigenetic changes are considered reversible, epigenetic mechanisms represent an attractive target for therapeutic intervention. Evidence of the efficacy of drugs that target epigenetic dysregulation was initially noted in clinical trials for cancer. Building on this approach, epigenetic therapeutics were trialed in mouse models of two neurodevelopmental disorders, specifically Rubinstein-Taybi syndrome (RTS) and Kabuki syndrome (KS), resulting from pathogenic variants in the epigenetic regulators CREB-binding protein (CREBBP) and KMT2D, respectively. In the mouse model of RTS, haploinsufficiency of CREBBP results in deficit chromatin acetylation as well as intellectual and memory deficits. Treatment using inhibition of histone deacetylase (HDAC) activity ameliorates both the chromatin acetylation and the memory deficit. In the case of KS, pathogenic variants in KMT2D cause a closed chromatin state impeding the transcription of genes critical for normal neurodevelopment. Treatment of KS mice with HDAC inhibitors restores the normal open chromatin state at these targets, resulting in improvement of long-term memory deficits. In addition, in a mouse model of KS, treatment with a ketogenic diet (which increases β-hydroxybutyrate levels) was noted to have a similar therapeutic effect. This positive outcome was attributed primarily to the HDAC inhibitor properties of β-hydroxybutyrate. These two models provide further evidence of the potential utility of epigenetic drugs or epigenetic-based therapies in treating neurodevelopment disorders postnatally. As of the writing of this chapter, some human clinical trials are being developed to evaluate the impact of epigenetic treatment approaches in neurodevelopmental disorders. There is a broad range of potential treatment options for neurodevelopmental disorders (see Chapter 14), including approaches that show promise other than epigenetic drugs. For example, the use of trofinetide, an IGF analog initially studied in mouse models, has now been shown to reduce repetitive behaviors and seizures in females with Rett syndrome (Case 40). Clinical trials are ongoing. Although these data are very promising, there remain many potential challenges that need to be addressed. One of particular importance is to define the critical brain regions that harbor cells that can be modulated postnatally. In this regard, studies in mouse models of both KS and RTS suggest that hippocampal cells in the dentate gyrus have self-renewal properties that could be channeled into partially rescuing memory and learning deficits in neurodevelopmental disorders. Other questions to be addressed include navigating the blood-brain barrier and windows of opportunity for successful treatment. Furthermore, current epigenetic agents do not target loci with aberrant epigenetic patterns but alter the epigenetic status at many sites across the genome, which may be associated with a number of adverse effects in unrelated cell types and pathways. Future progress in addressing these challenges is required to enable effective treatments of neurodevelopmental disorders. FUTURE DIRECTIONS In this chapter we have introduced many different facets of epigenetics and their relative applications, and a number of important themes clearly arise. Foremost, there is mounting evidence pointing to a role for epigenetic changes in health risk and disease in response to both genetic variation and environmental or lifestyle influences. Such epigenetic patterns have been shown to play a role not only in mendelian disorders but also in complex diseases and health outcomes that arise from certain environmental exposures. The dynamic and reversible nature of epigenetic changes permits a level of adaptability or plasticity that greatly exceeds the capacity of DNA sequence alone and thus is relevant both to the origins and the potential treatment of disease. Current obstacles to fully understanding the role of epigenetic aberrations in disease pathophysiology include (1) the sheer complexity of the epigenome, which consists of many interrelated and context-dependent chemical marks; (2) a unique epigenome that exists for each cell type and changes across the life span, especially during development; and (3) baseline levels of stochastic and nonstochastic variation among individuals, similar to that in the human genome. A number of large-scale epigenomics projects (akin to the original Human Genome Project) have been initiated to catalogue DNA methylation sites genome-wide (the so-called methylome), to evaluate Cp G landscapes across the genome, to discover new histone variants and modification patterns in various tissues, and to document positioning of nucleosomes ▶

CHAPTER 8 — Principles of Clinical Epigenetics 147 that four different subtypes are now recognized; these subtypes are associated with dramatic prognostic and therapeutic differences. We expect that o...

Ch8 · Pt14 148 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE around the genome in different cell types and in samples from both healthy individuals and those with cancer or other diseases. These analyses are part of a broad effort (called the ENCODE Project [Encyclopedia of DNA Elements]) to explore epigenetic patterns in chromatin genome-wide in order to better understand control of gene expression in different tissues or disease states. The data from such efforts can then be channeled into refining our understanding of the role of epigenetics in human health and disease and as a platform for improving personalized medicine approaches to diagnostics and therapeutics. GENERAL REFERENCES Bird A: Perceptions of epigenetics, Nature 447(7143):396–398, 2007. https://doi.org/10.1038/nature 05913 Greally JM: A user’s guide to the ambiguous word “epigenetics”, Nat Rev Mol Cell Biol 19(4):207–208, 2018. https://doi.org/10.1038/ nrm.2017.135 Smith ZD, Meissner A: DNA methylation: roles in mammalian development, Nat Rev Genet 14(3):204–220, 2013. https://doi.org/10.1038/ nrg 3354 Tucci V, Isles AR, Kelsey G, et al: Genomic imprinting and physiological processes in mammals, Cell 176(5):952–965, 2019. https://doi. org/10.1016/j.cell.2019.01.043 Ziller MJ, Gu H, Muller F, et al: Charting a dynamic DNA methylation landscape of the human genome, Nature 500(7463):477–481, 2013. https://doi.org/10.1038/nature 12433 SPECIFIC REFERENCES Aref-Eshghi E, Rodenhiser DI, Schenkel LC, et al: Genomic DNA methylation signatures enable concurrent diagnosis and clinical genetic variant classification in neurodevelopmental syndromes, Am J Hum Genet 102(1):156–174, 2018. https://doi.org/10.1016/j. ajhg.2017.12.008 Azzi S, Abi Habib W, Netchine I: Beckwith-Wiedemann and Russell- Silver syndromes: from new molecular insights to the comprehension of imprinting regulation, Curr Opin Endocrinol Diabetes Obes 21(1):30–38, 2014. https://doi.org/10.1097/MED.000000000 0000037 Barker DJ: The origins of the developmental origins theory, J Intern Med 261(5):412–417, 2007. https://doi.org/10.1111/j.1365-2796. 2007.01809.x Capper D, Jones DTW, Sill M, et al: DNA methylation-based classification of central nervous system tumours, Nature 555(7697):469–474, 2018. https://doi.org/10.1038/nature 26000 Chater-Diehl E, Goodman SJ, Cytrynbaum C, et al: Anatomy of DNA methylation signatures: emerging insights and applications, Am J Hum Genet 108(8):1359–1366, 2021. https://doi.org/10.1016/j. ajhg.2021.06.015 Cortessis VK, Azadian M, Buxbaum J: Comprehensive meta-analysis reveals association between multiple imprinting disorders and conception by assisted reproductive technology, J Assist Reprod Genet 35(6):943–952, 2018. https://doi.org/10.1007/s 10815-018-1173-x Dolinoy DC, Huang D, Jirtle RL: Maternal nutrient supplementation counteracts bisphenol A-induced DNA hypomethylation in early development, Proc Natl Acad Sci U S A 104(32):13056–13061, 2007. https://doi.org/10.1073/pnas.0703739104 Fahrner JA, Bjornsson HT: Mendelian disorders of the epigenetic machinery: postnatal malleability and therapeutic prospects, Hum Mol Genet 28(2):R254–R264, 2019. https://doi.org/10.1093/hmg/ddz 174 Fraga MF, Ballestar E, Paz MF: Epigenetic differences arise during the lifetime of monozygotic twins, Proc Natl Acad Sci U S A 102(30):10604– 10609, 2005. https://doi.org/10.1073/pnas.0500398102 Guo F, Yan L, Guo H, et al: The transcriptome and DNA methylome landscapes of human primordial germ cells, Cell 161(6):1437–1452, 2015. https://doi.org/10.1016/j.cell.2015.05.015 Heijmans BT, Tobi EW, Stein AD, et al: Persistent epigenetic differences associated with prenatal exposure to famine in humans, Proc Natl Acad Sci U S A 105(44):17046–17049, 2008. https://doi.org/10. 1073/pnas.0806560105 Horvath S, Raj K: DNA methylation-based biomarkers and the epigenetic clock theory of ageing, Nat Rev Genet 19(6):371–384, 2018. https://doi.org/10.1038/s 41576-018-0004-3 Kalish JM, Conlin LK, Bhatti TR, et al: Clinical features of three girls with mosaic genome-wide paternal uniparental isodisomy, Am J Med Genet A 161A(8):1929–1939, 2013. https://doi.org/10.1002/ajmg.a. 36045 Kraan CM, Godler DE, Amor DJ: Epigenetics of fragile X syndrome and fragile X-related disorders, Dev Med Child Neurol 61(2):121– 127, 2019. https://doi.org/10.1111/dmcn.13985 Lanni S, Pearson CE: Molecular genetics of congenital myotonic dystrophy, Neurobiol Dis 132:104533, 2019. https://doi.org/10.1016/j. nbd.2019.104533 Moran S, Martinez-Cardus A, Sayols S, et al: Epigenetic profiling to classify cancer of unknown primary: a multicentre, retrospective analysis, Lancet Oncol 17(10):1386–1395, 2016. https://doi. org/10.1016/S1470-2045(16)30297-2 PROBLEMS 1. a. When in human fetal development does genome-wide epigenetic reprogramming occur? b. How does this type of reprogramming differ by parent of origin? 2. Genetically identical Agouti mice heterozygous for the Avy or the viable yellow allele can display a range of phenotypes, including obesity and coat color differences. Describe the underlying epigenetic changes associated with expression of the Agouti gene and nutritional manipulations that can alter phenotypic presentation. 3. Assisted reproductive technologies (ART) increase the risk of a specific group of epigenetic disorders. Name this group of disorders, as well as two specific disorders within this category. 4. Mendelian disorders of the epigenetic machinery are characterized by a wide range of multisystem anomalies; however, these diverse disorders share some phenotypic features. a. List two common clinical features that are often observed. b. Describe two epigenetic functions of the proteins encoded by genes that cause such disorders when they carry pathogenic variants. 5. What is a DNA methylation signature? What is its application in clinical diagnostics for constitutional disorders of the epigenetic machinery and in the field of oncology? 6. Name a drug with an epigenetic mode of action. ▶

148 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE around the genome in different cell types and in samples from both healthy individuals and those with cancer or other diseases. These analys...

🧬 Part 4: Patterns of Inheritance and Epigenetics English

Chapter 7: Patterns of Single Gene Inheritance

Chapter 8: Principles of Clinical Epigenetics