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InternalMed_Harrison. Ataxia telangiectasia causes large telangiectatic lesions of the face, cerebellar ataxia, immunologic defects, and hypersensitivity to ionizing radiation (Chap. 450). 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. 132). Cells from these patients are susceptible to chromosomal breaks caused by a defect in genetic recombination. At least 13 different complementation groups have been identified, and the loci and genes associated with Fanconi’s anemia have been cloned. HNPCC (Lynch’s syndrome) is


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InternalMed_Harrison. in genetic recombination. At least 13 different complementation groups have been identified, and the loci and 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. 110). 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 (Chap. 84). Recognition


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InternalMed_Harrison. 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 (Chap. 84). Recognition of HNPCC allows early screening with colonoscopy and the implementation of prevention strategies using nonsteroidal anti-inflammatory drugs.


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InternalMed_Harrison. unstable Dna sequences 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 (Chap. 448), X-linked spinobulbar muscular atrophy (Chap. 452), and myotonic dystrophy (Chap. 462e). 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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InternalMed_Harrison. Functional Consequences of Mutations 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 82-2). Remarkably, the clinical features among patients with an identical mutation in a transcription factor often vary significantly. One mechanism underlying this variability consists in the influence of modifying genes.


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InternalMed_Harrison. clinical features among patients with an identical mutation in a transcription factor 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. 430).


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InternalMed_Harrison. 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. 410). 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. 427); (2) a mutant protein may occupy binding sites on proteins or promoter response elements, as illustrated by thyroid hormone resistance, a disorder in which inactivated thyroid hormone receptor β binds to target genes and functions as an antagonist of normal receptors (Chap. 405); or (3) a mutant protein can be cytotoxic as in α1 antitrypsin deficiency (Chap. 314) or autosomal dominant neurohypophyseal


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InternalMed_Harrison. genes and functions as an antagonist of normal receptors (Chap. 405); or (3) a mutant protein can be cytotoxic as in α1 antitrypsin deficiency (Chap. 314) or autosomal dominant neurohypophyseal diabetes insipidus (Chap. 404), in which the abnormally folded proteins are trapped within the endoplasmic reticulum and ultimately cause cellular damage.


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InternalMed_Harrison. Genotype and Phnotype • alleles, GenotyPes, anD HaPlotyPes 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), he or she can have only two alleles at a


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InternalMed_Harrison. 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), he or she 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. 82-10). 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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InternalMed_Harrison. 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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InternalMed_Harrison. A haplotype refers to a group of alleles that are closely linked together at a genomic locus (Fig. 82-4). 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. 82-6). 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


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InternalMed_Harrison. 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 through the HapMap Project is providing a novel tool for association studies designed to detect genes involved in the pathogenesis of complex disorders 435 (Table 82-1). The presence or absence of certain haplotypes may also become relevant for the customized choice of medical therapies (pharmacogenomics) or for preventive strategies.


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InternalMed_Harrison. 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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InternalMed_Harrison. allelic HeteroGeneity 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 82-3) (Fig. 82-9). 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


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InternalMed_Harrison. 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, there are currently 1963 reported mutations in the CFTR gene (Fig. 82-3). 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 generally 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 greatly facilitates concomitant mutational analyses of several genes


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InternalMed_Harrison. the coding regions, negative results need to be interpreted with caution. The advent of more comprehensive sequencing technologies 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.


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InternalMed_Harrison. PHenotyPic HeteroGeneity Phenotypic heterogeneity occurs when more than one phenotype is caused by allelic mutations (e.g., different mutations in the same gene) (Table 82-3). 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 four 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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InternalMed_Harrison. locus or nonallelic HeteroGeneity anD PHenocoPies Nonallelic or locus heterogeneity refers to the situation in which a similar disease phenotype results from mutations at different genetic loci (Table 82-3). 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 at least eight other genes (Chap. 427). The effects of inactivating mutations in these two genes are similar because the protein products comprise different subunits


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InternalMed_Harrison. Chapter 82 Principles of Human Genetics 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. 462e). 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


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InternalMed_Harrison. 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 also cause 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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InternalMed_Harrison. 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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InternalMed_Harrison. 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. 448). 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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InternalMed_Harrison. variable exPressivity anD incomPlete Penetrance 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, they can develop tumors of the parathyroid gland, endocrine pancreas, and the pituitary gland (Chap. 408). 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


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InternalMed_Harrison. pituitary gland (Chap. 408). 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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InternalMed_Harrison. 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. 428), and the course of phenylketonuria is affected by exposure to phenylalanine in the diet (Chap. 434e). 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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InternalMed_Harrison. 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. 283). 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


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InternalMed_Harrison. 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 Albright’s hereditary osteodystrophy, mutations in the Gsα subunit (GNAS1 gene) are expressed clinically only in individuals who inherit the mutation from their mother (Chap. 424).


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InternalMed_Harrison. sex-influenceD PHenotyPes 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. 141). 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. 432e). Because only males have a Y chromosome, mutations in genes such as SRY, which causes male-to-female sex reversal, or DAZ (deleted in


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InternalMed_Harrison. neuropathy, as well as other features of the disease (Chap. 432e). 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. 410).


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InternalMed_Harrison. 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. 411). 437 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. 411). 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


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InternalMed_Harrison. (Chap. 411). 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. 406). 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. 428).


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InternalMed_Harrison. Chromosomal Disorders Chromosomal or cytogenetic disorders are caused by numerical or structural aberrations in chromosomes. For a detailed discussion of disorders of chromosome number and structure, see Chap. 83e. Deviations in chromosome number are common causes of abortions, developmental disorders, and malformations. 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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InternalMed_Harrison. Monogenic Mendelian Disorders 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 82-1). 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. 82-11). The principles of allelic segregation, and the transmission of alleles from parents to children, are illustrated in Fig. 82-12. 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,


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InternalMed_Harrison. 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 increasingly important role in clinical medicine (Chap. 84).


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InternalMed_Harrison. autosomal Dominant DisorDers These disorders assume particular relevance because 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 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.


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InternalMed_Harrison. 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. 82-13A). 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 Chapter 82 Principles of Human Genetics Heterozygous Heterozygous Female male female carrier of X-linked trait FIGURE 82-11 Standard pedigree symbols.


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InternalMed_Harrison. Chapter 82 Principles of Human Genetics Heterozygous Heterozygous Female male female carrier of X-linked trait FIGURE 82-11 Standard pedigree symbols. clinical manifestations of autosomal dominant disorders may be variable. Because of these variations, it is sometimes challenging to determine the pattern of inheritance. 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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InternalMed_Harrison. autosomal recessive DisorDers 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 the offspring of


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InternalMed_Harrison. 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 the offspring of parents with one dominant (A) and one recessive (a) allele. The distribution of the parental alleles to their offspring depends on the combination present in the parents. Filled symbols = affected individuals.


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InternalMed_Harrison. B Autosomal recessive Autosomal recessive with pseudodominance FIGURE 82-13 (A) Dominant, (B) recessive, (C) X-linked, and (D)mitochondrial (matrilinear) inheritance. 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. 127).


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InternalMed_Harrison. 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. 82-10, 82-13B). 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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InternalMed_Harrison. x-linkeD DisorDers 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. 82-13C). 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


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InternalMed_Harrison. 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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InternalMed_Harrison. y-linkeD DisorDers 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. 410). 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


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InternalMed_Harrison. 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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InternalMed_Harrison. Exceptions to Simple Mendelian Inheritance Patterns • mitocHonDrial DisorDers 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. 85e). 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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InternalMed_Harrison. 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. 82-13D). 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 (Chap. 85e). 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


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InternalMed_Harrison. 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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InternalMed_Harrison. 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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InternalMed_Harrison. mosaicism Mosaicism refers to the presence of two or more genetically 439 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. 410). 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. 424). The clinical phenotype varies depending on the tissue distribution of the mutation; manifestations include ovarian cysts that secrete sex steroids and cause precocious puberty,


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InternalMed_Harrison. (Chap. 424). 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, growth hormone–secreting pituitary adenomas, and hypersecreting autonomous thyroid nodules (Chap. 412).


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InternalMed_Harrison. x-inactivation, imPrintinG, anD uniParental Disomy 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 (Chap. 83e). Prader-Willi syndrome is characterized by diminished fetal activity, obesity, hypotonia, mental retardation, short stature, and hypogonadotropic hypogonadism.


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InternalMed_Harrison. and Angelman’s syndrome (Chap. 83e). 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


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InternalMed_Harrison. 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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InternalMed_Harrison. 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 (Chap. 83e). 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) (Chap. 101e). 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


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InternalMed_Harrison. 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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InternalMed_Harrison. Chapter 82 Principles of Human Genetics somatic mutations Cancer can be considered a genetic disease at the cellular level (Chap. 101e). 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-offunction 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


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InternalMed_Harrison. 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 (Chaps. 101e and 102e). Genome-wide analyses of cancers using deep sequencing often reveal somatic rearrangements resulting in fusion genes and mutations in multiple genes. 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. The heterogeneity of mutations within a tumor


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InternalMed_Harrison. within the primary tumor and metastatic lesions result in diverse genetic and epigenetic alterations that require targeted (personalized) therapies. 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 novel strategy for treating advanced human cancers.


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InternalMed_Harrison. In many cancer syndromes, there is 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


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InternalMed_Harrison. 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. 408) and neurofibromatosis type 2 (Chap. 118).


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InternalMed_Harrison. nucleotiDe rePeat exPansion DisorDers Several diseases are associated with an increase in the number of nucleotide repeats above a certain threshold (Table 82-4). 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. 444e). Anticipation has also been documented in other diseases caused by


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InternalMed_Harrison. disease, for example, there is a correlation between age of onset and length of the triplet codon expansion (Chap. 444e). Anticipation has also been documented in other diseases caused by dynamic mutations in trinucleotide repeats (Table 82-4). 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. 462e).


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InternalMed_Harrison. Complex Genetic Disorders 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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InternalMed_Harrison. 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 82-5) (Chap. 417).


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InternalMed_Harrison. 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 82-2 and 82-5). Genome scans have identified numerous genes and loci that may be associated with susceptibility to development of diabetes mellitus in certain populations. 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


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InternalMed_Harrison. 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 are providing 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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InternalMed_Harrison. Linkage and Association Studies 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 82-6). Genetic linkaGe Genetic linkage refers to the fact that genes are physically connected, or linked, to one another along the chromosomes.


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InternalMed_Harrison. Genetic linkaGe 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. 82-6); 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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InternalMed_Harrison. Polymorphisms 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


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InternalMed_Harrison. 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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InternalMed_Harrison. 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 Abbreviation: GWAS, genome-wide association study. Suitable for identification of susceptibility genes in Requires large sample size and matched control polygenic and multifactorial disorders population Suitable for testing specific allelic variants of known False-positive results in the absence of suitable candidate loci control population


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InternalMed_Harrison. Suitable for testing specific allelic variants of known False-positive results in the absence of suitable candidate loci control population Facilitated by HapMap data, making GWAS more feasible Candidate gene approach does not permit detection of novel genes and pathways 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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InternalMed_Harrison. allelic association, linkaGe Disequilibrium, anD HaPlotyPes 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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InternalMed_Harrison. 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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InternalMed_Harrison. Detecting the genetic factors contributing to the pathogenesis of common complex disorders remains a great challenge. 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 82-1) (Fig. 82-14). 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. The data generated by the HapMap Project 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


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InternalMed_Harrison. by the HapMap Project 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. 82-4). 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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InternalMed_Harrison. PoPulation Genetics 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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InternalMed_Harrison. 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 (Chap. 127), 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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InternalMed_Harrison. APPROACH TO THE PATIENT: 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 443 Rare alleles Mendelian disease Low frequency variants with intermediate effect Typical: Common variants with low effect on complex disease Rare: Common variants with high effect on complex disease Rare variants with small effect: difficult to identify Effect size 50 3.0 1.5 1.1 High Intermediate Modest Low 0.001 0.005 0.05 FIGURE 82-14 Relationship between allele frequency and effect size in monogenic and polygenic disorders. In classic Mendelian disorders, the allele frequency is typically low but has a high impact (single gene disorder). This contrasts with polygenic disorders that require the combination of multiple low impact alleles that are frequently quite common in the general population.


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InternalMed_Harrison. 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. 82-11. 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


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InternalMed_Harrison. 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. 84).


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InternalMed_Harrison. 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 rapidly growing 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 82-1). 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.


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InternalMed_Harrison. 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.


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InternalMed_Harrison. Genomic 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.


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InternalMed_Harrison. 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


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InternalMed_Harrison. 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 are already commercially available. Occupational screening programs aim to detect individuals with increased risk for certain professional activities (e.g., α1 antitrypsin


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InternalMed_Harrison. causing hereditary deafness are already 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 may 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.


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InternalMed_Harrison. 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 (Chap. 84). Numerous techniques, Chapter 82 Principles of Human Genetics 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 cytogenetics, fluorescent in situ hybridization (FISH), and Southern blotting (Chap. 83e), as well as more sensitive novel techniques that search for multiple single exon deletions or duplications.


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InternalMed_Harrison. 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 all exons of the genome or selected chromosomes, or sequencing of numerous candidate genes in a single run, is now possible with next-generation sequencing platforms.


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InternalMed_Harrison. The majority of traditional diagnostic methods were gel-based. Novel technologies for the analysis of mutations, genotyping, large-scale sequencing, and mRNA expression profiles are undergoing rapid evolution. DNA chip technologies allow hybridization of DNA or RNA to hundreds of thousands of probes simultaneously. Microarrays are being used clinically for mutational analysis of several human disease genes, as well as for the identification of viral or bacterial sequence variations. With advances in high-throughput DNA sequencing technology, complete sequencing of the genome or an exome has entered the clinical realm. Although comprehensive sequencing of large genomic regions or multiple genes is already a reality, the subsequent bioinformatics analysis, assembly of sequence fragments, and comparative alignments remains a significant and commonly underestimated challenge. The discovery of incidental (or secondary) findings that are unrelated to the indication for the sequencing


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InternalMed_Harrison. and comparative alignments remains a significant and commonly underestimated challenge. 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.


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InternalMed_Harrison. A general algorithm for the approach to mutational analysis is outlined in Fig. 82-15. 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.


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InternalMed_Harrison. 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. The main indications for amniocentesis include advanced maternal age (>35 years), an abnormal serum triple marker test (α-fetoprotein, β human chorionic gonadotropin, pregnancy-associated plasma protein A, or 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


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InternalMed_Harrison. 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. Recently, the entire fetal genome has been determined prenatally from cells taken from the mother’s plasma through deep sequencing and the counting of parental haplotypes, or by inferring it from DNA sequences obtained from blood samples from the mother, father, and umbilical cord. 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


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InternalMed_Harrison. 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.


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InternalMed_Harrison. In combination with in vitro fertilization (IVF) techniques, it is even possible to perform genetic diagnoses in a single cell removed from the fourto 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. 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 mutations is an important diagnostic and FIGURE 82-15 Approach to genetic disease.


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InternalMed_Harrison. prognostic tool in leukemias and lymphomas. 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,


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InternalMed_Harrison. 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. 5).


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InternalMed_Harrison. 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, will fill this gap


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InternalMed_Harrison. 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, will fill 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.


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InternalMed_Harrison. 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 (Chap. 84). 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 (Chap. 84). 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


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InternalMed_Harrison. 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.


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InternalMed_Harrison. The impact of genetic testing on health care costs is currently unclear. It is likely to vary among disorders and depend 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 has not been defined, and there are numerous 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.


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InternalMed_Harrison. 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


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InternalMed_Harrison. 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 BRCA 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.


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InternalMed_Harrison. 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, 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. The World Health Organization has summarized the current issues and inequities surrounding genomic medicine in a detailed report titled “Genomics and World Health.”


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InternalMed_Harrison. 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. 84). The application of screening and prevention strategies will therefore require intensive patient and physician education, changes in health care financing, and legislation to protect patient’s rights. Chapter 82 Principles of Human Genetics


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InternalMed_Harrison. Chromosome Disorders Nancy B. Spinner, Laura K. Conlin CHROMOSOME DISORDERS Alterations of the chromosomes (numerical and structural) occur in about 1% of the general population, in 8% of stillbirths, and in close to 50% of spontaneously aborted fetuses. The 3 × 109 base pairs that 83e encode the human genome are packaged into 23 pairs of chromosomes, which consist of discrete portions of DNA, bound to several classes of regulatory proteins. Technical advances that led to the ability to analyze human chromosomes immediately translated into the revelation that human disorders can be caused by an abnormality of chromosome number. In 1959, the clinically recognizable disorder, Down syndrome, was demonstrated to result from having three copies of chromosome 21 (trisomy 21). Very soon thereafter, in 1960, a small, structurally abnormal chromosome was recognized in the cells of some patients with chronic myelogenous leukemia (CML), and this abnormal chromosome is now known as the


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InternalMed_Harrison. thereafter, in 1960, a small, structurally abnormal chromosome was recognized in the cells of some patients with chronic myelogenous leukemia (CML), and this abnormal chromosome is now known as the Philadelphia chromosome.


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InternalMed_Harrison. Since these early discoveries, the techniques for analysis of human chromosomes, and DNA in general, have gone through several revolutions, and with each technical advancement, our understanding of the role of chromosomal abnormalities in human disease has expanded. While early studies in the 1950s and 1960s easily identified abnormalities of chromosome number (aneuploidy) and large structural alterations such as deletions (chromosomes with missing regions), duplications (extra copies of chromosome regions), or translocations (where portions of the chromosomes are rearranged), many other types of structural alterations could only be identified as techniques improved. The first important technical advance was the introduction of chromosome banding in the late 1960s, a technique that allowed for the staining of the chromosomes, so that each chromosome could be recognized by its pattern of alternating dark and light (or fluorescent and nonfluorescent) bands. Other technical innovations


InternalMed_Harrison

InternalMed_Harrison. for the staining of the chromosomes, so that each chromosome could be recognized by its pattern of alternating dark and light (or fluorescent and nonfluorescent) bands. Other technical innovations ranged from the introduction of fluorescence in situ hybridization in the 1980s to use of array-based and sequencing technologies in the early 2000s. Currently, we can appreciate that many types of chromosome abnormalities contribute to human disease including aneuploidy; structural alterations such as deletions and duplications, translocations, or inversions; uniparental disomy, where two copies of one chromosome (or a portion of a chromosome) are inherited from one parent; complex alterations such as isochromosomes, markers, and rings; and mosaicism for all of the aforementioned abnormalities. The first chromosome disorders identified had very striking and generally severe phenotypes, because the abnormalities involved large regions of the genome, but as methods have become more sensitive,


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InternalMed_Harrison. The first chromosome disorders identified had very striking and generally severe phenotypes, because the abnormalities involved large regions of the genome, but as methods have become more sensitive, it is now possible to recognize many more subtle phenotypes, often involving smaller genomic regions.


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InternalMed_Harrison. Standard cytogenetic analysis refers to the examination of banded human chromosomes. Banded chromosome analysis allows for both the determination of the number and identity of chromosomes in the cell and recognition of abnormal banding patterns associated with a structural rearrangement. A stained band is defined as the part of a chromosome that is clearly distinguishable from its adjacent segments by appearing darker or lighter with one or more banding techniques. Cytogenetic analysis is most commonly carried out on cells in mitosis, requiring dividing cells. Actively growing cells are most often obtained from peripheral blood; however, it is only a small subset of the blood cells that are actually used for cytogenetic analysis. Often, chemicals, like phytohemagglutinin (PHA), are used to specially stimulate growth of T cells in a blood sample. Other sources of dividing cells include skin-derived fibroblasts, amniotic fluid or placental tissue (for prenatal diagnosis), or tumor


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InternalMed_Harrison. to specially stimulate growth of T cells in a blood sample. Other sources of dividing cells include skin-derived fibroblasts, amniotic fluid or placental tissue (for prenatal diagnosis), or tumor tissue (for cancer diagnosis). After culturing, cells are treated with a mitotic spindle inhibitor, which prevents the separation of the chromatids during metaphase. Halting mitosis 83e-1 in metaphase is essential, because chromosomes are at their most condensed state during this stage of mitosis. The banding pattern of a metaphase chromosome is easily recognizable and is ideal for karyotyping. There are several different types of chromosome staining techniques, including R-banding, C-banding, and quinacrine staining, but the most commonly used is G-banding. G-banding is accomplished by treatment of the chromosomes with a proteolytic enzyme, such as trypsin, which digests some of the proteins holding DNA in a three-dimensional structure, followed by staining with a dye (Giemsa) that binds


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InternalMed_Harrison. of the chromosomes with a proteolytic enzyme, such as trypsin, which digests some of the proteins holding DNA in a three-dimensional structure, followed by staining with a dye (Giemsa) that binds DNA. The resulting patterns have both dark and light bands; in general, the light bands occur in regions on the chromosome in which genes are actively being transcribed, and dark bands are in regions of less active transcription.


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InternalMed_Harrison. The banded human karyotype has now been standardized based on an internationally agreed upon system for designating not only individual chromosomes but also chromosome regions, providing a way in which structural rearrangements and variants can be described in terms of their composition. The normal human female karyotype is referred to as 46,XX (46 chromosomes, with 22 pairs of autosomes and two of the same type of sex chromosomes [two Xs], indicating this is a female); and the normal human male karyotype is referred to as 46,XY (46 chromosomes, with 22 pairs of autosomes and one of each type of sex chromosome [one X and one Y], indicating this is a male). The anatomy of a chromosome includes the central constriction, known as the centromere, which is critical for movement of the chromosomes during mitosis and meiosis; the two chromosome arms (p for the smaller or petite arm, and q for the longer arm); and the chromosome ends, which contain the telomeres. The telomeres are made up of


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InternalMed_Harrison. during mitosis and meiosis; the two chromosome arms (p for the smaller or petite arm, and q for the longer arm); and the chromosome ends, which contain the telomeres. The telomeres are made up of a hexanucleotide repeat (TTAGGG)n, and unlike the centromere, they are not visible at the light microscope level. Telomeres are functionally important because they confer stability to the end of the chromosome. Broken chromosomes tend to fuse end to end, whereas a normal chromosome with an intact telomere structure is stable. To create the standard chromosome-banding map, each chromosome is divided into segments that are numbered, and then further subdivided. The precise band names are recorded in an international document so that each band has a distinct number. Figure 83e-1 shows an ideogram (chromosome map with bands) of the X chromosome and a G-banded X chromosome. This system provides a way for a chromosome abnormality to be written, with an indication of which band is deleted,


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InternalMed_Harrison. (chromosome map with bands) of the X chromosome and a G-banded X chromosome. This system provides a way for a chromosome abnormality to be written, with an indication of which band is deleted, duplicated, or rearranged.


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InternalMed_Harrison. p22.3 p2 p21.3 p arm p11.2 centromere q1 q21.1 FIGuRE 83e-1 Ideogram of the X chromosome and a G-banded X chromosome. The labeling of the X ideogram shows the positioning of the p and q arms, the centromere, and the telomeres. The numbering of the bands is also demonstrated, indicating the broadest subbands (p1, p2, q1, q2) and the further subdivisions to the right. Numbering begins at the centromere and moves out along each arm toward the telomeres.


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InternalMed_Harrison. Molecular cytogenetics provides a link between chromosome and molecular analysis and overcomes some of the limitations of standard cytogenetics. Deletions smaller than several million base pairs are not routinely detectable by standard G-banding techniques, and chromosomal abnormalities with indistinct or novel banding patterns can be difficult or impossible to interpret. To carry out cytogenetic analysis, cells must be dividing, which is not always possible to obtain (e.g., in autopsy or tumor material that has already been fixed). Finally, growth selection or bias may occasionally cause the results of cytogenetic studies to be misleading because cells that proliferate in vitro may not be representative of the original population, as is often the case with tumor specimens.


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InternalMed_Harrison. Fluorescence in situ hybridization (FISH) is a combined cytogenetic-molecular technique that solves many of the aforementioned problems. FISH permits determination of the number and location of specific DNA sequences in human cells. FISH can be performed on metaphase chromosomes, as with G-banding, but can also be performed on cells not actively progressing through mitosis. FISH performed on nondividing cells is referred to as interphase or nuclear FISH (Fig. 83e-2). The FISH procedure relies on the complementarity between the two strands of the DNA double helix and uses a molecular probe, which can be a pool of sequences across an entire chromosome, a DNA sequence for a repetitive part of the genome (e.g., centromeres or telomeres), or a specific DNA sequence found only once in the genome (e.g., a disease-associated gene). The choice of probes for FISH studies is important and will vary with the information needed for the diagnosis of a particular disorder. The most common type of


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InternalMed_Harrison. (e.g., a disease-associated gene). The choice of probes for FISH studies is important and will vary with the information needed for the diagnosis of a particular disorder. The most common type of probes are locus-specific probes, which are used to determine if a critical gene or region is absent (indicating a deletion), or present in the normal number of copies, or if an additional copy of the region is present. FISH on metaphase chromosomes will give the additional information of the location of the additional copy, which is necessary information to determine whether a structural rearrangement, such as a translocation, is present. FISH can also be performed with probes that bind to repeated sequences, such as DNA found in centromeres or telomeres, or with probes that bind to an entire chromosome (“painting” probes), to determine the chromosome composition of an abnormal chromosome. Interphase FISH studies can also help to identify structural alterations when probes are used that map


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InternalMed_Harrison. (“painting” probes), to determine the chromosome composition of an abnormal chromosome. Interphase FISH studies can also help to identify structural alterations when probes are used that map to both sides of a translocation breakpoint. Each side of the breakpoint is labeled in a different color, and when no translocation is present the two probes appear to be overlapping. When a translocation is present, the two probes appear separate from one another. These set of probes, called “breakapart” probes, are commonly used to detect recurrent translocations in cancer cells.


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InternalMed_Harrison. Array-based methods were introduced into the clinical lab beginning in 2003 and quickly revolutionized the field of cytogenetics. These techniques used arrays (collections of DNA segments from the entire genome) which could be interrogated with respect to copy number. With standard cytogenetics, the missing or extra pieces of DNA have to be big enough to see in the microscope on banded chromosomes (usually larger than 5 Mb). FISH requires a preselection of an informative molecular probe prior to analysis. In contrast, array-based techniques permit analysis of many regions of the genome in a single analysis, with greatly increased resolution over standard cytogenetics. Array-based techniques allow for scanning of the genome for small deletions or duplications quickly and accurately. The resolution of the p13 –1.00 –0.75 –0.50 –0.25 0.00 0.25Log R ratio 0.50 0.75 1.00 p12 p11.2 q11.2 q14 q21.1 q21.3 q22.2 q23 q26.1 q26.2 q26.3 15


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InternalMed_Harrison. FIGuRE 83e-2 G-banding, fluorescence in situ hybridization (FISH), and single nucleotide polymorphism (SNP) array demonstrate an abnormal chromosome 15. A. G-banding shows an abnormal chromosome 15, with unrecognizable material in place of the p arm in the chromosome on the right (top arrow). B. Metaphase FISH (only chromosome 15s are shown) using a probe from the 15q telomere region (red) and a control probe that maps outside of the duplicated region (green). C. Interphase FISH demonstrates three copies of the 15q tel probe in red, and two copies of the 15q control probe (green). D. Genome-wide SNP array demonstrates the increased copy number for a portion of 15q. Note that the G-banding alone indicates the abnormal chromosome 15, but the origin of the extra material can only be demonstrated by FISH or array. The FISH analysis requires additional information about possible genetic causes to select the correct probe. The array can exactly identify the origin of the extra material, but


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InternalMed_Harrison. by FISH or array. The FISH analysis requires additional information about possible genetic causes to select the correct probe. The array can exactly identify the origin of the extra material, but by itself would not provide positional information.