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Lecture 3 Patterns of Single-Gene Inheritance Autosomal Dominant Inheritance More than half of all mendelian disorders are inherited as AD traits. The incidence of some autosomal dominant disorders is high, e.g., familial hypercholesterolemia, myotonic dystrophy, Huntington disease, neurofibromatosis, and polycystic kidney disease. AD disorders are individually much less common, in aggregate their total incidence is appreciable. The burden of autosomal dominant disorders is increased because of their hereditary nature; they become problems for whole kindreds, often through many generations. In some cases, the burden is compounded by social difficulties resulting from physical or mental disability. 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 strictly dominant or incompletely dominant. Denoting D as the mutant allele and d as the normal allele, matings that produce children with an autosomal dominant disease can be between two heterozygotes (D/d) for the mutation or, more frequently, between a heterozygote for the mutation (D/d) and a homozygote for a normal allele (d/d): Parental Mating Offspring Risk to Offspring Affected by unaffected D/d ×d/d 1/2 D/d, 1/2 d/d 1/2 affected 1/2 unaffected Affected by affected D/d ×D/d 1/4 D/D, 1/2 D/d, 1/4 d/d If strictly dominant: 3/4 affected 1/4 unaffected If incompletely dominant: 1/2 affected similarly to the parents 1/4 affected more severely than the parents 1/4 unaffected Offspring of D/d x d/d are approximately 50% D/d and 50% d/d. 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 theoretical expected ratio of 1:1, especially if the sibship is small. A pedigree showing typical inheritance of a form of progressive sensorineural deafness (DFNA1) inherited as an autosomal dominant trait. • Achondroplasia, an AD disorder that often occurs as a new mutation. • Note small stature with short limbs, large head, low nasal bridge, prominent forehead, and lumbar lordosis in this typical presentation. In medical practice, homozygotes for dominant phenotypes are not often seen because matings that could produce homozygous offspring are rare. Which mating can produce a D/D homozygote? Practically, only the mating of two heterozygotes need be considered because D/D homozygotes are very rare and generally too severely affected to reproduce (fitness =0). Incompletely Dominant Inheritance Achondroplasia: incompletely dominant skeletal disorder of short-limbed dwarfism and large head. Most achondroplastics have normal intelligence and lead normal lives within their physical capabilities. A homozygous child of two heterozygotes is often recognizable on clinical grounds alone; much more severely affected and commonly do not survive the immediate postnatal period. A pedigree of a mating between two individuals heterozygous for the mutation that causes achondroplasia. The deceased child, individual III-3, was a homozygote and died soon after birth. Another example is AD familial hypercholesterolemia, leading to premature coronary heart disease. The rare homozygotes have a more severe disease, with an earlier age at onset and much shorter life expectancy. Cutaneous xanthomas in a familial hypercholesterolemia homozygote. New Mutation in Autosomal Dominant Inheritance In typical AD 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 or until the occurrence of an original mutation. This is also true, for X-linked dominant pedigrees. In fact, most dominant conditions of any medical importance come about not only through transmission of the mutant allele but also through inheritance of a spontaneous, new mutation in a gamete. Relationship Between New Mutation and Fitness in Autosomal Dominant Disorders Once a new mutation has arisen, its survival in the population depends on the fitness of persons carrying it. There is an inverse relation between the fitness of a given AD disorder and the new mutation. At one extreme are disorders that have a fitness of zero, and the disorder is referred to as a genetic lethal. Must be due to new mutations. The affected individual will appear as an isolated case in the pedigree. If the fitness is normal, the disorder is rarely the result of fresh mutation; and the pedigree is likely to show multiple affected individuals. Sex-Limited Phenotype in Autosomal Dominant Disease AD phenotypes may also demonstrate a sex ratio that differs from 1:1. Extreme divergence of the sex ratio is seen in sexlimited phenotypes, in which the defect is autosomally transmitted but expressed in only one sex. An example is male-limited precocious puberty (familial testotoxicosis), an AD disorder in which affected boys develop secondary sexual characteristics and undergo an adolescent growth spurt at about 4 years of age. In some families, the defect is in the gene that encodes the receptor for luteinizing hormone (LCGR); these mutations constitutively activate the receptor's signaling action even in the absence of its hormone. The defect is not manifested in heterozygous females. Although the disease can be transmitted by unaffected females, it can also be transmitted directly from father to son, showing that it is autosomal, not X-linked. Males with precocious puberty due to activating LCGR mutations have normal fertility, and numerous multigeneration pedigrees are known. For disorders in which affected males do not reproduce, however, it is not always easy to distinguish sex-limited autosomal inheritance from X-linkage because the critical evidence, absence of male-to-male transmission, cannot be provided. In that case, other lines of evidence, especially gene mapping to learn whether the responsible gene maps to the X chromosome or to an autosome, can determine the pattern of inheritance and the consequent recurrence risk. Pedigree pattern (part of a much larger pedigree) of male-limited precocious puberty in the family of the child shown in Figure 7-14. This autosomal dominant disorder can be transmitted by affected males or by unaffected carrier females. Male-to-male transmission shows that the inheritance is autosomal, not X-linked. Because the trait is transmitted through unaffected carrier females, it cannot be Y-linked. Characteristics of Autosomal Dominant Inheritance The phenotype usually appears in every generation, each affected person having an affected parent. – Exceptions or apparent exceptions (1) cases originating from fresh 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 mutant allele. Any child of an affected parent has a 50% risk of inheriting the trait. – This is true for most families, in which the other parent is phenotypically normal. 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. – exceptions. Males and females are equally likely to transmit the phenotype, to children of either sex. In particular, male-to-male transmission can occur, and males can have unaffected daughters. A significant proportion of isolated cases are due to new mutation. The less the fitness, the greater is the proportion due to new mutation. X-LINKED INHERITANCE Phenotypes determined by genes on the X have a characteristic sex distribution and a pattern of inheritance that is usually easy to identify. Approximately 1100 genes are thought to be located on the X chromosome, of which approximately 40% are presently known to be associated with disease phenotypes There are only two possible genotypes in males and three in females with respect to a mutant allele at an X-linked locus. A male with a mutant allele at an X-linked locus is hemizygous for that allele, whereas females may be homozygous for either the wild-type or mutant allele or may be heterozygous. For example, if XH is the wild-type allele for the gene for coagulation factor VIII and a mutant allele, Xh, causes hemophilia A, the genotypes expected in males and females would be as follows: Genotypes Males Hemizygous XH Hemizygous Xh Females Homozygous XH/XH Heterozygous XH/Xh Homozygous Xh/Xh Phenotypes Unaffected Affected Unaffected Unaffected (usually) Affected X Inactivation, Dosage Compensation, and the Expression of X-Linked Genes The clinical relevance of X inactivation is profound. It leads to females having two cell populations, one in which one of the X chromosomes is active, the other in which the other X chromosome is active. For example, in Duchenne muscular dystrophy, female carriers exhibit typical mosaic expression, allowing carriers to be identified by dystrophin immunostaining. Depending on the pattern of random X inactivation of the two X chromosomes, two female heterozygotes for an Xlinked disease may have very different clinical presentations because they differ in the proportion of cells that have the mutant allele on the active X in a relevant tissue (as seen in manifesting heterozygotes). Immunostaining for dystrophin in muscle specimens. A, A normal female (magnification ×480). B, A male with Duchenne muscular dystrophy (×480). C, A carrier female (×240). Staining creates the bright lines 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, reflecting X inactivation Recessive and Dominant Inheritance of X-Linked Disorders X-linked "dominant" and "recessive" patterns of inheritance are distinguished on the basis of the phenotype in heterozygous females. Some Xlinked phenotypes are consistently expressed in carriers (dominant), whereas others usually are not (recessive). The difficulty in classifying an X-linked disorder as dominant or recessive arises because females who are heterozygous for the same mutant allele in the same 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 mutant allele on the active versus inactive chromosome. X-Linked Recessive Inheritance The inheritance of X-linked recessive phenotypes follows a well-defined and easily recognized pattern. An X-linked recessive mutation is typically expressed phenotypically in all males who receive it but only in those females who are homozygous for the mutation. X-linked recessive disorders are generally restricted to males and rarely seen among females (except for manifesting heterozygotes). Hemophilia A is a classic X-linked recessive disorder in which the blood fails to clot normally because of a deficiency of factor VIII. 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. If a hemophiliac mates with a normal female ? Now assume that a daughter of the affected male mates with an unaffected male ? 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. Homozygous Affected Females Relevant for X-linked color-blindness, a relatively common X-linked disorder (an affected male x a carrier female). Most X-linked diseases are so rare, unusual for a female to be homozygous unless parents are consanguineous Affected male x carrier female Homozygous affected female Consanguinity in an X-linked recessive pedigree for redgreen color blindness, resulting in a homozygous affected female Manifesting Heterozygotes and Unbalanced Inactivation for X-linked Disease Rare, a female carrier of a recessive X-linked allele has phenotypic expression of disease = manifesting heterozygote. Have been described for many X-linked recessive disorders, e.g., color-blindness, hemophilia A & B, DMD, Wiskott-Aldrich syndrome (an X-linked immunodeficiency), etc. Whether a female heterozygote will be a manifesting heterozygote depends on a number of factors: First, the fraction of cells in which the normal/mutant allele happens to remain active (unbalanced or skewed X-inactivation). Second, depending on the disorder in question, females can have very different degrees of disease penetrance and expression, even if their degree of skewed inactivation is the same, because of underlying physiological functioning of genes e.g., – In Hunter syndrome (iduronate sulfatase deficiency), cells with normal allele on active X can export enzyme to extracellular space, picked up by cells in which mutant allele on active X and defect is corrected in those cells – So, penetrance for Hunter syndrome in heterozygous females is extremely low even when X-inactivation deviates from expected random 50%:50% pattern On the other hand, nearly half of all female heterozygotes for fragile-X syndrome show developmental abnormalities. In addition to manifesting heterozygotes, the opposite pattern of skewed inactivation can also occur. Characteristics of X-Linked Recessive Inheritance The incidence of the trait is much higher in males. Heterozygous females are usually unaffected, exception? The gene responsible is transmitted from an affected man through all his daughters. Any of his daughters' sons has a 50% chance of inheriting it. The mutant allele is ordinarily never transmitted directly from father to son. The mutant allele may be transmitted 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. X-linked Dominant Inheritance Regularly expressed in heterozygotes No male-to-male transmission For a fully penetrant XD pedigree, all daughters and none of sons of affected males are affected. Pattern of inheritance through female is no different from AD. The expression is usually milder in females, who are almost always heterozygotes. Thus, most XD disorders are incompletely dominant. Only a few genetic disorders are classified as XD. E.g., X-linked hypophosphatemic rickets (a.k.a. vitamin D-resistant rickets) Defective gene product is one of the endopeptidases that activate or degrade a variety of peptide hormones Both sexes are affected but, serum phosphate level is less depressed and rickets less severe in heterozgous females. Pedigree pattern demonstrating X-linked dominant inheritance X-linked Dominant Disorders with Male Lethality Some rare genetic defects expressed exclusively or almost exclusively in females appear to be XD lethal in males before birth Typical pedigrees: transmission by affected female affected daughters, normal daughters, normal sons in equal proportions (1:1:1) Rett syndrome meets criteria for an XD that is usually lethal in hemizygous males. The syndrome is characterized by normal prenatal and neonatal growth and development, followed by rapid onset of neurological symptoms and loss of milestones between 6 and 18 months of age. Rett syndrome cont. Children become spastic and ataxic, develop autistic features and irritable behavior with outbursts of crying, and demonstrate characteristic purposeless wringing or flapping movements of hands and arms. Head growth slows and microcephaly develops. Seizures are common (~50%) Surprisingly, mental deterioration stops after a few years and the patients can then survive for many decades with a stable but severe neurological disability. Most cases caused by spontaneous mutations in an Xlinked MECP2 gene encoding methyl CpG binding protein 2. ? Thought to reflect abnormalities in regulation of genes in developing brain. Typical appearance and hand posture of girls with Rett syndrome Rett syndrome cont. Males who survive with the syndrome usually have two X chromosomes (as in 47,XXY or in a 46,X,der(X) male with the male determining SRY gene translocated to an X) or are mosaic for a mutation that is absent in most of their cells There are a few apparently unaffected women who have given birth to more than one child with Rett syndrome. ? X-inactivation pattern in a heterozygous female. ? Germline mosaic ? Pedigree pattern demonstrating an X-linked dominant disorder, lethal in males during the prenatal period. Characteristics of X-Linked Dominant Inheritance Affected males with normal mates have no affected sons and no normal daughters. Both male and female offspring of a heterozygous female have a 50% risk of inheriting the phenotype. The pedigree pattern is similar to that seen with autosomal dominant inheritance. Affected females are about twice as common as affected males, but affected females typically have milder (although variable) expression of the phenotype. New Mutation in X-Linked Disorders In males, genes for 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. Patients with hemophilia have only about 70% as many offspring as unaffected males do; that is, the fitness of affected males is about 0.70. Selection against mutant alleles is more dramatic for X-linked disorders such as DMD. DMD is currently a genetic lethal because affected males usually fail to reproduce. It may, of course, be transmitted by carrier females, who themselves rarely show any clinical manifestation of the disease. New mutations constitute a significant fraction of isolated cases of many X-linked diseases. When patients are affected with a severe X-linked recessive disease, such as DMD, they cannot reproduce (i.e., selection is complete), and therefore the mutant alleles they carry are lost from the population. Because the incidence of DMD is not changing, mutant alleles lost through failure of the affected males to reproduce are continually replaced by new mutations. PSEUDOAUTOSOMAL INHERITANCE Pseudoautosomal inheritance describes the inheritance pattern seen with genes in the pseudoautosomal region. Alleles for genes in the pseudoautosomal region can show male-to-male transmission, and therefore mimic autosomal inheritance, because they can cross over from the X to the Y during male gametogenesis and be passed on from a father to his male offspring. Dyschondrosteosis, a dominantly inherited skeletal dysplasia with disproportionate short stature and deformity of the forearm, is an example of a pseudoautosomal condition inherited in a dominant manner. A greater prevalence of the disease was seen in females as compared with males, suggesting an Xlinked dominant disorder, but the presence of male-to-male transmission clearly ruled out strict X-linked inheritance. Mutations in the SHOX gene encoding a homeodomain-containing transcription factor have been found responsible for this condition. SHOX is located in the pseudoautosomal region on Xp and Yp and escapes X inactivation. Figure 7-22 Pedigree showing inheritance of dyschondrosteosis due to mutations in 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 MOSAICISM Mosaicism is the presence in an individual or a tissue of at least two cell lines that differ genetically but are derived from a single zygote. Mosaicism due to X inactivation is a wellknown phenomenon. More generally, mutations arising in a single cell in either prenatal or postnatal life can give rise to mosaicism. Mosaicism for numerical or structural abnormalities of chromosomes is a clinically important phenomenon, and somatic mutation is recognized as a major contributor to many types of cancer. Mosaicism for mutations in single genes, in either somatic or germline cells, explains a number of unusual clinical observations, such as segmental neurofibromatosis, in which skin manifestations are not uniform and occur in a patchy distribution, and the recurrence of osteogenesis imperfecta, a highly penetrant autosomal dominant disease, in two or more children born to unaffected parents. The population of cells that carry a mutation in a mosaic individual could theoretically be present in some tissues of the body but not in the gametes (pure somatic mosaicism), be restricted to the gamete lineage only and nowhere else (pure germline mosaicism), or be present in both somatic lineages and the germline, depending on when the mutation occurred in embryological development. Whether mosaicism for a mutation involves only somatic tissues, the germline, or both depends on whether during embryogenesis the mutation occurred before or after the separation of germline cells from somatic cells. If before, both somatic and germline cell lines would be mosaic and the mutation could be transmitted to the offspring as well as being expressed somatically in mosaic form. Thus, e.g., if a mutation were to occur in a germline precursor cell, a proportion of the gametes would carry the mutation. There are about 30 mitotic divisions in the cells of the germline before meiosis in the female and several hundred in the male, allowing ample opportunity for mutations to occur during the mitotic stages of gamete development. Schematic presentation of mitotic cell divisions. A mutation occurring during cell proliferation, in somatic cells or during gametogenesis, leads to a proportion of cells carrying the mutation-that is, to either somatic or germline mosaicism. Determining whether mosaicism for a mutation is present only in the germline or only in somatic tissues may be difficult because failure to find a mutation in a subset of cells from a readily accessible somatic tissue (such as peripheral white blood cells, skin, or buccal cells) does not ensure that the mutation is not present elsewhere in the body, including the germline. Characterizing the extent of somatic mosaicism is made more difficult when the mutant allele in a mosaic fetus occurs exclusively in the extraembryonic tissues (i.e., the placenta) and is not present in the fetus itself. Somatic 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 is sometimes segmental, affecting only one part of the body. Segmental NF1 is caused by mosaicism for a mutation that occurred after conception. In such cases, the patient has normal parents, but if he or she has an affected child, the child's phenotype is typical for NF1, that is, not segmental. Germline Mosaicism There are well-documented examples where parents who are phenotypically normal and test negative for being carriers have more than one child affected with a highly penetrant autosomal dominant or X-linked disorder. Such unusual pedigrees can be explained by germline mosaicism. Germline mosaicism is well documented in as many as 6% of severe, lethal forms of the AD osteogenesis imperfecta, in which mutations in type I collagen genes lead to abnormal collagen, brittle bones, and frequent fractures. Pedigrees that could be explained by germline mosaicism have also been reported for several other well-known disorders, such as hemophilia A, hemophilia B, and DMD, but have only very rarely been seen in other dominant diseases, such as achondroplasia. Accurate measurement of the frequency of germline mosaicism is difficult, but estimates suggest that the highest incidence is in DMD, in which up to 15% of the mothers of isolated cases show no evidence of the mutation in their somatic tissues and yet carry the mutation in their germline. Pedigree demonstrating recurrence of the autosomal dominant disorder osteogenesis imperfecta. Both affected children have the same point mutation in a collagen gene. Their father (arrow) is unaffected and has no such mutation in DNA from examined somatic tissues. He must have been a mosaic for the mutation in his germline. Geneticists and genetic counselors are aware of the potential inaccuracy of predicting that a specific autosomal dominant or X-linked phenotype that appears by every test to be a new mutation must have a negligible recurrence risk in future offspring. Obviously, in diseases known to show germline mosaicism, phenotypically normal parents of a child whose disease is believed to be due to a new mutation should be informed that the recurrence risk is not negligible! Furthermore, apparently non-carrier parents of a child with any autosomal dominant or X-linked disorder in which mosaicism is possible but unproven may have a recurrence risk that may be as high as 3% to 4%; these couples should be offered whatever prenatal diagnostic tests are appropriate. The exact recurrence risk is difficult to assess, however, because it depends on what proportion of gametes contains the mutation IMPRINTING IN PEDIGREES Unusual Inheritance Patterns due to Genomic imprinting In some genetic disorders such as PWS and AS, the expression of the disease phenotype depends on whether the mutant allele has been inherited from the father or from the mother, a phenomenon known as genomic imprinting. Imprinting can cause unusual inheritance patterns in pedigrees, as clearly demonstrated by a rare condition known as Albright hereditary osteodystrophy (AHO). AHO is characterized by obesity, short stature, subcutaneous calcifications, and brachydactyly, particularly of the fourth and fifth metacarpal bones. A, Characteristic appearance of a patient with Albright hereditary osteodystrophy. B, Hand radiograph showing shortened metacarpals and distal phalanges, especially and characteristically the fourth metacarpal AHO is inherited as a fully penetrant autosomal dominant trait. What is unusual, however, is that in families of individuals affected by AHO, some but not all of the affected patients have an additional clinical disorder known as pseudohypoparathyroidism (PHP). In PHP, an abnormality of calcium metabolism typically seen with a deficiency of parathyroid hormone occurs but with elevated levels of parathyroid hormone (hence the use of the prefix pseudo) that is secondary to renal tubular resistance to the effects of parathyroid hormone. PHP in an individual with the AHO phenotype is known as pseudohypoparathyroidism type 1a (PHP1a). AHO with or without PHP is caused by a defect in the GNAS gene. GNAS is involved in transmitting the parathyroid hormone signal from the surface of renal cells to inside the cell. A careful examination of PHP1a pedigrees shows that some individuals have AHO only, without the calcium and renal problems, whereas others have the physical characteristics as a component of PHP1a. When AHO occurs without the renal tubular dysfunction in families in which other relatives have PHP1a, it is often referred to as pseudopseudohypoparathyroidism (PPHP). Interestingly, when PPHP and PHP1a occur within the same family, affected brothers and sisters in any one sibship either all have PPHP or all have PHP1a; what does not happen is that one sib will have one condition while another has the other. This unusual pattern of inheritance can be explained by the fact that the defective gene (GNAS) in PHP1a and PPHP is imprinted only in certain tissues, including renal tubular cells, so that only the GNAS allele inherited from the mother is expressed in these cells while the father's allele is normally silent. PHP1a therefore occurs only when an individual inherits an inactivating mutation in GNAS from his or her mother; since the paternal copy is not expressed anyway, these tissues have no normal, functioning copy of GNAS, and resistance to the effects of parathyroid hormone ensues. There is no imprinting, however, in most of the tissues of the body. In the tissues without GNAS imprinting, heterozygotes for one mutant GNAS allele all develop AHO, which is passed on as a simple autosomal dominant trait. Pedigrees of pseudohypoparathyroidism. A, Family with pseudohypoparathyroidism 1a (PHP1a, solid-blue symbols) and pseudopseudohypoparathyroidism (PPHP, halfblue symbols), showing that all PHP1a patients inherit the mutant GNAS gene from their mothers, whereas all PPHP patients have a paternally derived mutant allele. The effect of imprinting is also seen in another form of AD pseudo-hypoparathyroidism, known as PHP type 1b. PHP1b has the calcium abnormalities seen in PHP1a but without the physical signs of AHO. PHP1b is caused by a mutation in upstream regulatory elements (the "imprinting center") that control the imprinting of the GNAS gene; the normal function of these regulatory elements is to specify that the maternally inherited GNAS allele, and only that allele, will be expressed in renal tubules. When a mutation of the imprinting control region is inherited from the mother, both the paternal allele, which is normally silent in kidney tubules, and the maternal allele, which is silenced in these tissues because of the deletion, fail to be expressed, and PHP1b ensues. Individuals who inherit the mutation from their fathers, however, are asymptomatic heterozygotes because their maternal copy of GNAS, with its imprinting control region intact, is expressed normally in these tissues. Outside of the kidney and a few other tissues, both maternal and paternal GNAS alleles are expressed independently of any imprinting, and AHO therefore does not occur. B, Pedigree of family with PHP1b (solid-blue symbols) due to a deletion in the imprinting control region. All affected patients inherit the deletion allele from their mothers; heterozygotes with a paternal allele are unaffected. Heterozygotes for a deletion mutation in the imprinting regulatory region of the GNAS gene are indicated by the blue dots. UNSTABLE REPEAT EXPANSIONS In all of the types of inheritance presented earlier in this chapter, the responsible mutation, once it occurs, is stable from generation to generation. In contrast, an entirely new class of genetic disease has been recognized, diseases due to unstable repeat expansions. By definition, these conditions are characterized by an expansion within the affected gene of a segment of DNA consisting of repeating units of three or more nucleotides in tandem (i.e., adjacent to each other). For example, the repeat unit often consists of three nucleotides, such as CAG or CCG, and the repeat will be CAGCAGCAG … CAG or CCGCCGCCG … CCG. In general, the genes associated with these diseases all have wild-type alleles that are polymorphic; that is, there is a variable but relatively low number of repeat units in the normal population. As the gene is passed from generation to generation, however, the number of repeats can increase (undergoes expansion), far beyond the normal polymorphic range, leading to abnormalities in gene expression and function. The molecular mechanisms by which such expansions occur are not clearly understood but are likely to be due to a type of DNA replication error known as slipped mispairing. The discovery of this unusual group of conditions has dispelled the orthodox notions of germline stability and provided a biological basis for such eccentric genetic phenomena as anticipation and parental transmission bias. Table 7-3. Four Representative Examples of Unstable Repeat Expansion Diseases Repeat Number Disease Inheritance pattern Repeat Gene Affected Location in Gene Normals intermediate Affected Huntington disease Autosomal dominant CAG HD coding region <36 36-39 usually affected >40 Fragile X X-linked CGG FMR1 5' untranslated <60 60-200 usually unaffected* Myotonic dystrophy Autosomal dominant CTG DMPK 3' untranslated <30 50-80 may be mildly affected Friedreich ataxia Autosomal recessive AAG FRDA intron <34 36-100 *May have tremor-ataxia syndrome or premature ovarian failure. >200 80-2000 >100 More than a dozen diseases are known to result from unstable repeat expansions. All of these conditions are primarily neurological. A dominant inheritance pattern occurs in some, Xlinked in others, and recessive inheritance in still others. The degree of expansion of the repeat unit that causes disease is sometimes subtle (as in the rare disorder oculopharyngeal muscular dystrophy) and sometimes explosive (as in congenital myotonic dystrophy or severe fragile X syndrome). Other differences between the various unstable repeat expansion diseases include: - the length and base sequence of the repeated - unit; the number of repeated units in normal, presymptomatic and fully affected individuals; the location of the repeated unit within genes; the pathogenesis of the disease; the degree to which the repeated units are unstable during meiosis or mitosis; and parental bias in when expansion occurs. Polyglutamine Disorders Huntington Disease Huntington disease (HD) is a well-known disorder that illustrates many of the common genetic features of the polyglutamine disorders caused by expansion of an unstable repeat. HD was first described by the physician George Huntington in 1872 in an American kindred of English descent. The neuropathology is dominated by degeneration of the striatum and the cortex. Patients first present clinically in midlife and manifest 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, AD. Although homozygotes may have a more rapid course of their disease. There are, however, obvious peculiarities in its inheritance that could not be explained by simple AD inheritance. First, the age at onset of HD is variable; only about half the individuals who carry a mutant HD allele show symptoms by the age of 40 years. Second, the disease appears to develop at an earlier and earlier age when it is transmitted through the pedigree, a phenomenon referred to as anticipation, but only when it is transmitted by an affected father and not by an affected mother. These are now readily explained by the discovery that the mutation is composed of an abnormally long expansion of a stretch of the nucleotides CAG, the codon specifying glutamine, in the coding region of a gene for a protein of unknown function called huntingtin. Normal individuals carry between 9 and 35 CAG repeats in their HD gene, with the average being 18 or 19. Individuals affected with HD have 40 or more repeats, with the average being around 46. 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. Once an expansion increases to greater than 39, however, disease always occurs, and the larger the expansion, the earlier the onset of the disease. Figure 7-27 Graph correlating approximate age at onset of Huntington disease with the number of CAG repeats found in the HD gene. The solid line is the average age at onset, and the shaded area shows the range of age at onset for any given number of repeats Figure 7-28 Pedigree of family with Huntington disease. Shown beneath the pedigree is a Southern blot analysis for CAG repeat expansions in the huntingtin 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. I-1, who developed HD at the age of 64 years and is now deceased, had an abnormal repeat length of 37. He has three affected children, two of whom have repeat lengths of 55 and 70 and developed disease in their 40s, and a son with juvenile HD and 103 CAG repeats in his huntingtin gene. Individual II-1 is unaffected at the age of 50 years but will develop the disease later in life. Individuals I-2 and II-3 have two alleles of normal length (25). Repeat lengths were confirmed by PCR analysis. How, then, does an individual come to have an expanded CAG repeat in his or her HD gene? Most commonly, he or she inherits it as a straightforward autosomal dominant trait from an affected parent who already has an expanded repeat (>36). In contrast to stable mutations, however, the size of the repeat may expand on transmission, resulting in earlier onset disease in later generations; on the other hand, repeat numbers in the range of 40 to 50 may not result in disease until later in life, thereby explaining the agedependent penetrance. In this pedigree, individual I-1, now deceased, was diagnosed with HD at the age of 64 years and had an expansion of 37 CAG repeats. Four of his children inherited the expanded allele, and in all four of them, the expansion increased over that found in individual I-1 Individual II-5, in particular, has the largest number of repeats and became symptomatic during adolescence. Individual II-1, in contrast, inherited an expanded allele but remains asymptomatic and will likely develop the disease sometime later in life. On occasion, unaffected individuals carry alleles with repeat lengths at the upper limit of the normal range (29 to 35 CAG repeats) that, however, can expand during meiosis to 40 or more repeats. 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 premutations. Expansion in HD shows a paternal transmission bias and occurs most frequently during male gametogenesis, which is why the severe earlyonset juvenile form of the disease, seen with the largest expansions (70 to 121 repeats), is always paternally inherited. Expanded repeats may continue to be unstable during mitosis in somatic cells, resulting in some degree of somatic mosaicism for the number of repeats in different tissues from the same patient. The largest known group of HD patients lives in the region of Lake Maracaibo, Venezuela; these patients are descendants of a single individual who introduced the gene into the population early in the 19th century. About 100 living affected persons and another 900, each at 50% risk, are currently known in the Lake Maracaibo community. High frequency of a disease in a local population descended from a small number of individuals, one of whom carried the gene responsible for the disease, is an example of founder effect. Spinobulbar Muscular Atrophy and Other Polyglutamine Disorders In addition to HD, other neurological diseases are caused by CAG expansions encoding polyglutamine, such as X-linked recessive spinobulbar muscular atrophy and the various autosomal dominant spinocerebellar ataxias. These conditions differ in the gene involved, the normal range of the repeat, the threshold for clinical disease caused by expansion, and the regions of the brain affected. They all share with HD the fundamental characteristic that results from instability of a stretch of repeated CAG nucleotides leading to expansion of a glutamine tract in a protein. Fragile X Syndrome The fragile X syndrome is the most common heritable form of moderate MR and is second only to Down syndrome among all causes of MR in males. The name refers to a cytogenetic marker on the X chromosome at Xq27.3, a "fragile site" 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 at least 1 in 4000 male births and is so common that it requires consideration in the differential diagnosis of MR in both males and females. Testing for the fragile X syndrome is among the most frequent indications for DNA analysis, genetic counseling, and prenatal diagnosis. Figure 7-30 The fragile site at Xq27.3 associated with X-linked mental retardation The disorder is caused by another unstable repeat expansion, a massive expansion of another triplet repeat, CGG, located in the 5' untranslated region of the first exon of a gene called FMR1 (fragile X mental retardation 1). The normal number of repeats is up to 60, whereas as many as several thousand repeats are found in patients with the "full" fragile X syndrome mutation. More than 200 copies of the repeat lead to excessive methylation of cytosines in the promoter of FMR1; this interferes with replication or chromatin condensation or both, producing the characteristic chromosomal fragile site, a form of DNA modification that prevents normal promoter function or blocks translation. Triplet repeat numbers between 60 and 200 constitute a special 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), with the risk of expansion increasing dramatically with increasing premutation size. Carriers of premutations can develop an adult-onset neurological disorder of cerebellar dysfunction and neurological deterioration, known as the fragile Xassociated tremor/ataxia syndrome. In addition, approximately one quarter of female carriers of premutations will experience premature ovarian failure by the age of 40 years. Figure 7-29 Characteristic facial appearance of a patient with the fragile X syndrome Figure 7-31 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 of fragile X syndrome to her sons is approximately half this frequency, since there is a 50% chance a son will inherit the expanded allele. The risk of fragile X syndrome to her daughters is approximately one-fourth this frequency, since there is a 50% chance a daughter would inherit the full mutation, and penetrance of the full mutation in a female is approximately 50% Myotonic Dystrophy Myotonic dystrophy (dystrophia myotonica, or DM) is inherited as an autosomal dominant myopathy characterized by myotonia, muscular dystrophy, cataracts, hypogonadism, diabetes, frontal balding, and changes in the electroencephalogram. The disease is notorious for lack of penetrance, pleiotropy, and its variable expression in both clinical severity and age at onset. The DM congenital form, is particularly severe and may be life-threatening as well as a cause of MR. Virtually every child with the congenital form is the offspring of an affected mother, who herself may have only a mild expression of the disease and may not even know that she is affected. Thus, pedigrees of DM, like those of HD and fragile X syndrome, show clear evidence of anticipation. DM is also associated with amplification of a triplet repeat, in this case a CTG triplet located in the 3' untranslated region of a protein kinase gene (DMPK). The normal range for repeats in DMPK is 5 to 30; carriers of repeats in the range of 38 to 54 (premutations) are usually asymptomatic but have an increased risk of passing on fully expanded repeats. Mildly affected individuals have about 50 to 80 copies; the severity increases and age at onset decreases the longer the expansion. Myotonic dystrophy, an autosomal dominant condition with variable expression in clinical severity and age at onset. The grandmother in this family (left) had bilateral cataracts but has no facial weakness or muscle symptoms; her daughter was thought to be unaffected until after the birth of her severely affected child, but she now has moderate facial weakness and ptosis, with myotonia, and has had cataract extraction. The child has congenital myotonic dystrophy Severely affected individuals can have more than 2000 copies. Either parent can transmit an amplified copy, but males can pass on up to 1000 copies of repeat, whereas really massive expansions containing many thousands of repeats occur only in female gametogenesis. Because congenital DM is due to huge expansions in the many thousands, this form of myotonic dystrophy is therefore almost always inherited from an affected mother. Friedreich Ataxia Friedreich ataxia (FRDA), a spinocerebellar ataxia, constitutes a fourth category of triplet repeat disease. The disease is inherited in an AR pattern, in contrast to HD, DM, and fragile X syndrome. The disorder is usually manifested before adolescence and is generally characterized by incoordination of limb movements, difficulty with speech, diminished or absent tendon reflexes, impairment of position and vibratory senses, cardiomyopathy, scoliosis, and foot deformities. In most cases, Friedreich ataxia is caused by amplification of still another triplet repeat, AAG, located this time in an intron of a gene that encodes a mitochondrial protein called frataxin, which is involved in iron metabolism. In normal individuals, the repeat length varies from 7 to 34 copies, whereas repeat expansions in the patients are typically between 100 and 1200 copies. Expansion within the intron interferes with normal expression of the frataxin gene; because Friedreich ataxia is recessive, loss of expression from both alleles is required to produce the disease. In fact, 1% to 2% of FRDA patients are known to be compound heterozygotes in whom one allele is the common amplified intronic AAG repeat mutation and the other a nucleotide mutation Similarities and Differences Among Unstable Repeat Expansion Disorders A comparison of HD (and the other polyglutamine neurodegeneration diseases) with the fragile X syndrome, DM, and FRDA reveals some similarities but also many differences Although unstable repeat expansions of a trinucleotide are involved in all four types of disease, the expansion in the polyglutamine diseases is in the coding region and ranges from 40 to 120 copies of the CAG, whereas the repeat expansions in fragile X syndrome, DM, and FRDA involve different triplet nucleotides, contain hundreds to thousands of repeated triplets, and are located in untranslated portions of the FMR1, DMPK, and FRDA genes, respectively. Premutation expansions causing an increased risk for passing on full mutations are the rule in all four of these disorders, and anticipation is commonly seen in pedigrees of the dominant and X-linked diseases (HD, fragile X syndrome, and DM). However, the number of repeats in premutation alleles in HD is 29 to 35, similar to what is seen in DM but far less than in fragile X syndrome. Premutation carriers can develop significant disease in fragile X syndrome but are, by definition, disease-free in HD and DM. The expansion of premutation alleles occurs in the female primarily in FRDA, DM, and fragile X syndrome; the largest expansions causing juvenile onset HD occur in the male germline. Finally, the degree of mitotic instability in fragile X syndrome, DM, and FRDA is far greater than that seen in HD and results in much greater variability in the numbers of repeats found among cells of the same tissue and between different somatic tissues in a single individual. CONDITIONS THAT MAY MIMIC MENDELIAN INHERITANCE OF SINGLE-GENE DISORDERS A pedigree pattern sometimes simulates a single-gene pattern even though the disorder does not have a single-gene basis. It is easy to be misled in this way by teratogenic effects; by certain types of inherited chromosome disorders, such as balanced translocations; or by environmental exposures shared among family members. Inherited single-gene disorders can usually be distinguished from these other types of familial disorders by their typical mendelian segregation ratios within kindreds. Confirmation that a familial disease is due to mutations in a single gene eventually requires demonstration of defects at the level of the gene product, or the gene itself. There is also a class of disorders called segmental aneusomies, in which there is a deficiency or excess of two or more genes at neighboring loci on a chromosome, due to a deletion or a duplication or triplication of an entire segment of DNA. Here the phenotype, referred to as a contiguous gene syndrome, results from alterations in the copy number of more than one gene and yet shows typical mendelian segregation ratios, with a usually dominant inheritance pattern, because the segmental aneusomy is passed on as if it were a single mutant allele. Examples include: – autosomal dominant Parkinson disease due to a triplication of an approximately 2-Mb region of chromosome 4q; – autosomal dominant velocardiofacial syndrome, where the phenotype is caused by deletions of millions of base pairs of DNA encoding multiple genes at 22q11.2; and – the X-linked syndrome of choroideremia (a retinal degeneration), deafness, and mental retardation, caused by a deletion of at least three loci in band Xq21 MATERNAL INHERITANCE OF DISORDERS CAUSED BY MUTATIONS IN THE MITOCHONDRIAL GENOME Some pedigrees of inherited diseases that could not be explained by typical mendelian inheritance of nuclear genes are now known to be caused by mutations of the mitochondrial genome and to manifest maternal inheritance. Disorders caused by mutations in mitochondrial DNA demonstrate a number of unusual features that result from the unique characteristics of mitochondrial biology and function. The Mitochondrial Genome The mt genome consists of a circular chr., 16.5 kb. Most cells contain at least 1000 mtDNA molecules, distributed among hundreds of individual mt. A remarkable exception is the mature oocyte, which has more than 100,000 copies of mtDNA, composing about one third of the total DNA content of these cells. Mitochondrial DNA (mtDNA) contains 37 genes. The genes encode 13 polypeptides that are subunits of enzymes of oxidative phosphorylation, two types of rRNA, and 22 tRNAs required for translating the transcripts of the mitochondria-encoded polypeptides. More than 100 different rearrangements and 100 different point mutations have been identified in mtDNA that can cause human disease, often involving the central nervous and musculoskeletal systems (e.g., myoclonic epilepsy with ragged-red fibers). The diseases that result from these mutations show a distinctive pattern of inheritance because of three unusual features of mitochondria: replicative segregation, homoplasmy and heteroplasmy, and maternal inheritance. Replicative Segregation The first unique feature of the mt. chromosome is the absence of the tightly controlled segregation seen during mitosis and meiosis of the 46 nuclear chromosomes. At cell division, the multiple copies of mtDNA in each of the mitochondria in a cell replicate and sort randomly among newly synthesized mitochondria. The mitochondria, in turn, are distributed randomly between the two daughter cells. This process is known as replicative segregation. Homoplasmy-Heteroplasmy The second feature arises from the fact that most cells contain many copies of mtDNA molecules. When a mutation arises in the mtDNA, it is at first present in only one of the mtDNA molecules in a mitochondrion. With replicative segregation, however, a mitochondrion containing a mutant mtDNA will acquire multiple copies of the mutant molecule. With cell division, a cell containing a mixture of normal and mutant mtDNAs can distribute very different proportions of mutant and wild-type mitochondrial DNA to its daughter cells. One daughter cell may, by chance, receive mitochondria that contain only a pure population of normal mtDNA or a pure population of mutant mtDNA (a situation known as homoplasmy). Alternatively, the daughter cell may receive a mixture of mitochondria, some with and some without mutation (heteroplasmy). Because the phenotypic expression of a mutation in mtDNA depends on the relative proportions of normal and mutant mtDNA in the cells making up different tissues, reduced penetrance, variable expression, and pleiotropy are all typical features of mitochondrial disorders. Homoplasmy and Heteroplasmy Figure 7-33 Replicative segregation of a heteroplasmic mitochondrial mutation. Random partitioning of mutant and wild-type mitochondria through multiple rounds of mitosis produces a collection of daughter cells with wide variation in the proportion of mutant and wild-type mitochondria carried by each cell. Cell and tissue dysfunction results when the fraction of mitochondria that are carrying a mutation exceeds a threshold level. N, nucleus. Maternal Inheritance of mtDNA The final mtDNA is its maternal inheritance. Sperm mitochondria are generally eliminated from the embryo, so that mtDNA is inherited from the mother. Thus, all the children of a female who is homoplasmic for a mtDNA mutation will inherit the mutation, whereas none of the offspring of a male carrying the same mutation will inherit the defective DNA. 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 huge total seen in mature oocytes. This restriction and subsequent amplification of mtDNA during oogenesis is termed the mitochondrial genetic bottleneck. Consequently, the variability in the percentage of mutant mtDNA molecules seen in the offspring of a mother with heteroplasmy for a mtDNA mutation arises, at least in part, from the sampling of only a subset of the mtDNAs during oogenesis. As might be expected, mothers with a high proportion of mutant mtDNA molecules are more likely to produce eggs with a higher proportion of mutant mtDNA and therefore are more likely to have clinically affected offspring than are mothers with a lower proportion. One exception to maternal inheritance occurs when the mother is heteroplasmic for deletion mutation in her mtDNA; for unknown reasons, deleted mtDNA molecules are generally not transmitted from clinically affected mothers to their children. Figure 7-34 Pedigree of Leber hereditary optic neuropathy, a form of spontaneous 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. No affected male transmits the disease. Although mitochondria are almost always inherited exclusively through the mother, at least one instance of paternal inheritance of mtDNA has occurred in a patient with a mitochondrial myopathy. Consequently, in patients with apparently sporadic mtDNA mutations, the rare occurrence of paternal mtDNA inheritance must be considered. Characteristics of Mitochondrial Inheritance All children of females homoplasmic for a mutation will inherit the mutation; the children of males carrying a similar mutation will not. Females heteroplasmic for point mutations and duplications will pass them on to all of their children. However, the fraction of mutant mitochondria in the offspring, and therefore the risk and severity of disease, can vary considerably, depending on the fraction of mutant mitochondria in their mother as well as on random chance operating on small numbers of mitochondria per cell at the oocyte bottleneck. Heteroplasmic deletions are generally not heritable. The fraction of mutant mitochondria in different tissues of an individual heteroplasmic for a mutation can vary tremendously, thereby causing a spectrum of disease among the members of a family in which there is heteroplasmy for a mitochondrial mutation. Pleiotropy and variable expressivity in different affected family members are frequent. FAMILY HISTORY AS PERSONALIZED MEDICINE An accurate determination of the family pedigree is an important part of the work-up of every patient. Pedigrees may demonstrate a straightforward, typical mendelian inheritance pattern; one that is more atypical, as is seen with mitochondrial mutations and germline mosaicism; or a complex pattern of familial occurrence that matches no obvious inheritance pattern. Not only is a determination of the inheritance pattern important for making a diagnosis in the proband, but it also identifies other individuals in the family who may be at risk and in need of evaluation and counseling. Despite the sophisticated cytogenetic and molecular testing available to geneticists, an accurate family history, including the family pedigree, remains a fundamental tool for all physicians and genetic counselors to use in designing an individualized management and treatment plan for their patients.