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Origins and Types of Mutations
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The term mutation is used to designate the process of generating genetic variations as well as the outcome of these alterations. A mutation can be defined as any change in the primary nucleotide sequence of DNA regardless of its functional consequences, although it often has a negative connotation. The more neutral term variation is now increasingly used to describe sequence changes and is recommended by several professional organizations and guidelines instead of mutation. Some variations may be lethal, others are less deleterious, and some may confer an evolutionary advantage. Variations can occur in the germline (sperm or oocytes); these can be transmitted to progeny. Alternatively, variations can occur during embryogenesis or in somatic tissues. Variations that occur during development lead to mosaicism, a situation in which tissues are composed of cells with different genetic constitutions. If the germline is mosaic, a mutation can be transmitted to some progeny but not others, which sometimes leads to confusion in assessing the pattern of inheritance. Somatic mutations that do not affect cell survival can sometimes be detected because of variable phenotypic effects in tissues (e.g., pigmented lesions in McCune-Albright syndrome). Other somatic mutations are associated with neoplasia because they confer a growth advantage to cells. Epigenetic events may also influence gene expression or facilitate genetic damage. With the exception of triplet nucleotide repeats, which can expand (see below), variations are usually stable.
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Mutations are structurally diverse—they can involve the entire genome, as in triploidy (one extra set of chromosomes), or gross numerical or structural alterations in chromosomes or individual genes. Large deletions may affect a portion of a gene or an entire gene, or, if several genes are involved, they may lead to a contiguous gene syndrome. Unequal crossing-over between homologous genes can result in fusion gene mutations, as illustrated by color blindness. Variations involving single nucleotides are referred to as point mutations. Substitutions are called transitions if a purine is replaced by another purine base (A ↔ G) or if a pyrimidine is replaced by another pyrimidine (C ↔ T). Changes from a purine to a pyrimidine, or vice versa, are referred to as transversions. If the DNA sequence change occurs in a coding region and alters an amino acid, it is called a missense mutation. Depending on the functional consequences of such a missense mutation, amino acid substitutions in different regions of the protein can lead to distinct phenotypes.
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Variations can occur in all domains of a gene (Fig. 456-9). A point mutation occurring within the coding region leads to an amino acid substitution if the codon is altered (Fig. 456-10). Point mutations that introduce a premature stop codon result in a truncated or missing protein. Large deletions may affect a portion of a gene or an entire gene, whereas small deletions and insertions alter the reading frame if they do not represent a multiple of three bases. These “frameshift” mutations, now also designated as amphigoric amino acid changes, lead to an entirely altered carboxy terminus. Mutations in intronic sequences or in exon junctions may destroy or create splice donor or splice acceptor sites. Variations may also be found in the regulatory sequences of genes, resulting in reduced or enhanced gene transcription.
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Certain DNA sequences are particularly susceptible to mutagenesis. Successive pyrimidine residues (e.g., T-T or C-C) are subject to the formation of ultraviolet light–induced photoadducts. If these pyrimidine dimers are not repaired by the nucleotide excision repair pathway, mutations will be introduced after DNA synthesis. The dinucleotide C-G, or CpG, is also a hot spot for a specific type of mutation. In this case, methylation of the cytosine is associated with an enhanced rate of deamination to uracil, which is then replaced with thymine. This C → T transition (or G → A on the opposite strand) accounts for at least one-third of point mutations associated with polymorphisms and mutations. In addition to the fact that certain types of mutations (C → T or G → A) are relatively common, the nature of the genetic code also results in overrepresentation of certain amino acid substitutions.
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Polymorphisms are sequence variations that have a frequency of at least 1%. Usually, they do not result in a perceptible phenotype but because allele frequency and functional consequences are often not known, the term variation is now increasingly recommended for the description of these sequence changes. Often they consist of single base-pair substitutions that do not alter the protein coding sequence because of the degenerate nature of the genetic code (synonymous polymorphism), although it is possible that some might alter mRNA stability, translation, or the amino acid sequence (nonsynonymous polymorphism) (Fig. 456-10). The detection of sequence variants poses a practical problem because it is often unclear whether it creates a change with functional consequences or a benign variation. In this situation, the sequence alteration is also described as variant of unknown significance (VUS).
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Mutations represent an important cause of genetic diversity as well as disease. Mutation rates are difficult to determine in humans because many mutations are silent and because testing is often not adequate to detect the phenotypic consequences. Mutation rates vary in different genes but are estimated to occur at a rate of ~10−10/bp per cell division. Germline mutation rates (as opposed to somatic mutations) are relevant in the transmission of genetic disease. Because the population of oocytes is established very early in development, only ~20 cell divisions are required for completed oogenesis, whereas spermatogenesis involves ~30 divisions by the time of puberty and 20 cell divisions each year thereafter. Consequently, the probability of acquiring new point mutations is much greater in the male germline than the female germline, in which rates of aneuploidy are increased. Thus, the incidence of new point mutations in spermatogonia increases with paternal age (e.g., achondrodysplasia, Marfan’s syndrome, neurofibromatosis). It is estimated that about 1 in 10 sperm carries a new deleterious mutation. The rates for new mutations are calculated most readily for autosomal dominant and X-linked disorders and are ~10−5−10−6/locus per generation. Because most monogenic diseases are relatively rare, new mutations account for a significant fraction of cases. This is important in the context of genetic counseling, because a new mutation can be transmitted to the affected individual but does not necessarily imply that the parents are at risk to transmit the disease to other children. An exception to this is when the new mutation occurs early in germline development, leading to gonadal mosaicism.
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UNEQUAL CROSSING-OVER
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Normally, DNA recombination in germ cells occurs with remarkable fidelity to maintain the precise junction sites for the exchanged DNA sequences (Fig. 456-6). However, mispairing of homologous sequences leads to unequal crossover, with gene duplication on one of the chromosomes and gene deletion on the other chromosome. A significant fraction of growth hormone (GH) gene deletions, for example, involve unequal crossing-over (Chap. 372). The GH gene is a member of a large gene cluster that includes a GH variant gene as well as several structurally related chorionic somatomammotropin genes and pseudogenes (highly homologous but functionally inactive relatives of a normal gene). Because such gene clusters contain multiple homologous DNA sequences arranged in tandem, they are particularly prone to undergo recombination and, consequently, gene duplication or deletion. On the other hand, duplication of the PMP22 gene because of unequal crossing-over results in increased gene dosage and type IA Charcot-Marie-Tooth disease. Unequal crossing-over resulting in deletion of PMP22 causes a distinct neuropathy called hereditary liability to pressure palsy (Chap. 438).
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Glucocorticoid-remediable aldosteronism (GRA) is caused by a gene fusion or rearrangement involving the genes that encode aldosterone synthase (CYP11B2) and steroid 11β-hydroxylase (CYP11B1), normally arranged in tandem on chromosome 8q. These two genes are 95% identical, predisposing to gene duplication and deletion by unequal crossing-over. The rearranged gene product contains the regulatory regions of 11β-hydroxylase fused to the coding sequence of aldosterone synthetase. Consequently, the latter enzyme is expressed in the adrenocorticotropic hormone (ACTH)–dependent zona fasciculata of the adrenal gland, resulting in overproduction of mineralocorticoids and hypertension (Chap. 379).
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Gene conversion refers to a nonreciprocal exchange of homologous genetic information. It has been used to explain how an internal portion of a gene is replaced by a homologous segment copied from another allele or locus; these genetic alterations may range from a few nucleotides to a few thousand nucleotides. As a result of gene conversion, it is possible for short DNA segments of two chromosomes to be identical, even though these sequences are distinct in the parents. A practical consequence of this phenomenon is that nucleotide substitutions can occur during gene conversion between related genes, often altering the function of the gene. In disease states, gene conversion often involves intergenic exchange of DNA between a gene and a related pseudogene. For example, the 21-hydroxylase gene (CYP21A2) is adjacent to a nonfunctional pseudogene (CYP21A1P). Many of the nucleotide substitutions that are found in the CYP21A2 gene in patients with congenital adrenal hyperplasia correspond to sequences that are present in the CYP21A1P pseudogene, suggesting gene conversion as one cause of mutagenesis. In addition, mitotic gene conversion has been suggested as a mechanism to explain revertant mosaicism in which an inherited mutation is “corrected” in certain cells. For example, patients with autosomal recessive generalized atrophic benign epidermolysis bullosa have acquired reverse mutations in one of the two mutated COL17A1 alleles, leading to clinically unaffected patches of skin.
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INSERTIONS AND DELETIONS
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Although many instances of insertions and deletions occur as a consequence of unequal crossing-over, there is also evidence for internal duplication, inversion, or deletion of DNA sequences. The fact that certain deletions or insertions appear to occur repeatedly as independent events indicates that specific regions within the DNA sequence predispose to these errors. For example, certain regions of the DMD gene, which encodes dystrophin, appear to be hot spots for deletions and result in muscular dystrophy (Chap. 441). Some regions within the human genome are rearrangement hot spots and lead to CNVs.
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Because mutations caused by defects in DNA repair accumulate as somatic cells divide, these types of mutations are particularly important in the context of neoplastic disorders. Several genetic disorders involving DNA repair enzymes underscore their importance. Patients with xeroderma pigmentosum have defects in DNA damage recognition or in the nucleotide excision and repair pathway (Chap. 72). Exposed skin is dry and pigmented and is extraordinarily sensitive to the mutagenic effects of ultraviolet irradiation. More than 10 different genes have been shown to cause the different forms of xeroderma pigmentosum. This finding is consistent with the earlier classification of this disease into different complementation groups in which normal function is rescued by the fusion of cells derived from two different forms of xeroderma pigmentosum.
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Ataxia telangiectasia causes large telangiectatic lesions of the face, cerebellar ataxia, immunologic defects, and hypersensitivity to ionizing radiation (Chap. 431). The discovery of the ataxia telangiectasia mutated (ATM) gene reveals that it is homologous to genes involved in DNA repair and control of cell cycle checkpoints. Mutations in the ATM gene give rise to defects in meiosis as well as increasing susceptibility to damage from ionizing radiation. Fanconi’s anemia is also associated with an increased risk of multiple acquired genetic abnormalities. It is characterized by diverse congenital anomalies and a strong predisposition to develop aplastic anemia and acute myelogenous leukemia (Chap. 100). Cells from these patients are susceptible to chromosomal breaks caused by a defect in genetic recombination. Currently, at least 16 different complementation groups have been identified, and the genes associated with Fanconi’s anemia have been cloned. HNPCC (Lynch’s syndrome) is characterized by autosomal dominant transmission of colon cancer, young age (<50 years) of presentation, predisposition to lesions in the proximal large bowel, and associated malignancies such as uterine cancer and ovarian cancer. HNPCC is predominantly caused by mutations in one of several different mismatch repair (MMR) genes including MutS homologue 2 (MSH2), MutL homologue 1 and 6 (MLH1, MLH6), MSH6, PMS1, and PMS2 (Chap. 77). These proteins are involved in the detection of nucleotide mismatches and in the recognition of slipped-strand trinucleotide repeats. Germline mutations in these genes lead to microsatellite instability and a high mutation rate in colon cancer. Genetic screening tests for this disorder are now being used for families considered to be at risk. Recognition of HNPCC allows early screening with colonoscopy and the implementation of prevention strategies using nonsteroidal anti-inflammatory drugs.
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UNSTABLE DNA SEQUENCES
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Trinucleotide repeats may be unstable and expand beyond a critical number. Mechanistically, the expansion is thought to be caused by unequal recombination and slipped mispairing. A premutation represents a small increase in trinucleotide copy number. In subsequent generations, the expanded repeat may increase further in length and result in an increasingly severe phenotype, a process called dynamic mutation (see below for discussion of anticipation). Trinucleotide expansion was first recognized as a cause of the fragile X syndrome, one of the most common causes of intellectual disability. Other disorders arising from a similar mechanism include Huntington’s disease, X-linked spinobulbar muscular atrophy, and myotonic dystrophy. Malignant cells are also characterized by genetic instability, indicating a breakdown in mechanisms that regulate DNA repair and the cell cycle.
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Functional Consequences of Mutations
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Functionally, mutations can be broadly classified as gain-of-function and loss-of-function mutations. Gain-of-function mutations are typically dominant (e.g., they result in phenotypic alterations when a single allele is affected). Inactivating mutations are usually recessive, and an affected individual is homozygous or compound heterozygous (e.g., carrying two different mutant alleles of the same gene) for the disease-causing mutations. Alternatively, mutation in a single allele can result in haploinsufficiency, a situation in which one normal allele is not sufficient to maintain a normal phenotype. Haploinsufficiency is a commonly observed mechanism in diseases associated with mutations in transcription factors (Table 456-2). Remarkably, the clinical features among patients with an identical mutation often vary significantly. One mechanism underlying this variability consists in the influence of modifying genes. Haploinsufficiency can also affect the expression of rate-limiting enzymes. For example, haploinsufficiency in enzymes involved in heme synthesis can cause porphyrias (Chap. 409).
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An increase in dosage of a gene product may also result in disease, as illustrated by the duplication of the DAX1 gene in dosage-sensitive sex reversal (Chap. 383). Mutation in a single allele can also result in loss of function due to a dominant-negative effect. In this case, the mutated allele interferes with the function of the normal gene product by one of several different mechanisms: (1) a mutant protein may interfere with the function of a multimeric protein complex, as illustrated by mutations in type 1 collagen (COL1A1, COL1A2) genes in osteogenesis imperfecta (Chap. 406); (2) a mutant protein may occupy binding sites on proteins or promoter response elements, as illustrated by thyroid hormone resistance β, a disorder in which inactivated thyroid hormone receptor β binds to target genes and functions as an antagonist of normal receptors (Chap. 375); or (3) a mutant protein can be cytotoxic as in α1 antitrypsin deficiency (Chap. 286) or autosomal dominant neurohypophyseal diabetes insipidus (Chap. 374), in which the abnormally folded proteins are trapped within the endoplasmic reticulum and ultimately cause cellular damage.
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Genotype and Phenotype
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ALLELES, GENOTYPES, AND HAPLOTYPES
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An observed trait is referred to as a phenotype; the genetic information defining the phenotype is called the genotype. Alternative forms of a gene or a genetic marker are referred to as alleles. Alleles may be polymorphic variants of nucleic acids that have no apparent effect on gene expression or function. In other instances, these variants may have subtle effects on gene expression, thereby conferring adaptive advantages associated with genetic diversity. On the other hand, allelic variants may reflect mutations that clearly alter the function of a gene product. The common Glu6Val (E6V) sickle cell mutation in the β-globin gene and the ΔF508 deletion of phenylalanine (F) in the CFTR gene are examples of allelic variants of these genes that result in disease. Because each individual has two copies of each chromosome (one inherited from the mother and one inherited from the father), an individual can have only two alleles at a given locus. However, there can be many different alleles in the population. The normal or common allele is usually referred to as wild type. When alleles at a given locus are identical, the individual is homozygous. Inheriting identical copies of a mutant allele occurs in many autosomal recessive disorders, particularly in circumstances of consanguinity or isolated populations. If the alleles are different on the maternal and the paternal copy of the gene, the individual is heterozygous at this locus (Fig. 456-10). If two different mutant alleles are inherited at a given locus, the individual is said to be a compound heterozygote. Hemizygous is used to describe males with a mutation in an X chromosomal gene or a female with a loss of one X chromosomal locus.
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Genotypes describe the specific alleles at a particular locus. For example, there are three common alleles (E2, E3, E4) of the apolipoprotein E (APOE) gene. The genotype of an individual can therefore be described as APOE3/4 or APOE4/4 or any other variant. These designations indicate which alleles are present on the two chromosomes in the APOE gene at locus 19q13.2. In other cases, the genotype might be assigned arbitrary numbers (e.g., 1/2) or letters (e.g., B/b) to distinguish different alleles.
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A haplotype refers to a group of alleles that are closely linked together at a genomic locus (Fig. 456-4). Haplotypes are useful for tracking the transmission of genomic segments within families and for detecting evidence of genetic recombination, if the crossover event occurs between the alleles (Fig. 456-6). As an example, various alleles at the histocompatibility locus antigen (HLA) on chromosome 6p are used to establish haplotypes associated with certain disease states. For example, 21-hydroxylase deficiency, complement deficiency, and hemochromatosis are each associated with specific HLA haplotypes. It is now recognized that these genes lie in close proximity to the HLA locus, which explains why HLA associations were identified even before the disease genes were cloned and localized. In other cases, specific HLA associations with diseases such as ankylosing spondylitis (HLA-B27) or type 1 diabetes mellitus (HLA-DR4) reflect the role of specific HLA allelic variants in susceptibility to these autoimmune diseases. The characterization of common SNP haplotypes in numerous populations from different parts of the world has provided the necessary tools for association studies designed to detect genes involved in the pathogenesis of complex disorders (Table 456-1). The presence or absence of certain haplotypes can also be relevant for the customized choice of medical therapies (pharmacogenomics) or may have value for preventive strategies.
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Genotype-phenotype correlation describes the association of a specific mutation and the resulting phenotype. The phenotype may differ depending on the location or type of the mutation in some genes. For example, in von Hippel–Lindau disease, an autosomal dominant multisystem disease that can include renal cell carcinoma, hemangioblastomas, and pheochromocytomas, among others, the phenotype varies greatly and the identification of the specific mutation can be clinically useful in order to predict the phenotypic spectrum.
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ALLELIC HETEROGENEITY
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Allelic heterogeneity refers to the fact that different mutations in the same genetic locus can cause an identical or similar phenotype. For example, many different mutations of the β-globin locus can cause β thalassemia (Table 456-3) (Fig. 456-9). In essence, allelic heterogeneity reflects the fact that many different mutations are capable of altering protein structure and function. For this reason, maps of inactivating mutations in genes usually show a near-random distribution. Exceptions include (1) a founder effect, in which a particular mutation that does not affect reproductive capacity can be traced to a single individual; (2) “hot spots” for mutations, in which the nature of the DNA sequence predisposes to a recurring mutation; and (3) localization of mutations to certain domains that are particularly critical for protein function. Allelic heterogeneity creates a practical problem for genetic testing because one must often examine the entire genetic locus for mutations, because these can differ in each patient. For example, about 2000 variants have been identified in the CFTR gene to date, although some of them are very rare and some may not be disease-causing (Fig. 456-3). Mutational analysis may initially focus on a panel of mutations that are particularly frequent (often taking the ethnic background of the patient into account), but a negative result does not exclude the presence of a mutation elsewhere in the gene. One should also be aware that mutational analyses tend to focus on the coding region of a gene without considering regulatory and intronic regions. Because disease-causing mutations may be located outside the coding regions, negative results need to be interpreted with caution. The advent of more comprehensive sequencing technologies now greatly facilitates concomitant mutational analyses of several genes after targeted enrichment, or even mutational analysis of the whole exome or genome. However, comprehensive sequencing can result in significant diagnostic challenges because the detection of a sequence alteration alone is not always sufficient to establish that it has a causal role (variants of unknown significance, VUS).
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PHENOTYPIC HETEROGENEITY
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Phenotypic heterogeneity occurs when more than one phenotype is caused by allelic mutations (e.g., different mutations in the same gene) (Table 456-3). For example, laminopathies are monogenic multisystem disorders that result from mutations in the LMNA gene, which encodes the nuclear lamins A and C. Twelve autosomal dominant and five autosomal recessive disorders are caused by mutations in the LMNA gene. They include several forms of lipodystrophies, Emery-Dreifuss muscular dystrophy, progeria syndromes, a form of neuronal Charcot-Marie-Tooth disease (type 2B1), and a group of overlapping syndromes. Remarkably, hierarchical cluster analysis has revealed that the phenotypes vary depending on the position of the mutation (genotype-phenotype correlation). Similarly, identical mutations in the FGFR2 gene can result in very distinct phenotypes: Crouzon’s syndrome (craniofacial synostosis) or Pfeiffer’s syndrome (acrocephalopolysyndactyly).
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LOCUS OR NONALLELIC HETEROGENEITY AND PHENOCOPIES
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Nonallelic or locus heterogeneity refers to the situation in which a similar disease phenotype results from mutations at different genetic loci (Table 456-3). This often occurs when more than one gene product produces different subunits of an interacting complex or when different genes are involved in the same genetic cascade or physiologic pathway. For example, osteogenesis imperfecta can arise from mutations in two different procollagen genes (COL1A1 or COL1A2) that are located on different chromosomes, and can involve multiple other genes (Chap. 406). The effects of inactivating mutations in these two genes are similar because the protein products comprise different subunits of the helical collagen fiber. Similarly, muscular dystrophy syndromes can be caused by mutations in various genes, consistent with the fact that it can be transmitted in an X-linked (Duchenne or Becker), autosomal dominant (limb-girdle muscular dystrophy type 1), or autosomal recessive (limb-girdle muscular dystrophy type 2) manner (Chap. 441). Mutations in the X-linked DMD gene, which encodes dystrophin, are the most common cause of muscular dystrophy. This feature reflects the large size of the gene as well as the fact that the phenotype is expressed in hemizygous males because they have only a single copy of the X chromosome. Dystrophin is associated with a large protein complex linked to the membrane-associated cytoskeleton in muscle. Mutations in several different components of this protein complex can also cause muscular dystrophy syndromes. Although the phenotypic features of some of these disorders are distinct, the phenotypic spectrum caused by mutations in different genes overlaps, thereby leading to nonallelic heterogeneity. It should be noted that mutations in dystrophin are also associated with allelic heterogeneity. For example, mutations in the DMD gene can cause either Duchenne’s or the less severe Becker’s muscular dystrophy, depending on the severity of the protein defect.
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Recognition of nonallelic heterogeneity is important for several reasons: (1) the ability to identify disease loci in linkage studies is reduced by including patients with similar phenotypes but different genetic disorders; (2) genetic testing is more complex because several different genes need to be considered along with the possibility of different mutations in each of the candidate genes; and (3) novel information is gained about how genes or proteins interact, providing unique insights into molecular physiology.
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Phenocopies refer to circumstances in which nongenetic conditions mimic a genetic disorder. For example, features of toxin- or drug-induced neurologic syndromes can resemble those seen in Huntington’s disease, and vascular causes of dementia share phenotypic features with familial forms of Alzheimer’s dementia (Chap. 423). As in nonallelic heterogeneity, the presence of phenocopies has the potential to confound linkage studies and genetic testing. Patient history and subtle differences in phenotype can often provide clues that distinguish these disorders from related genetic conditions.
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VARIABLE EXPRESSIVITY AND INCOMPLETE PENETRANCE
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The same genetic mutation may be associated with a phenotypic spectrum in different affected individuals, thereby illustrating the phenomenon of variable expressivity. This may include different manifestations of a disorder variably involving different organs (e.g., multiple endocrine neoplasia [MEN]), the severity of the disorder (e.g., cystic fibrosis), or the age of disease onset (e.g., Alzheimer’s dementia). MEN 1 illustrates several of these features. In this autosomal dominant tumor syndrome, affected individuals carry an inactivating germline mutation that is inherited in an autosomal dominant fashion. After somatic inactivation of the alternate allele (loss of heterozygosity; Knudson two-hit model), they can develop tumors of the parathyroid gland, endocrine pancreas, and the pituitary gland (Chap. 381). However, the pattern of tumors in the different glands, the age at which tumors develop, and the types of hormones produced vary among affected individuals, even within a given family. In this example, the phenotypic variability arises, in part, because of the requirement for a second somatic mutation in the normal copy of the MEN1 gene, as well as the large array of different cell types that are susceptible to the effects of MEN1 gene mutations. In part, variable expression reflects the influence of modifier genes, or genetic background, on the effects of a particular mutation. Even in identical twins, in whom the genetic constitution is essentially the same, one can occasionally see variable expression of a genetic disease.
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Interactions with the environment can also influence the course of a disease. For example, the manifestations and severity of hemochromatosis can be influenced by iron intake (Chap. 407), and the course of phenylketonuria is affected by exposure to phenylalanine in the diet (Chap. 413). Other metabolic disorders, such as hyperlipidemias and porphyria, also fall into this category. Many mechanisms, including genetic effects and environmental influences, can therefore lead to variable expressivity. In genetic counseling, it is particularly important to recognize this variability, because one cannot always predict the course of disease, even when the mutation is known.
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Penetrance refers to the proportion of individuals with a mutant genotype that express the phenotype. If all carriers of a mutant express the phenotype, penetrance is complete, whereas it is said to be incomplete or reduced if some individuals do not exhibit features of the phenotype. Dominant conditions with incomplete penetrance are characterized by skipping of generations with unaffected carriers transmitting the mutant gene. For example, hypertrophic obstructive cardiomyopathy (HCM) caused by mutations in the myosin-binding protein C gene is a dominant disorder with clinical features in only a subset of patients who carry the mutation (Chap. 254). Patients who have the mutation but no evidence of the disease can still transmit the disorder to subsequent generations. In many conditions with postnatal onset, the proportion of gene carriers who are affected varies with age. Thus, when describing penetrance, one has to specify age. For example, for disorders such as Huntington’s disease or familial amyotrophic lateral sclerosis, which present later in life, the rate of penetrance is influenced by the age at which the clinical assessment is performed. Imprinting can also modify the penetrance of a disease. For example, in patients with AHO, mutations in the Gsα subunit (GNAS1 gene) are expressed clinically only in individuals who inherit the mutation from their mother (Chap. 403).
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SEX-INFLUENCED PHENOTYPES
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Certain mutations affect males and females quite differently. In some instances, this is because the gene resides on the X or Y sex chromosomes (X-linked disorders and Y-linked disorders). As a result, the phenotype of mutated X-linked genes will be expressed fully in males but variably in heterozygous females, depending on the degree of X-inactivation and the function of the gene. For example, most heterozygous female carriers of factor VIII deficiency (hemophilia A) are asymptomatic because sufficient factor VIII is produced to prevent a defect in coagulation (Chap. 112). On the other hand, some females heterozygous for the X-linked lipid storage defect caused by α-galactosidase A deficiency (Fabry’s disease) experience mild manifestations of painful neuropathy, as well as other features of the disease (Chap. 411). Because only males have a Y chromosome, mutations in genes such as SRY, which causes male-to-female sex reversal, or DAZ (deleted in azoospermia), which causes abnormalities of spermatogenesis, are unique to males (Chap. 383).
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Other diseases are expressed in a sex-limited manner because of the differential function of the gene product in males and females. Activating mutations in the luteinizing hormone receptor cause dominant male-limited precocious puberty in boys (Chap. 384). The phenotype is unique to males because activation of the receptor induces testosterone production in the testis, whereas it is functionally silent in the immature ovary. Biallelic inactivating mutations of the follicle-stimulating hormone (FSH) receptor cause primary ovarian failure in females because the follicles do not develop in the absence of FSH action. In contrast, affected males have a more subtle phenotype, because testosterone production is preserved (allowing sexual maturation) and spermatogenesis is only partially impaired (Chap. 384). In congenital adrenal hyperplasia, most commonly caused by 21-hydroxylase deficiency, cortisol production is impaired and ACTH stimulation of the adrenal gland leads to increased production of androgenic precursors (Chap. 379). In females, the increased androgen level causes ambiguous genitalia, which can be recognized at the time of birth. In males, the diagnosis may be made on the basis of adrenal insufficiency at birth, because the increased adrenal androgen level does not alter sexual differentiation, or later in childhood, because of the development of precocious puberty. Hemochromatosis is more common in males than in females, presumably because of differences in dietary iron intake and losses associated with menstruation and pregnancy in females (Chap. 407).
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Chromosomal Disorders
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Chromosomal disorders and the techniques used for their characterization have been discussed in detail in a chapter in previous editions of this textbook. Chromosomal or cytogenetic disorders are caused by numerical (aneuploidy) or structural aberrations (deletions, duplications, translocations, inversions, dicentric and ring chromosomes, Robertsonian translocations) in chromosomes. They occur in about 1% of the general population, in 8% of stillbirths, and in close to 50% of spontaneously aborted fetuses. Indications for cytogenetic and cytogenomic chromosome analyses are summarized in Table 456-4. Contiguous gene syndromes (e.g., large deletions affecting several genes) have been useful for identifying the location of new disease-causing genes. Because of the variable size of gene deletions in different patients, a systematic comparison of phenotypes and locations of deletion breakpoints allows positions of particular genes to be mapped within the critical genomic region.
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Monogenic Mendelian Disorders
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Monogenic human diseases are frequently referred to as Mendelian disorders because they obey the principles of genetic transmission originally set forth in Gregor Mendel’s classic work. The continuously updated OMIM catalogue lists several thousand of these disorders and provides information about the clinical phenotype, molecular basis, allelic variants, and pertinent animal models (Table 456-1). The mode of inheritance for a given phenotypic trait or disease is determined by pedigree analysis. All affected and unaffected individuals in the family are recorded in a pedigree using standard symbols (Fig. 456-11). The principles of allelic segregation, and the transmission of alleles from parents to children, are illustrated in Fig. 456-12. One dominant (A) allele and one recessive (a) allele can display three Mendelian modes of inheritance: autosomal dominant, autosomal recessive, and X-linked. About 65% of human monogenic disorders are autosomal dominant, 25% are autosomal recessive, and 5% are X-linked. Genetic testing is now available for many of these disorders and plays an important role in clinical medicine (Chap. 457).
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AUTOSOMAL DOMINANT DISORDERS
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In autosomal dominant disorders, mutations in a single allele are sufficient to cause the disease. In contrast to recessive disorders, in which disease pathogenesis is relatively straightforward because there is a biallelic loss of gene function, dominant disorders can be caused by various disease mechanisms, many of which are unique to the function of the genetic pathway involved. Mechanistically, the mutation may confer constitutive activation (gain-of-function), exert a dominant negative effect, or result in loss-of-function and haploinsufficiency.
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In autosomal dominant disorders, individuals are affected in successive generations; the disease does not occur in the offspring of unaffected individuals. Males and females are affected with equal frequency because the defective gene resides on one of the 22 autosomes (Fig. 456-13A). Autosomal dominant mutations alter one of the two alleles at a given locus. Because the alleles segregate randomly at meiosis, the probability that an offspring will be affected is 50%. Unless there is a new germline mutation, an affected individual has an affected parent. Children with a normal genotype do not transmit the disorder. Due to differences in penetrance or expressivity (see above), the clinical manifestations of autosomal dominant disorders may be variable. Because of these variations, it is sometimes challenging to determine the pattern of inheritance.
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It should be recognized, however, that some individuals acquire a mutated gene from an unaffected parent. De novo germline mutations occur more frequently during later cell divisions in gametogenesis, which explains why siblings are rarely affected. As noted before, new germline mutations occur more frequently in fathers of advanced age. For example, the average age of fathers with new germline mutations that cause Marfan’s syndrome is ~37 years, whereas fathers who transmit the disease by inheritance have an average age of ~30 years.
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AUTOSOMAL RECESSIVE DISORDERS
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In recessive disorders, the mutated alleles result in a complete or partial loss of function. They frequently involve enzymes in metabolic pathways, receptors, or proteins in signaling cascades. In an autosomal recessive disease, the affected individual, who can be of either sex, is a homozygote or compound heterozygote for a single-gene defect. With a few important exceptions, autosomal recessive diseases are rare and often occur in the context of parental consanguinity. The relatively high frequency of certain recessive disorders such as sickle cell anemia, cystic fibrosis, and thalassemia, is partially explained by a selective biologic advantage for the heterozygous state (see below). Although heterozygous carriers of a defective allele are usually clinically normal, they may display subtle differences in phenotype that only become apparent with more precise testing or in the context of certain environmental influences. In sickle cell anemia, for example, heterozygotes are normally asymptomatic. However, in situations of dehydration or diminished oxygen pressure, sickle cell crises can also occur in heterozygotes (Chap. 94).
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In most instances, an affected individual is the offspring of heterozygous parents. In this situation, there is a 25% chance that the offspring will have a normal genotype, a 50% probability of a heterozygous state, and a 25% risk of homozygosity for the recessive alleles (Figs. 456-10, 456-13B). In the case of one unaffected heterozygous and one affected homozygous parent, the probability of disease increases to 50% for each child. In this instance, the pedigree analysis mimics an autosomal dominant mode of inheritance (pseudodominance). In contrast to autosomal dominant disorders, new mutations in recessive alleles are rarely manifest because they usually result in an asymptomatic carrier state.
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Males have only one X chromosome; consequently, a daughter always inherits her father’s X chromosome in addition to one of her mother’s two X chromosomes. A son inherits the Y chromosome from his father and one maternal X chromosome. Thus, the characteristic features of X-linked inheritance are (1) the absence of father-to-son transmission, and (2) the fact that all daughters of an affected male are obligate carriers of the mutant allele (Fig. 456-13C). The risk of developing disease due to a mutant X-chromosomal gene differs in the two sexes. Because males have only one X chromosome, they are hemizygous for the mutant allele; thus, they are more likely to develop the mutant phenotype, regardless of whether the mutation is dominant or recessive. A female may be either heterozygous or homozygous for the mutant allele, which may be dominant or recessive. The terms X-linked dominant or X-linked recessive are therefore only applicable to expression of the mutant phenotype in women. In addition, the expression of X-chromosomal genes is influenced by X chromosome inactivation.
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The Y chromosome has a relatively small number of genes. One such gene, the sex-region determining Y factor (SRY), which encodes the testis-determining factor (TDF), is crucial for normal male development. Normally there is infrequent exchange of sequences on the Y chromosome with the X chromosome. The SRY region is adjacent to the pseudoautosomal region, a chromosomal segment on the X and Y chromosomes with a high degree of homology. A crossing-over event occasionally involves the SRY region with the distal tip of the X chromosome during meiosis in the male. Translocations can result in XY females with the Y chromosome lacking the SRY gene or XX males harboring the SRY gene on one of the X chromosomes (Chap. 383). Point mutations in the SRY gene may also result in individuals with an XY genotype and an incomplete female phenotype. Most of these mutations occur de novo. Men with oligospermia/azoospermia frequently have microdeletions on the long arm of the Y chromosome that involve one or more of the azoospermia factor (AZF) genes.
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Exceptions to Simple Mendelian Inheritance Patterns
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MITOCHONDRIAL DISORDERS
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Mendelian inheritance refers to the transmission of genes encoded by DNA contained in the nuclear chromosomes. In addition, each mitochondrion contains several copies of a small circular chromosome (Chap. 472). The mitochondrial DNA (mtDNA) is ~16.5 kb and encodes transfer and ribosomal RNAs and 13 core proteins that are components of the respiratory chain involved in oxidative phosphorylation and ATP generation. The mitochondrial genome does not recombine and is inherited through the maternal line because sperm does not contribute significant cytoplasmic components to the zygote. A noncoding region of the mitochondrial chromosome, referred to as D-loop, is highly polymorphic. This property, together with the absence of mtDNA recombination, makes it a valuable tool for studies tracing human migration and evolution, and it is also used for specific forensic applications.
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Inherited mitochondrial disorders are transmitted in a matrilineal fashion; all children from an affected mother will inherit the disease, but it will not be transmitted from an affected father to his children (Fig. 456-13D). Alterations in the mtDNA that involves enzymes required for oxidative phosphorylation lead to reduction of ATP supply, generation of free radicals, and induction of apoptosis. Several syndromic disorders arising from mutations in the mitochondrial genome are known in humans and they affect both protein-coding and tRNA genes. The broad clinical spectrum often involves (cardio) myopathies and encephalopathies because of the high dependence of these tissues on oxidative phosphorylation. The age of onset and the clinical course are highly variable because of the unusual mechanisms of mtDNA transmission, which replicates independently from nuclear DNA. During cell replication, the proportion of wild-type and mutant mitochondria can drift among different cells and tissues. The resulting heterogeneity in the proportion of mitochondria with and without a mutation is referred to as heteroplasmia and underlies the phenotypic variability that is characteristic of mitochondrial diseases.
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Acquired somatic mutations in mitochondria are thought to be involved in several age-dependent degenerative disorders affecting predominantly muscle and the peripheral and central nervous system (e.g., Alzheimer’s and Parkinson’s diseases). Establishing that an mtDNA alteration is causal for a clinical phenotype is challenging because of the high degree of polymorphism in mtDNA and the phenotypic variability characteristic of these disorders. Certain pharmacologic treatments may have an impact on mitochondria and/or their function. For example, treatment with the antiretroviral compound azidothymidine (AZT) causes an acquired mitochondrial myopathy through depletion of muscular mtDNA.
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Mosaicism refers to the presence of two or more genetically distinct cell lines in the tissues of an individual. It results from a mutation that occurs during embryonic, fetal, or extrauterine development. The developmental stage at which the mutation arises will determine whether germ cells and/or somatic cells are involved. Chromosomal mosaicism results from nondisjunction at an early embryonic mitotic division, leading to the persistence of more than one cell line, as exemplified by some patients with Turner’s syndrome (Chap. 383). Somatic mosaicism is characterized by a patchy distribution of genetically altered somatic cells. The McCune-Albright syndrome, for example, is caused by activating mutations in the stimulatory G protein α (Gsα) that occur early in development (Chap. 403). The clinical phenotype varies depending on the tissue distribution of the mutation; manifestations include ovarian cysts that secrete sex steroids and cause precocious puberty, polyostotic fibrous dysplasia, café-au-lait skin pigmentation, GH–secreting pituitary adenomas, and hypersecreting autonomous thyroid nodules.
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X-INACTIVATION, IMPRINTING, AND UNIPARENTAL DISOMY
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According to traditional Mendelian principles, the parental origin of a mutant gene is irrelevant for the expression of the phenotype. There are, however, important exceptions to this rule. X-inactivation prevents the expression of most genes on one of the two X chromosomes in every cell of a female. Gene inactivation through genomic imprinting occurs on selected chromosomal regions of autosomes and leads to inheritable preferential expression of one of the parental alleles. It is of pathophysiologic importance in disorders where the transmission of disease is dependent on the sex of the transmitting parent and, thus, plays an important role in the expression of certain genetic disorders. Two classic examples are the Prader-Willi syndrome and Angelman’s syndrome. Prader-Willi syndrome is characterized by diminished fetal activity, obesity, hypotonia, mental retardation, short stature, and hypogonadotropic hypogonadism. Deletions of the paternal copy of the Prader-Willi locus located on the short arm of chromosome 15 result in a contiguous gene syndrome involving missing paternal copies of the necdin and SNRPN genes, among others. In contrast, patients with Angelman’s syndrome, characterized by mental retardation, seizures, ataxia, and hypotonia, have deletions involving the maternal copy of this region on chromosome 15. These two syndromes may also result from uniparental disomy. In this case, the syndromes are not caused by deletions on chromosome 15 but by the inheritance of either two maternal chromosomes (Prader-Willi syndrome) or two paternal chromosomes (Angelman’s syndrome). Lastly, the two distinct phenotypes can also be caused by an imprinting defect that impairs the resetting of the imprint during zygote development (defect in the father leads to Prader-Willi syndrome; defect in the mother leads to Angelman’s syndrome).
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Imprinting and the related phenomenon of allelic exclusion may be more common than currently documented, because it is difficult to examine levels of mRNA expression from the maternal and paternal alleles in specific tissues or in individual cells. Genomic imprinting, or uniparental disomy, is involved in the pathogenesis of several other disorders and malignancies. For example, hydatidiform moles contain a normal number of diploid chromosomes, but they are all of paternal origin. The opposite situation occurs in ovarian teratomata, with 46 chromosomes of maternal origin. Expression of the imprinted gene for insulin-like growth factor II (IGF-II) is involved in the pathogenesis of the cancer-predisposing Beckwith-Wiedemann syndrome (BWS). These children show somatic overgrowth with organomegalies and hemihypertrophy, and they have an increased risk of embryonal malignancies such as Wilms’ tumor. Normally, only the paternally derived copy of the IGF-II gene is active and the maternal copy is inactive. Imprinting of the IGF-II gene is regulated by H19, which encodes an RNA transcript that is not translated into protein. Disruption or lack of H19 methylation leads to a relaxation of IGF-II imprinting and expression of both alleles. Alterations of the epigenome through gain and loss of DNA methylation, as well as altered histone modifications, play an important role in the pathogenesis of malignancies.
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Cancer can be considered a genetic disease at the cellular level (Chap. 67). Cancers are monoclonal in origin, indicating that they have arisen from a single precursor cell with one or several mutations in genes controlling growth (proliferation or apoptosis) and/or differentiation. These acquired somatic mutations are restricted to the tumor and its metastases and are not found in the surrounding normal tissue. The molecular alterations include dominant gain-of-function mutations in oncogenes, recessive loss-of-function mutations in tumor-suppressor genes and DNA repair genes, gene amplification, and chromosome rearrangements. Rarely, a single mutation in certain genes may be sufficient to transform a normal cell into a malignant cell. In most cancers, however, the development of a malignant phenotype requires several genetic alterations for the gradual progression from a normal cell to a cancerous cell, a phenomenon termed multistep carcinogenesis. Genome-wide analyses of cancers using deep sequencing often reveal somatic rearrangements resulting in fusion genes and mutations in multiple genes (Fig. 456-14). Comprehensive sequence analyses provide further insight into genetic heterogeneity within malignancies; these include intratumoral heterogeneity among the cells of the primary tumor, intermetastatic and intrametastatic heterogeneity, and interpatient differences. These analyses further support the notion of cancer as an ongoing process of clonal evolution, in which successive rounds of clonal selection within the primary tumor and metastatic lesions result in diverse genetic and epigenetic alterations that require targeted (personalized) therapies (precision medicine). The heterogeneity of mutations within a tumor can also lead to resistance to target therapies because cells with mutations that are resistant to the therapy, even if they are a minor part of the tumor population, will be selected as the more sensitive cells are killed. Most human tumors express telomerase, an enzyme formed of a protein and an RNA component, which adds telomere repeats at the ends of chromosomes during replication. This mechanism impedes shortening of the telomeres, which is associated with senescence in normal cells and is associated with enhanced replicative capacity in cancer cells. Telomerase inhibitors provide a strategy for treating advanced human cancers.
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In many cancer syndromes, there is frequently an inherited predisposition to tumor formation. In these instances, a germline mutation is inherited in an autosomal dominant fashion inactivating one allele of an autosomal tumor-suppressor gene. If the second allele is inactivated by a somatic mutation or by epigenetic silencing in a given cell, this will lead to neoplastic growth (Knudson two-hit model). Thus, the defective allele in the germline is transmitted in a dominant mode, although tumorigenesis results from a biallelic loss of the tumor-suppressor gene in an affected tissue. The classic example to illustrate this phenomenon is retinoblastoma, which can occur as a sporadic or hereditary tumor. In sporadic retinoblastoma, both copies of the retinoblastoma (RB) gene are inactivated through two somatic events. In hereditary retinoblastoma, one mutated or deleted RB allele is inherited in an autosomal dominant manner and the second allele is inactivated by a subsequent somatic mutation. This two-hit model applies to other inherited cancer syndromes such as MEN 1 (Chap. 381) and neurofibromatosis type 2 (Chap. 86). In contrast, in the autosomal dominant MEN2 syndrome, the predisposition for tumor formation in various organs is caused by a gain-of-function mutation in a single allele of the RET gene (Chap. 381).
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NUCLEOTIDE REPEAT EXPANSION DISORDERS
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Several diseases are associated with an increase in the number of nucleotide repeats above a certain threshold (Table 456-5). The repeats are sometimes located within the coding region of the genes, as in Huntington’s disease or the X-linked form of spinal and bulbar muscular atrophy (SBMA; Kennedy’s syndrome). In other instances, the repeats probably alter gene regulatory sequences. If an expansion is present, the DNA fragment is unstable and tends to expand further during cell division. The length of the nucleotide repeat often correlates with the severity of the disease. When repeat length increases from one generation to the next, disease manifestations may worsen or be observed at an earlier age; this phenomenon is referred to as anticipation. In Huntington’s disease, for example, there is a correlation between age of onset and length of the triplet codon expansion (Chap. 417). Anticipation has also been documented in other diseases caused by dynamic mutations in trinucleotide repeats (Table 456-5). The repeat number may also vary in a tissue-specific manner. In myotonic dystrophy, the CTG repeat may be tenfold greater in muscle tissue than in lymphocytes (Chap. 441).
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Complex Genetic Disorders
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The expression of many common diseases such as cardiovascular disease, hypertension, diabetes, asthma, psychiatric disorders, and certain cancers is determined by a combination of genetic background, environmental factors, and lifestyle. A trait is called polygenic if multiple genes contribute to the phenotype or multifactorial if multiple genes are assumed to interact with environmental factors. Genetic models for these complex traits need to account for genetic heterogeneity and interactions with other genes and the environment. Complex genetic traits may be influenced by modifier genes that are not linked to the main gene involved in the pathogenesis of the trait. This type of gene-gene interaction, or epistasis, plays an important role in polygenic traits that require the simultaneous presence of variations in multiple genes to result in a pathologic phenotype.
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Type 2 diabetes mellitus provides a paradigm for considering a multifactorial disorder, because genetic, nutritional, and lifestyle factors are intimately interrelated in disease pathogenesis (Table 456-6) (Chap. 396). The identification of genetic variations and environmental factors that either predispose to or protect against disease is essential for predicting disease risk, designing preventive strategies, and developing novel therapeutic approaches. The study of rare monogenic diseases may provide insight into some of the genetic and molecular mechanisms important in the pathogenesis of complex diseases. For example, the identification of the genes causing monogenic forms of permanent neonatal diabetes mellitus or maturity-onset diabetes defined them as candidate genes in the pathogenesis of diabetes mellitus type 2 (Tables 456-2 and 456-6) (Fig. 456-15). Genome scans have identified numerous genes and loci that may be associated with susceptibility to development of diabetes mellitus in certain populations (Fig. 456-16). Efforts to identify susceptibility genes require very large sample sizes, and positive results may depend on ethnicity, ascertainment criteria, and statistical analysis. Association studies analyzing the potential influence of (biologically functional) SNPs and SNP haplotypes on a particular phenotype have revealed new insights into the genes involved in the pathogenesis of these common disorders. Large variants ([micro]deletions, duplications, and inversions) present in the human population also contribute to the pathogenesis of complex disorders, but their contributions remain poorly understood.
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Linkage and Association Studies
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There are two primary strategies for mapping genes that cause or increase susceptibility to human disease: (1) classic linkage can be performed based on a known genetic model or, when the model is unknown, by studying pairs of affected relatives; or (2) disease genes can be mapped using allelic association studies (Table 456-7).
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Genetic linkage refers to the fact that genes are physically connected, or linked, to one another along the chromosomes. Two fundamental principles are essential for understanding the concept of linkage: (1) when two genes are close together on a chromosome, they are usually transmitted together, unless a recombination event separates them (Figs. 456-6); and (2) the odds of a crossover, or recombination event, between two linked genes is proportional to the distance that separates them. Thus, genes that are farther apart are more likely to undergo a recombination event than genes that are very close together. The detection of chromosomal loci that segregate with a disease by linkage can be used to identify the gene responsible for the disease (positional cloning) and to predict the odds of disease gene transmission in genetic counseling.
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Polymorphic variants are essential for linkage studies because they provide a means to distinguish the maternal and paternal chromosomes in an individual. On average, 1 out of every 1000 bp varies from one person to the next. Although this degree of variation seems low (99.9% identical), it means that >3 million sequence differences exist between any two unrelated individuals and the probability that the sequence at such loci will differ on the two homologous chromosomes is high (often >70–90%). These sequence variations include variable number of tandem repeats (VNTRs), short tandem repeats (STRs), and SNPs. Most STRs, also called polymorphic microsatellite markers, consist of di-, tri-, or tetranucleotide repeats that can be characterized readily using the polymerase chain reaction (PCR). Characterization of SNPs, using DNA chips or beads, permits comprehensive analyses of genetic variation, linkage, and association studies. Although these sequence variations often have no apparent functional consequences, they provide much of the basis for variation in genetic traits.
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In order to identify a chromosomal locus that segregates with a disease, it is necessary to characterize polymorphic DNA markers from affected and unaffected individuals of one or several pedigrees. One can then assess whether certain marker alleles cosegregate with the disease. Markers that are closest to the disease gene are less likely to undergo recombination events and therefore receive a higher linkage score. Linkage is expressed as a lod (logarithm of odds) score—the ratio of the probability that the disease and marker loci are linked rather than unlinked. Lod scores of +3 (1000:1) are generally accepted as supporting linkage, whereas a score of –2 is consistent with the absence of linkage.
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ALLELIC ASSOCIATION, LINKAGE DISEQUILIBRIUM, AND HAPLOTYPES
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Allelic association refers to a situation in which the frequency of an allele is significantly increased or decreased in individuals affected by a particular disease in comparison to controls. Linkage and association differ in several aspects. Genetic linkage is demonstrable in families or sibships. Association studies, on the other hand, compare a population of affected individuals with a control population. Association studies can be performed as case-control studies that include unrelated affected individuals and matched controls or as family-based studies that compare the frequencies of alleles transmitted or not transmitted to affected children.
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Allelic association studies are particularly useful for identifying susceptibility genes in complex diseases. When alleles at two loci occur more frequently in combination than would be predicted (based on known allele frequencies and recombination fractions), they are said to be in linkage disequilibrium. Evidence for linkage disequilibrium can be helpful in mapping disease genes because it suggests that the two loci are tightly linked.
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Detecting the genetic factors contributing to the pathogenesis of common complex disorders is challenging. In many instances, these are low-penetrance alleles (e.g., variations that individually have a subtle effect on disease development, and they can only be identified by unbiased GWAS) (Catalog of published Genome-Wide Association Studies; Table 456-1) (Fig. 456-16). Most variants occur in noncoding or regulatory sequences but do not alter protein structure. The analysis of complex disorders is further complicated by ethnic differences in disease prevalence, differences in allele frequencies in known susceptibility genes among different populations, locus and allelic heterogeneity, gene-gene and gene-environment interactions, and the possibility of phenocopies. Catalogues of human variation and genotype data (HapMap, International Genome Sample Resource) are greatly facilitating GWAS for the characterization of complex disorders. Adjacent SNPs are inherited together as blocks, and these blocks can be identified by genotyping selected marker SNPs, so-called Tag SNPs, thereby reducing cost and workload (Fig. 456-4). The availability of this information permits the characterization of a limited number of SNPs to identify the set of haplotypes present in an individual (e.g., in cases and controls). This, in turn, permits performing GWAS by searching for associations of certain haplotypes with a disease phenotype of interest, an essential step for unraveling the genetic factors contributing to complex disorders.
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In population genetics, the focus changes from alterations in an individual’s genome to the distribution pattern of different genotypes in the population. In a case where there are only two alleles, A and a, the frequency of the genotypes will be p2 + 2pq + q2 = 1, with p2 corresponding to the frequency of AA, 2pq to the frequency of Aa, and q2 to aa. When the frequency of an allele is known, the frequency of the genotype can be calculated. Alternatively, one can determine an allele frequency if the genotype frequency has been determined.
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Allele frequencies vary among ethnic groups and geographic regions. For example, heterozygous mutations in the CFTR gene are relatively common in populations of European origin but are rare in the African population. Allele frequencies may vary because certain allelic variants confer a selective advantage. For example, heterozygotes for the sickle cell mutation, which is particularly common in West Africa, are more resistant to malarial infection because the erythrocytes of heterozygotes provide a less favorable environment for Plasmodium parasites. Although homozygosity for the sickle cell mutation is associated with severe anemia and sickle crises, heterozygotes have a higher probability of survival because of the reduced morbidity and mortality from malaria; this phenomenon has led to an increased frequency of the mutant allele. Recessive conditions are more prevalent in geographically isolated populations because of the more restricted gene pool.
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APPROACH TO THE PATIENT
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APPROACH TO THE PATIENT Inherited Disorders
For the practicing clinician, the family history remains an essential step in recognizing the possibility of a hereditary predisposition to disease. When taking the history, it is useful to draw a detailed pedigree of the first-degree relatives (e.g., parents, siblings, and children), because they share 50% of genes with the patient. Standard symbols for pedigrees are depicted in Fig. 456-11. The family history should include information about ethnic background, age, health status, and deaths, including infants. Next, the physician should explore whether there is a family history of the same or related illnesses to the current problem. An inquiry focused on commonly occurring disorders such as cancers, heart disease, and diabetes mellitus should follow. Because of the possibility of age-dependent expressivity and penetrance, the family history will need intermittent updating. If the findings suggest a genetic disorder, the clinician should assess whether some of the patient’s relatives may be at risk of carrying or transmitting the disease. In this circumstance, it is useful to confirm and extend the pedigree based on input from several family members. This information may form the basis for genetic counseling, carrier detection, early intervention, and disease prevention in relatives of the index patient (Chap. 457).
In instances where a diagnosis at the molecular level may be relevant, it is important to identify an appropriate laboratory that can perform the appropriate test. Genetic testing is available for a large number of monogenic disorders through commercial laboratories. For uncommon disorders, the test may only be performed in a specialized research laboratory. Approved laboratories offering testing for inherited disorders can be identified in continuously updated online resources (e.g., GeneTests; Table 456-1). If genetic testing is considered, the patient and the family should be counseled about the potential implications of positive results, including psychological distress and the possibility of discrimination. The patient or caretakers should be informed about the meaning of a negative result, technical limitations, and the possibility of false-negative and inconclusive results. For these reasons, genetic testing should only be performed after obtaining informed consent. Published ethical guidelines address the specific aspects that should be considered when testing children and adolescents. Genetic testing should usually be limited to situations in which the results may have an impact on medical management.
IDENTIFYING THE DISEASE-CAUSING GENE Precision medicine aims to enhance the quality of medical care through the use of genotypic analysis (DNA testing) to identify genetic predisposition to disease, to select more specific pharmacotherapy, and to design individualized medical care based on genotype. Genotype can be deduced by analysis of protein (e.g., hemoglobin, apoprotein E), mRNA, or DNA. However, technologic advances have made DNA analysis particularly useful because it can be readily applied.
DNA testing is performed by mutational analysis or linkage studies in individuals at risk for a genetic disorder known to be present in a family. Mass screening programs require tests of high sensitivity and specificity to be cost-effective. Prerequisites for the success of genetic screening programs include the following: that the disorder is potentially serious; that it can be influenced at a presymptomatic stage by changes in behavior, diet, and/or pharmaceutical manipulations; and that the screening does not result in any harm or discrimination. Screening in Jewish populations for the autosomal recessive neurodegenerative storage disease Tay-Sachs has reduced the number of affected individuals. In contrast, screening for sickle cell trait/disease in African Americans has led to unanticipated problems of discrimination by health insurers and employers. Mass screening programs harbor additional potential problems. For example, screening for the most common genetic alteration in cystic fibrosis, the ΔF508 mutation with a frequency of ~70% in northern Europe, is feasible and seems to be effective. One has to keep in mind, however, that there is pronounced allelic heterogeneity and that the disease can be caused by about 2000 other mutations. The search for these less common mutations would substantially increase costs, but not the effectiveness of the screening program as a whole. Next-generation genome sequencing permits comprehensive and cost-effective mutational analyses after selective enrichment of candidate genes. For example, tests that sequence all the common genes causing hereditary deafness or hereditary pheochromocytomas are commercially available. Occupational screening programs aim to detect individuals with increased risk for certain professional activities (e.g., α1 antitrypsin deficiency and smoke or dust exposure). Integrating genomic data into electronic medical records is evolving and can provide significant decision support at the point of care, for example, by providing the clinician with genomic data and decision algorithms for the prescription of drugs that are subject to pharmacogenetic influences.
Mutational Analyses DNA sequence analysis is now widely used as a diagnostic tool and has significantly enhanced diagnostic accuracy. It is used for determining carrier status and for prenatal testing in monogenic disorders. Numerous techniques, discussed in previous versions of this chapter, are available for the detection of mutations. In a very broad sense, one can distinguish between techniques that allow for screening of known mutations (screening mode) or techniques that definitively characterize mutations. Analyses of large alterations in the genome are possible using classic methods such as karyotype analysis, cytogenetics, fluorescent in situ hybridization (FISH), as well as more sensitive array- or bead-based techniques that search for multiple single exon deletions or duplications.
More discrete sequence alterations rely heavily on the use of PCR, which allows rapid gene amplification and analysis. Moreover, PCR makes it possible to perform genetic testing and mutational analysis with small amounts of DNA extracted from leukocytes or even from single cells, buccal cells, or hair roots. DNA sequencing can be performed directly on PCR products or on fragments cloned into plasmid vectors amplified in bacterial host cells. Sequencing of the whole genome, exome, selected chromosomes, or sequencing of numerous candidate genes in a single run, is now possible with next-generation sequencing platforms and has entered the clinical realm. Genomic tests are also widely used for the detection of pathogens and for the identification of viral or bacterial sequence variations. The integration of genomic tests into clinical medicine is, however, associated with a number of ongoing challenges related to costs, variable sensitivities of the tests, bioinformatics analyses, storage and sharing of data, and the difficulty of interpreting all genetic variants identified with comprehensive testing. The discovery of incidental (or secondary) findings that are unrelated to the indication for the sequencing analysis but indicators of other disorders of potential relevance for patient care can pose a difficult ethical dilemma. It can lead to the detection of undiagnosed medically actionable genetic conditions, but can also reveal deleterious mutations that cannot be influenced, as numerous sequence variants are of unknown significance.
A general algorithm for the approach to mutational analysis is outlined in Fig. 456-17. The importance of a detailed clinical phenotype cannot be overemphasized. This is the step where one should also consider the possibility of genetic heterogeneity and phenocopies. If obvious candidate genes are suggested by the phenotype, they can be analyzed directly. After identification of a mutation, it is essential to demonstrate that it segregates with the phenotype. The functional characterization of novel mutations is labor intensive and may require analyses in vitro or in transgenic models in order to document the relevance of the genetic alteration.
Prenatal diagnosis of numerous genetic diseases in instances with a high risk for certain disorders is now possible by direct DNA analysis. Amniocentesis involves the removal of a small amount of amniotic fluid, usually at 16 weeks of gestation. Cells can be collected and submitted for karyotype analyses, FISH, and mutational analysis of selected genes Table 456-4). The main indications for amniocentesis include advanced maternal age (>35 years), presence of an abnormality of the fetus on ultrasound examination, an abnormal serum “quad” test (α-fetoprotein, β human chorionic gonadotropin, inhibin-A, and unconjugated estriol), a family history of chromosomal abnormalities, or a Mendelian disorder amenable to genetic testing. Prenatal diagnosis can also be performed by chorionic villus sampling (CVS), in which a small amount of the chorion is removed by a transcervical or transabdominal biopsy. Chromosomes and DNA obtained from these cells can be submitted for cytogenetic and mutational analyses. CVS can be performed earlier in gestation (weeks 9–12) than amniocentesis, an aspect that may be of relevance when termination of pregnancy is a consideration. Later in pregnancy, beginning at about 18 weeks of gestation, percutaneous umbilical blood sampling (PUBS) permits collection of fetal blood for lymphocyte culture and analysis. These approaches enable screening for clinically relevant and deleterious alleles inherited from the parents, as well as for de novo germline mutations, and they may have the potential to change the diagnosis of genetic disorders in the prenatal setting. Although genomic sequencing of fetal DNA circulating in the bloodstream of the mother has been achieved, it is not commonly used for non-invasive prenatal testing.
In combination with in vitro fertilization (IVF) techniques, it is possible to perform genetic diagnoses in a single cell removed from the four- to eight-cell embryo or to analyze the first polar body from an oocyte. Preconceptual diagnosis thereby avoids therapeutic abortions but is costly and labor intensive. It should be emphasized that excluding a specific disorder by any of these approaches is never equivalent to the assurance of having a normal child. Postnatal indications for cytogenetic analyses in infants or children include multiple congenital anomalies, suspicion of a known cytogenetic syndrome, developmental delay, dysmorphic features, autism, short stature and disorders of sexual development, among others (Table 456-4).
Mutations in certain cancer susceptibility genes such as BRCA1 and BRCA2 may identify individuals with an increased risk for the development of malignancies and result in risk-reducing interventions. The detection of cytogenetic alterations and mutations is an important diagnostic and prognostic tool in leukemias and somatic mutational analysis is transforming oncology by providing diagnostic and prognostic information, and it informs the choice of targeted therapies. However, the cancer may recur due to treatment resistance of subclones due to the continuously evolving genomic landscape. The demonstration of the presence or absence of mutations and polymorphisms is also relevant for the rapidly evolving field of pharmacogenomics, including the identification of differences in drug treatment response or metabolism as a function of genetic background. For example, the thiopurine drugs 6-mercaptopurine and azathioprine are commonly used cytotoxic and immunosuppressive agents. They are metabolized by thiopurine methyltransferase (TPMT), an enzyme with variable activity associated with genetic polymorphisms in 10% of whites and complete deficiency in about 1 in 300 individuals. Patients with intermediate or deficient TPMT activity are at risk for excessive toxicity, including fatal myelosuppression. Characterization of these polymorphisms allows mercaptopurine doses to be modified based on TPMT genotype. Pharmacogenomics may increasingly permit individualized drug therapy, improve drug effectiveness, reduce adverse side effects, and provide cost-effective pharmaceutical care (Chap. 64).
ETHICAL ISSUES Determination of the association of genetic defects with disease, comprehensive data of an individual’s genome, and studies of genetic variation raise many ethical and legal issues. Genetic information is generally regarded as sensitive information that should not be readily accessible without explicit consent (genetic privacy). The disclosure of genetic information may risk possible discrimination by insurers or employers. The scientific components of the Human Genome Project have been paralleled by efforts to examine ethical, social, and legal implications. An important milestone emerging from these endeavors consists in the Genetic Information Nondiscrimination Act (GINA), signed into law in 2008, which aims to protect asymptomatic individuals against the misuse of genetic information for health insurance and employment. It does not, however, protect the symptomatic individual. Provisions of the U.S. Patient Protection and Affordable Care Act, effective in 2014, have, in part, filled this gap and prohibit exclusion from, or termination of, health insurance based on personal health status. Potential threats to the maintenance of genetic privacy consist in the emerging integration of genomic data into electronic medical records, compelled disclosures of health records, and direct-to-consumer genetic testing.
It is widely accepted that identifying disease-causing genes can lead to improvements in diagnosis, treatment, and prevention. However, the information gleaned from genotypic results can have quite different impacts, depending on the availability of strategies to modify the course of disease. For example, the identification of mutations that cause MEN 2 or hemochromatosis allows specific interventions for affected family members. On the other hand, at present, the identification of an Alzheimer’s or Huntington’s disease gene does not currently alter therapy and outcomes. Most genetic disorders are likely to fall into an intermediate category where the opportunity for prevention or treatment is significant but limited. However, the progress in this area is unpredictable, as underscored by the finding that angiotensin II receptor blockers may slow disease progression in Marfan’s syndrome. Genetic test results can generate anxiety in affected individuals and family members. Comprehensive sequence analyses are particularly challenging because most individuals can be expected to harbor several serious recessive gene mutations. Moreover, the sensitivity of comprehensive sequence analyses are not always greater, for example, if CNV analysis is not integrated, but can be associated with higher costs.
The impact of genetic testing on health care costs remain unclear. It does vary among disorders and depends on the availability of effective therapeutic modalities. A significant problem arises from the marketing of genetic testing directly to consumers by commercial companies. The validity of these tests is, in part, not well defined, and there are persisting concerns about the lack of appropriate regulatory oversight, the accuracy and confidentiality of genetic information, the availability of counseling, and the handling of these results.
Many issues raised by the genome project are familiar, in principle, to medical practitioners. For example, an asymptomatic patient with increased low-density lipoprotein (LDL) cholesterol, high blood pressure, or a strong family history of early myocardial infarction is known to be at increased risk of coronary heart disease. In such cases, it is clear that the identification of risk factors and an appropriate intervention are beneficial. Likewise, patients with phenylketonuria, cystic fibrosis, or sickle cell anemia are often identified as having a genetic disease early in life. These precedents can be helpful for adapting policies that relate to genetic information. We can anticipate similar efforts, whether based on genotypes or other markers of genetic predisposition, to be applied to many disorders. One confounding aspect of the rapid expansion of information is that our ability to make clinical decisions often lags behind initial insights into genetic mechanisms of disease. For example, when genes that predispose to breast cancer such as BRCA1 are described, they generate tremendous public interest in the potential to predict disease, but many years of clinical research are still required to rigorously establish genotype and phenotype correlations.
Genomics may contribute to improvements in global health by providing a better understanding of pathogens and diagnostics, and through contributions to drug development. There is, however, ongoing concern about the development of a “genomics divide” because of the costs associated with these developments and uncertainty as to whether these advances will be accessible to the populations of developing countries faced with pressing health needs associated with poverty, infectious diseases, and the relative lack of essential infrastructure. The World Health Organization has summarized these issues and inequities surrounding genomic medicine in a detailed report titled “Genomics and World Health.”
Whether related to informed consent, participation in research, or the management of a genetic disorder that affects an individual or his or her family, there is a great need for more information about fundamental principles of genetics. The pervasive nature of the role of genetics in medicine makes it important for physicians and other health care professionals to become more informed about genetics and to provide advice and counseling in conjunction with trained genetic counselors (Chap. 457). The application of screening and prevention strategies does therefore require continuing patient and physician education, changes in health care financing, and legislation to protect patient’s rights.
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Acknowledgment
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Selected sections and Table 456-4 have been integrated from the chapter on Chromosome Disorders by Dr. Nancy B. Spinner and Dr. Laura K. Conlin, published in the 19th edition of Harrison’s Principles in Internal Medicine. The data and concept for Figure 456-16 have been graciously provided by Dr. Miriam Udler and Dr. Jose Florez, Massachusetts General Hospital and Harvard Medical School, Boston.