Chromosomes and DNA Replication
Organization of DNA into Chromosomes
The human genome is divided into 23 different chromosomes, including 22 autosomes (numbered 1–22) and the X and Y sex chromosomes. Adult cells are diploid, meaning they contain two homologous sets of 22 autosomes and a pair of sex chromosomes. Females have two X chromosomes (XX), whereas males have one X and one Y chromosome (XY). As a consequence of meiosis, germ cells (sperm or oocytes) are haploid and contain one set of 22 autosomes and one of the sex chromosomes. At the time of fertilization, the diploid genome is reconstituted by pairing of the homologous chromosomes from the mother and father. With each cell division (mitosis), chromosomes are replicated, paired, segregated, and divided into two daughter cells (Chap. 62).
Although the exact number of genes encoded by the human genome is still unknown, current estimates predict about 23,000 to 25,000 protein-coding genes, a number that is substantially smaller than initially predicted. A gene is a functional unit that is regulated by transcription (see below) and encodes an RNA product, which is most commonly, but not always, translated into a protein that exerts activity within or outside the cell. Historically, genes were identified because they conferred specific traits that are transmitted from one generation to the next. Increasingly, they are characterized based on expression in various tissues (transcriptome). The number of genes greatly underestimates the complexity of genetic expression, as single genes can generate multiple spliced messenger RNA (mRNA) products, which are translated into proteins that are subject to complex posttranslational modification such as phosphorylation. Proteomics, the study of the proteome using technologies of large-scale protein separation and identification, is focused on protein variation and function. Similarly, the field of metabolomics aims at determining the composition and modifications of the metabolome, the complement of low-molecular-weight molecules, many of which participate in various metabolic functions. The human microbiome refers to the constellation of viruses, bacteria, and fungi that colonize various human tissues (Chap. 64). Comprehensive characterization of the microbiome has been made feasible by the availability of high-throughput DNA-sequencing methods. Analyses of genomics, proteomics, metabolomics, and the microbiome are heavily dependent on bioinformatics, and they are beginning to reveal how physiologic or pathologic alterations affect modular networks rather than linear pathways (Chap. e19).
Human DNA consists of ∼3 billion base pairs (bp) of DNA per haploid genome. DNA length is normally measured in units of 1000 bp (kilobases, kb) or 1,000,000 bp (megabases, Mb). Not all DNA encodes genes. In fact, genes account for only ∼10–15% of DNA. Much of the remaining DNA consists of highly repetitive sequences, the function of which is poorly understood. These repetitive DNA regions, along with nonrepetitive sequences that do not encode genes, may serve a structural role in the packaging of DNA into chromatin [i.e., DNA bound to histone proteins, and chromosomes (Fig. 61-1)]. If only 10% of DNA is expressed and there are 25,000 genes, the average gene would be ∼12 kb in length. Although many genes are about this size, the range is quite broad. For example, some genes are only a few hundred bp, whereas others such as the DMD gene, are extraordinarily large (2 Mb).
Structure of chromatin and chromosomes. Chromatin is composed of double-strand DNA that is wrapped around histone and non–histone proteins forming nucleosomes. The nucleosomes are further organized into solenoid structures. Chromosomes assume their characteristic structure, with short (p) and long (q) arms at the metaphase stage of the cell cycle.
Each gene is composed of a linear polymer of DNA. DNA is a double-stranded helix composed of four different bases: adenine (A), thymidine (T), guanine (G), and cytosine (C). Adenine is paired to thymidine, and guanine is paired to cytosine, by hydrogen bond interactions that span the double helix. DNA has several remarkable features that make it ideal for the transmission of genetic information. It is relatively stable, at least in comparison to RNA or proteins. The double-stranded nature of DNA and its feature of strict base-pair complementarity permit faithful replication during cell division. As described below, complementarity also allows the transmission of genetic information from DNA → RNA → protein (Fig. 61-2). mRNA is encoded by the so-called sense or coding strand of the DNA double helix and is translated into proteins by ribosomes.
Flow of genetic information. Multiple extracellular signals activate intracellular signal cascades that result in altered regulation of gene expression through the interaction of transcription factors with regulatory regions of genes. RNA polymerase transcribes DNA into RNA that is processed to mRNA by excision of intronic sequences. The mRNA is translated into a polypeptide chain to form the mature protein after undergoing posttranslational processing. HAT, histone acetyl transferase; CBP, CREB-binding protein; CREB, cyclic AMP response element–binding protein; CRE, cyclic AMP responsive element; CoA, Co activator; TAF, TBP-associated factors; GTF, general transcription factors; TBP, TATA-binding protein; TATA, TATA box; RE, response element; NH2, aminoterminus; COOH, carboxyterminus.
The presence of four different bases provides surprising genetic diversity. In the protein-coding regions of genes, the DNA bases are arranged into codons, a triplet of bases that specifies a particular amino acid. It is possible to arrange the four bases into 64 different triplet codons (43). Each codon specifies 1 of the 20 different amino acids, or a regulatory signal such as initiation and stop of translation. Because there are more codons than amino acids, the genetic code is degenerate; that is, most amino acids can be specified by several different codons. By arranging the codons in different combinations and in various lengths, it is possible to generate the tremendous diversity of primary protein structure.
Replication of DNA and Mitosis
Genetic information in DNA is transmitted to daughter cells under two different circumstances: (1) somatic cells divide by mitosis, allowing the diploid (2n) genome to replicate itself completely in conjunction with cell division; and (2) germ cells (sperm and ova) undergo meiosis, a process that enables the reduction of the diploid (2n) set of chromosomes to the haploid state (1n) (Chap. 62).
Prior to mitosis, cells exit the resting, or G0 state, and enter the cell cycle (Chap. 84). After traversing a critical checkpoint in G1, cells undergo DNA synthesis (S phase), during which the DNA in each chromosome is replicated, yielding two pairs of sister chromatids (2n → 4n). The process of DNA synthesis requires stringent fidelity in order to avoid transmitting errors to subsequent generations of cells. Genetic abnormalities of DNA mismatch/repair include xeroderma pigmentosum, Bloom's syndrome, ataxia telangiectasia, and hereditary nonpolyposis colon cancer (HNPCC), among others. Many of these disorders strongly predispose to neoplasia because of the rapid acquisition of additional mutations (Chap. 83). After completion of DNA synthesis, cells enter G2 and progress through a second checkpoint before entering mitosis. At this stage, the chromosomes condense and are aligned along the equatorial plate at metaphase. The two identical sister chromatids, held together at the centromere, divide and migrate to opposite poles of the cell (Fig. 61-3). After formation of a nuclear membrane around the two separated sets of chromatids, the cell divides and two daughter cells are formed, thus restoring the diploid (2n) state.
Crossing-over and genetic recombination. During chiasma formation, either of the two sister chromatids on one chromosome pairs with one of the chromatids of the homologous chromosome. Genetic recombination occurs through crossing-over and results in recombinant and nonrecombinant chromosome segments in the gametes. Together with the random segregation of the maternal and paternal chromosomes, recombination contributes to genetic diversity and forms the basis of the concept of linkage.
Assortment and Segregation of Genes during Meiosis
Meiosis occurs only in germ cells of the gonads. It shares certain features with mitosis but involves two distinct steps of cell division that reduce the chromosome number to the haploid state. In addition, there is active recombination that generates genetic diversity. During the first cell division, two sister chromatids (2n → 4n) are formed for each chromosome pair and there is an exchange of DNA between homologous paternal and maternal chromosomes. This process involves the formation of chiasmata, structures that correspond to the DNA segments that cross over between the maternal and paternal homologues (Fig. 61-3). Usually there is at least one crossover on each chromosomal arm; recombination occurs more frequently in female meiosis than in male meiosis. Subsequently, the chromosomes segregate randomly. Because there are 23 chromosomes, there exist 223 (>8 million) possible combinations of chromosomes. Together with the genetic exchanges that occur during recombination, chromosomal segregation generates tremendous diversity, and each gamete is genetically unique. The process of recombination, and the independent segregation of chromosomes, provide the foundation for performing linkage analyses, whereby one attempts to correlate the inheritance of certain chromosomal regions (or linked genes) with the presence of a disease or genetic trait (see below).
After the first meiotic division, which results in two daughter cells (2n), the two chromatids of each chromosome separate during a second meiotic division to yield four gametes with a haploid state (1n). When the egg is fertilized by sperm, the two haploid sets are combined, thereby restoring the diploid state (2n) in the zygote.
Implications of the Human Genome Project
The HGP was initiated in the mid-1980s as an ambitious effort to characterize the human genome, culminating in a complete DNA sequence. The initial main goals were (1) creation of genetic maps, (2) development of physical maps, and (3) determination of the complete human DNA sequence. Some analogies help in appreciating the scope of the HGP. The 23 pairs of human chromosomes encode ∼23,000–30,000 genes. The total length of DNA is ∼3 billion bp, which is nearly 1000-fold greater than that of the E. coli genome. If the human DNA sequence were printed out, it would correspond to about 120 volumes of Harrison's Principles of Internal Medicine.
The identification of the ∼10 million SNPs estimated to occur in the human genome has generated a catalogue of common genetic variants that occur in human beings from distinct ethnic backgrounds (Fig. 61-7). SNPs that are in close proximity are inherited together (i.e., they are linked) and are referred to as haplotypes, hence the name HapMap (Fig. 61-8). The HapMap describes the nature and location of these SNP haplotypes and how they are distributed among individuals within and among populations. The HapMap information is greatly facilitating GWAS designed to elucidate the complex interactions among multiple genes and lifestyle factors in multifactorial disorders (see below). Moreover, haplotype analyses will be useful to assess variations in responses to medications (pharmacogenomics) and environmental factors, as well as the prediction of disease predisposition.
Chromosome 7 is shown with the density of single nucleotide polymorphisms (SNPs) and genes above. A 200-kb region in 7q31.2 containing the CFTR gene is shown below. The CFTR gene contains 27 exons. More than 1790 mutations in this gene have been found in patients with cystic fibrosis. A 20-kb region encompassing exons 4–9 is shown further amplified in order to illustrate the SNPs in this region.
The origin of haplotypes is due to repeated recombination events occurring in multiple generations. Over time, this leads to distinct haplotypes. These haplotype blocks can often be characterized by genotyping selected Tag single-nucleotide polymorphisms, an approach that now facilitates performing genome-wide association studies (GWAS).
The complete DNA sequence of each chromosome provides the highest-resolution physical map. The primary focus of the HGP was to obtain DNA sequence for the entire human genome as well as model organisms. Although the prospect of determining the complete sequence of the human genome seemed daunting several years ago, technical advances in DNA sequencing and bioinformatics led to the completion of a draft human sequence in June 2000, well in advance of the original goal year of 2003. High-quality reference sequences, completed in 2003, further closed gaps and reduced remaining ambiguities, and the HGP announced the completion of the DNA sequence for the last of the human chromosomes in May 2006. The Personal Genome Project (PGP) launched in 2006 aims to completely sequence the genomes from multiple individuals and to associate them with health and physical information to enhance our understanding of human (patho)physiology. A variety of technical advances in DNA sequencing and computational analyses may soon reduce the cost of obtaining a complete human genome sequence to close to US $1,000. In addition to the human genome, the genomes of numerous organisms have been sequenced completely (∼ 1000) or partially (∼ 5500) [Genomes Online Database (GOLD); Table 61-1]. They include, among others, eukaryotes such as man and mouse; S. cerevisiae, C. elegans, and D. melanogaster; bacteria (e.g., E. coli); and archeae, viruses, organelles (mitochondriae, chloroplasts), and plants (e.g., Arabidopsis thaliana). This information, together with technological advances and refinement of computational bioinformatics, has led to a fast-paced transition from the study of single genes to whole genomes. The current directions arising from the HGP include, among others, (1) the comparison of entire genomes (comparative genomics), (2) the study of large-scale expression of RNAs (functional genomics) and proteins (proteomics) in order to detect differences between various tissues in health and disease, (3) the characterization of the variation among individuals by establishing catalogues of sequence variations and SNPs (HapMap project) and the Personal Genome Project, and (4) the identification of genes that play critical roles in the development of polygenic and multifactorial disorders.
Implicit in the HGP is the concept that identifying disease-causing genes can lead to improvements in diagnosis, treatment, and prevention. Most individuals can be expected to harbor several serious recessive gene mutations. Completion of the human genome sequence, determination of the association of genetic defects with disease, and studies of genetic variation raise many new issues with implications for the individual and mankind. The controversies concerning the cloning of mammals, the establishment of human ES cells, and the creation of synthetic organisms underscore the relevance of these questions. Moreover, the information gleaned from genotypic results can have quite different impacts, depending on the availability of strategies to modify the course of disease. For example, the identification of mutations that cause multiple endocrine neoplasia (MEN) type 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 alter therapy and outcomes. However, the progress in this area is unpredictable, as underscored by the finding that angiotensin II receptor blockers may slow disease progression in Marfan's syndrome.
Genetic test results can generate anxiety in affected individuals and family members, and there is the possibility of discrimination on the basis of the test results. Most genetic disorders are likely to fall into an intermediate category where the opportunity for prevention or treatment is significant but limited (Chap. 63). For these reasons, the scientific components of the HGP have been paralleled by efforts to examine ethical, social, and legal implications as new issues arise.
The Genetic Information Nondiscrimination Act (GINA), signed into law in 2008, aims to protect individuals against the misuse of genetic information for health insurance and employment. 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.
Many issues raised by the genome project are familiar, in principle, to medical practitioners. For example, an asymptomatic patient with increased low-density lipoprotein (LDL) cholesterol, high blood pressure, or a strong family history of early myocardial infarction is known to be at increased risk of coronary heart disease. In such cases, it is clear that the identification of risk factors and an appropriate intervention are beneficial. Likewise, patients with phenylketonuria, cystic fibrosis, or sickle cell anemia are often identified as having a genetic disease early in life. These precedents can be helpful for adapting policies that relate to genetic information. We can anticipate similar efforts, whether based on genotypes or other markers of genetic predisposition, to be applied to many disorders. One confounding aspect of the rapid expansion of information is that our ability to make clinical decisions often lags behind initial insights into genetic mechanisms of disease. For example, when genes that predispose to breast cancer such as BRCA1 are described, they generate tremendous public interest in the potential to predict disease, but many years of clinical research are still required to rigorously establish genotype and phenotype correlations.
Whether related to informed consent, participation in research, or the management of a genetic disorder that affects an individual or their families, 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 imperative 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. 63). 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.
Genomics and Global Health
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 on Genomics and World Health.
Transmission of Genetic Disease
Origins and Types of Mutations
A mutation can be defined as any change in the primary nucleotide sequence of DNA regardless of its functional consequences. Some mutations may be lethal, others are less deleterious, and some may confer an evolutionary advantage. Mutations can occur in the germline (sperm or oocytes); these can be transmitted to progeny. Alternatively, mutations can occur during embryogenesis or in somatic tissues. Mutations that occur during development lead to mosaicism, a situation in which tissues are composed of cells with different genetic constitutions. If the germline is mosaic, a mutation can be transmitted to some progeny but not others, which sometimes leads to confusion in assessing the pattern of inheritance. Somatic mutations that do not affect cell survival can sometimes be detected because of variable phenotypic effects in tissues (e.g., pigmented lesions in McCune-Albright syndrome). Other somatic mutations are associated with neoplasia because they confer a growth advantage to cells. Epigenetic events, heritable changes that do not involve changes in gene sequence (e.g., altered DNA methylation), may influence gene expression or facilitate genetic damage. With the exception of triplet nucleotide repeats, which can expand (see below), mutations are usually stable.
Mutations are structurally diverse'they can involve the entire genome, as in triploidy (one extra set of chromosomes), or gross numerical or structural alterations in chromosomes or individual genes (Chap. 62). Large deletions may affect a portion of a gene or an entire gene, or, if several genes are involved, they may lead to a contiguous gene syndrome. Unequal crossing-over between homologous genes can result in fusion gene mutations, as illustrated by color blindness (Chap. 28). Mutations involving single nucleotides are referred to as point mutations. Substitutions are called transitions if a purine is replaced by another purine base (A ↔ G) or if a pyrimidine is replaced by another pyrimidine (C ↔ T). Changes from a purine to a pyrimidine, or vice versa, are referred to as transversions. If the DNA sequence change occurs in a coding region and alters an amino acid, it is called a missense mutation. Depending on the functional consequences of such a missense mutation, amino acid substitutions in different regions of the protein can lead to distinct phenotypes. Polymorphisms are sequence variations that have a frequency of at least 1%. Usually, they do not result in a perceptible phenotype. Often they consist of single base-pair substitutions that do not alter the protein coding sequence because of the degenerate nature of the genetic code (synonymous polymorphism), although it is possible that some might alter mRNA stability, translation, or the amino acid sequence (nonsynonymous polymorphism) (Fig. 61-7). These types of base substitutions are encountered frequently during genetic testing and must be distinguished from true mutations that alter protein expression or function. Small nucleotide deletions or insertions cause a shift of the codon reading frame (frameshift). Most commonly, reading frame alterations result in an abnormal protein segment of variable length before termination of translation occurs at a stop codon (nonsense mutation) (Fig. 61-6). Mutations in intronic sequences or in exon junctions may destroy or create splice donor or splice acceptor sites. Mutations may also be found in the regulatory sequences of genes, resulting in reduced gene transcription.
As noted before, mutations represent an important cause of genetic diversity as well as disease. Mutation rates are difficult to determine in humans because many mutations are silent and because testing is often not adequate to detect the phenotypic consequences. Mutation rates vary in different genes but are estimated to occur at a rate of ∼10−10/bp per cell division. Germline mutation rates (as opposed to somatic mutations) are relevant in the transmission of genetic disease. Because the population of oocytes is established very early in development, only ∼20 cell divisions are required for completed oogenesis, whereas spermatogenesis involves ∼30 divisions by the time of puberty and 20 cell divisions each year thereafter. Consequently, the probability of acquiring new point mutations is much greater in the male germline than the female germline, in which rates of aneuploidy are increased (Chap. 62). Thus, the incidence of new point mutations in spermatogonia increases with paternal age (e.g., achondrodysplasia, Marfan's syndrome, neurofibromatosis). It is estimated that about 1 in 10 sperm carries a new deleterious mutation. The rates for new mutations are calculated most readily for autosomal dominant and X-linked disorders and are ∼10−5−10−6/locus per generation. Because most monogenic diseases are relatively rare, new mutations account for a significant fraction of cases. This is important in the context of genetic counseling, as a new mutation can be transmitted to the affected individual but does not necessarily imply that the parents are at risk to transmit the disease to other children. An exception to this is when the new mutation occurs early in germline development, leading to gonadal mosaicism.
Normally, DNA recombination in germ cells occurs with remarkable fidelity to maintain the precise junction sites for the exchanged DNA sequences (Fig. 61-3). However, mispairing of homologous sequences leads to unequal crossover, with gene duplication on one of the chromosomes and gene deletion on the other chromosome. A significant fraction of growth hormone (GH) gene deletions, for example, involve unequal crossing-over (Chap. 339). The GH gene is a member of a large gene cluster that includes a gh variant gene as well as several structurally related chorionic somatomammotropin genes and pseudogenes (highly homologous but functionally inactive relatives of a normal gene). Because such gene clusters contain multiple homologous DNA sequences arranged in tandem, they are particularly prone to undergo recombination and, consequently, gene duplication or deletion. On the other hand, duplication of the PMP22 gene because of unequal crossing-over results in increased gene dosage and type IA Charcot-Marie-Tooth disease. Unequal crossing-over resulting in deletion of PMP22 causes a distinct neuropathy called hereditary liability to pressure palsy (Chap. 384).
Glucocorticoid-remediable aldosteronism (GRA) is caused by a rearrangement involving the genes that encode aldosterone synthase (CYP11B2) and steroid 11β-hydroxylase (CYP11B1), normally arranged in tandem on chromosome 8q. These two genes are 95% identical, predisposing to gene duplication and deletion by unequal crossing-over. The rearranged gene product contains the regulatory regions of 11β-hydroxylase fused to the coding sequence of aldosterone synthetase. Consequently, the latter enzyme is expressed in the adrenocorticotropic hormone (ACTH)–dependent zona fasciculata of the adrenal gland, resulting in overproduction of mineralocorticoids and hypertension (Chap. 342).
Gene conversion refers to a nonreciprocal exchange of homologous genetic information; it is probably more common than generally recognized. In human genetics, gene conversion has been used to explain how an internal portion of a gene is replaced by a homologous segment copied from another allele or locus; these genetic alterations may range from a few nucleotides to a few thousand nucleotides. As a result of gene conversion, it is possible for short DNA segments of two chromosomes to be identical, even though these sequences are distinct in the parents. A practical consequence of this phenomenon is that nucleotide substitutions can occur during gene conversion between related genes, often altering the function of the gene. In disease states, gene conversion often involves intergenic exchange of DNA between a gene and a related pseudogene. For example, the 21-hydroxylase gene (CYP21A2) is adjacent to a nonfunctional pseudogene (CYP21A1P). Many of the nucleotide substitutions that are found in the CYP21A2 gene in patients with congenital adrenal hyperplasia correspond to sequences that are present in the CYP21A1P pseudogene, suggesting gene conversion as a mechanism of mutagenesis. In addition, mitotic gene conversion has been suggested as a mechanism to explain revertant mosaicism in which an inherited mutation is "corrected" in certain cells. For example, patients with autosomal recessive generalized atrophic benign epidermolysis bullosa have acquired reverse mutations in one of the two mutated COL17A1 alleles, leading to clinically unaffected patches of skin.
Although many instances of insertions and deletions occur as a consequence of unequal crossing-over, there is also evidence for internal duplication, inversion, or deletion of DNA sequences. The fact that certain deletions or insertions appear to occur repeatedly as independent events suggests that specific regions within the DNA sequence predispose to these errors. For example, certain regions of the DMD gene appear to be hot spots for deletions. Some regions within the human genome are rearrangement hot spots and lead to copy number variations (CNVs).
Because mutations caused by defects in DNA repair accumulate as somatic cells divide, these types of mutations are particularly important in the context of neoplastic disorders (Chap. 84). Several genetic disorders involving DNA repair enzymes underscore their importance. Patients with xeroderma pigmentosum have defects in DNA damage recognition or in the nucleotide excision and repair pathway (Chap. 87). Exposed skin is dry and pigmented and is extraordinarily sensitive to the mutagenic effects of ultraviolet irradiation. More than 10 different genes have been shown to cause the different forms of xeroderma pigmentosum. This finding is consistent with the earlier classification of this disease into different complementation groups in which normal function is rescued by the fusion of cells derived from two different forms of xeroderma pigmentosum.
Ataxia telangiectasia causes large telangiectatic lesions of the face, cerebellar ataxia, immunologic defects, and hypersensitivity to ionizing radiation (Chap. 373). 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. 109). 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 several loci and genes associated with Fanconi's anemia have been mapped or 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. 91). 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. 63). Recognition of HNPCC allows early screening with colonoscopy and the implementation of prevention strategies using nonsteroidal anti-inflammatory drugs.
Dipyrimidine and CpG Sequences
Certain DNA sequences are particularly susceptible to mutagenesis. Successive pyrimidine residues (e.g., T-T or C-C) are subject to the formation of ultraviolet light–induced photoadducts. If these pyrimidine dimers are not repaired by the nucleotide excision repair pathway, mutations will be introduced after DNA synthesis. The dinucleotide C-G, or CpG, is also a hot spot for a specific type of mutation. In this case, methylation of the cytosine is associated with an enhanced rate of deamination to uracil, which is then replaced with thymine. This C → T transition (or G → A on the opposite strand) accounts for at least one-third of point mutations associated with polymorphisms and mutations. Many of the MSH2 mutations in HNPCC, for example, involve CpG sequences. In addition to the fact that certain types of mutations (C → T or G → A) are relatively common, the nature of the genetic code also results in overrepresentation of certain amino acid substitutions.
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 mental retardation. Other disorders arising from a similar mechanism include Huntington's disease (Chap. 371), X-linked spinobulbar muscular atrophy (Chap. 374), and myotonic dystrophy (Chap. 387). Malignant cells are also characterized by genetic instability, indicating a breakdown in mechanisms that regulate DNA repair and the cell cycle.
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 (i.e., 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 61-2). 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. Haploinsufficiency can also affect the expression of rate-limiting enzymes. For example, haploinsufficiency in enzymes involved in heme synthesis can cause porphyrias (Chap. 358).
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. 349). 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. 363); (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 binds to target genes and functions as an antagonist of normal receptors (Chap. 341); or (3) a mutant protein can be cytotoxic as in α1 antitrypsin deficiency (Chap. 260) or autosomal dominant neurohypophyseal diabetes insipidus (Chap. 340), in which the abnormally folded proteins are trapped within the endoplasmic reticulum and ultimately cause cellular damage.
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 the adaptive advantages associated with genetic diversity. On the other hand, allelic variants may reflect mutations in a gene that clearly alter its function. 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 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. If the alleles are different on the maternal and the paternal copy of the gene, the individual is heterozygous at this locus (Fig. 61-6). 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.
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.
A haplotype refers to a group of alleles that are closely linked together at a genomic locus (Fig. 61-8). 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. 61-3). 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 vicinity to the HLA locus, which explains why HLA associations were identified even before the disease genes were cloned and localized. In other cases, specific HLA associations with diseases such as ankylosing spondylitis (HLA-B27) or type 1 diabetes mellitus (HLA-DR4) reflect the role of specific HLA allelic variants in susceptibility to these autoimmune diseases. The characterization of common SNP haplotypes in numerous populations from different parts of the world through the HapMap project is providing a novel tool for association studies designed to detect genes involved in the pathogenesis of complex disorders (Table 61-1). The presence or absence of certain haplotypes may also become relevant for the customized choice of medical therapies (pharmacogenomics) or for preventive strategies.
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 61-4) (Fig. 61-5). In essence, allelic heterogeneity reflects the fact that many different mutations are capable of altering protein structure and function. For this reason, maps of inactivating mutations in genes usually show a near-random distribution. Exceptions include (1) a founder effect, in which a particular mutation that does not affect reproductive capacity can be traced to a single individual; (2) "hot spots" for mutations, in which the nature of the DNA sequence predisposes to a recurring mutation; and (3) localization of mutations to certain domains that are particularly critical for protein function. Allelic heterogeneity creates a practical problem for genetic testing because one must often examine the entire genetic locus for mutations, as these can differ in each patient. For example, there are currently 1795 reported mutations in the CFTR gene (Fig. 61-7). The mutational analysis initially focuses 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 should be interpreted with caution. It is expected that the advent of more comprehensive sequencing technologies will greatly facilitate mutational analyses. It will, however, also introduce challenges because the detection of a sequence alteration alone is not always sufficient to establish that it has a causal role.
Table 61-4 Selected Examples of Locus Heterogeneity and Phe-Notypic Heterogeneity |Favorite Table|Download (.pdf)
Table 61-4 Selected Examples of Locus Heterogeneity and Phe-Notypic Heterogeneity
|LMNA, Lamin A/C||Emery-Dreifuss muscular dystrophy (AD)||AD||181350|
|Familial partial lipodystrophy Dunnigan||AD||151660|
|Atypical Werner's syndrome||AD||150330|
|Early-onset atrial fibrillation||AD||607554|
|Emery-Dreifuss muscular dystrophy (AR)||AR||604929|
|Limb-girdle muscular dystrophy type 1B||AR||159001|
|Charcot-Marie-Tooth type 2B1||AR||605588|
|Familial hypertrophic cardiomyopathy|
Myosin heavy chain beta
| Genes encoding sarcomeric proteins|
Myosin-binding protein C
Myosin light chain 2
Myosin light chain 3
Cardiac alpha actin
Myosin heavy chain alpha
Myosin light-peptide kinase
| Genes encoding nonsarcomeric proteins||MTT1||Mitochondrial||tRNA isoleucine|
|PRKAG2||7q35-q36||AMP-activated protein kinase γ2 subunit|
|DMPK||19q13.2-13.3||Myotonin protein kinase (myotonic dystrophy)|
|FRDA||9q13||Frataxin (Friedreich ataxia)|
|Polycystic kidney disease||PKD1||16p13.3-13.12||Polycystin 1 (AD)|
|PKD2||4q21.-23||Polycystin 2 (AD)|
|Noonan syndrome||PTPN11||12q24.1||Protein-tyrosine phosphatase 2c|
Phenotypic heterogeneity occurs when more than one phenotype is caused by allelic mutations (i.e., different mutations in the same gene) (Table 61-4). 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. Similarly, identical mutations in the FGFR2 gene can result in very distinct phenotypes: Crouzon's syndrome (craniofacial synostosis) or Pfeiffer's syndrome (acrocephalopolysyndactyly).
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. 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 (Chap. 363). The effects of inactivating mutations in these two genes are similar because the protein products comprise different subunits of the helical collagen fiber. Similarly, muscular dystrophy syndromes can be caused by mutations in various genes, consistent with the fact that it can be transmitted in an X-linked (Duchenne or Becker), autosomal dominant (limb-girdle muscular dystrophy type 1), or autosomal recessive (limb-girdle muscular dystrophy type 2) manner (Chap. 387). Mutations in the X-linked DMD gene, which encodes dystrophin, are the most common cause of muscular dystrophy. This feature reflects the large size of the gene as well as the fact that the phenotype is expressed in hemizygous males because they have only a single copy of the X chromosome. Dystrophin is associated with a large protein complex linked to the membrane-associated cytoskeleton in muscle. Mutations in several different components of this protein complex can also cause muscular dystrophy syndromes. Although the phenotypic features of some of these disorders are distinct, the phenotypic spectrum caused by mutations in different genes overlaps, thereby leading to nonallelic heterogeneity. It should be noted that mutations in dystrophin 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.
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.
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. 371). Children born with activating mutations of the thyroid-stimulating hormone receptor (TSH-R) exhibit goiter and thyrotoxicosis similar to that seen in neonatal Graves' disease, which is caused by the transfer of maternal autoantibodies to the fetus (Chap. 341). 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.
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., 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. Families with this autosomal dominant disorder develop tumors of the parathyroid gland, endocrine pancreas, and the pituitary gland (Chap. 351). 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 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.
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. 357), and the course of phenylketonuria is affected by exposure to phenylalanine in the diet (Chap. 364). 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, as one cannot always predict the course of disease, even when the mutation is known.
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 have any 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. 237). Patients who have the mutation but no evidence of the disease can still transmit the disorder to subsequent generations. In many conditions with postnatal onset, the proportion of gene carriers who are affected varies with age. Thus, when describing penetrance, one has to specify age. For example, for disorders such as Huntington's disease or familial amyotrophic lateral sclerosis, which present late 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 (see below). 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. 353).
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. 116). 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. 361). 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. 349).
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. 346). 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. 346). 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. 342). 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. 357).
Chromosomal or cytogenetic disorders are caused by numerical or structural aberrations in chromosomes. Deviations in chromosome number are common causes of abortions, developmental disorders, and malformations. Contiguous gene syndromes (i.e., 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.
For discussion of disorders of chromosome number and structure, see Chap. 62.
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 61-1). 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. 61-9). The principles of allelic segregation, and the transmission of alleles from parents to children, are illustrated in Fig. 61-10. One dominant (A) allele and one recessive (a) allele can display three Mendelian modes of inheritance: autosomal dominant, autosomal recessive, and X-chromosomal. 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. 63).
Standard pedigree symbols.
Segregation of alleles. Segregation of genotypes in 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 represent affected individuals.
Autosomal Dominant Disorders
Autosomal dominant 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.
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. 61-11A). Autosomal dominant mutations alter one of the two alleles at a given locus. Because the alleles segregate randomly at meiosis, the probability that an offspring will be affected is 50%. Unless there is a new germline mutation, an affected individual has an affected parent. Children with a normal genotype do not transmit the disorder. Due to differences in penetrance or expressivity (see above), the clinical manifestations of autosomal dominant disorders may be variable. Because of these variations, it is sometimes challenging to determine the pattern of inheritance.
Dominant, recessive, X-linked, and mitochondrial (matrilinear) 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.
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). Though heterozygous carriers of a defective allele are usually clinically normal, they may display subtle differences in phenotype that only become apparent with more precise testing or in the context of certain environmental influences. In sickle cell anemia, for example, heterozygotes are normally asymptomatic. However, in situations of dehydration or diminished oxygen pressure, sickle cell crises can also occur in heterozygotes (Chap. 104).
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. 61-10, 61-11B). 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.
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. 61-11C). The risk of developing disease due to a mutant X-chromosomal gene differs in the two sexes. Because males have only one X chromosome, they are hemizygous for the mutant allele; thus, they are more likely to develop the mutant phenotype, regardless of whether the mutation is dominant or recessive. A female may be either heterozygous or homozygous for the mutant allele, which may be dominant or recessive. The terms X-linked dominant or X-linked recessive are therefore only applicable to expression of the mutant phenotype in women. In addition, the expression of X-chromosomal genes is influenced by X chromosome inactivation (see below).
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 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. 349). Point mutations in the SRY gene may also result in individuals with an XY genotype and an incomplete female phenotype. Most of these mutations occur de novo. Men with oligospermia/azoospermia frequently have microdeletions on the long arm of the Y chromosome that involve one or more of the azoospermia factor (AZF) genes.
Exceptions to Simple Mendelian Inheritance Patterns
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. The mitochondrial DNA (mtDNA) is ∼16.5 kb and encodes transfer and ribosomal RNAs and 13 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.
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. 61-11D). Alterations in the mtDNA affecting 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 (Tables 61-1 and 61-5). The broad clinical spectrum often involves (cardio)myopathies and encephalopathies because of the high dependence of these tissues on oxidative phosphorylation. The age of onset and the clinical course are highly variable because of the unusual mechanisms of mtDNA transmission, which replicates independently from nuclear DNA. During cell replication, the proportion of wild-type and mutant mitochondria can drift among different cells and tissues. The resulting heterogeneity in the proportion of mitochondria with and without a mutation is referred to as heteroplasmia and underlies the phenotypic variability that is characteristic of mitochondrial diseases.
Table 61-5 Selected Mitochondrial Diseases |Favorite Table|Download (.pdf)
Table 61-5 Selected Mitochondrial Diseases
|MELAS syndrome: mitochondrial myopathy with encephalopathy, lactacidosis, and stroke||540000|
|Leber's optic atrophy: hereditary optical neuropathy||535000|
|Kearns-Sayre syndrome (KSS): ophthalmoplegia, pigmental degeneration of the retina, cardiomyopathy||530000|
|MERRF syndrome: myoclonic epilepsy and ragged-red fibers||545000|
|Neurogenic muscular weakness with ataxia and retinitis pigmentosa (NARP)||551500|
|Chronic progressive external ophthalmoplegia (CEOP)||258470|
|Pearson's syndrome (PEAR): bone marrow and pancreatic failure||557000|
|Autosomal dominant inherited mitochondrial myopathy with mitochondrial deletion (ADMIMY)||157640|
|Somatic mutations in cytochrome b gene: exercise intolerance, lactic acidosis, complex III deficiency, muscle pain, ragged-red fibers||516020|
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.
Mosaicism refers to the presence of two or more genetically distinct cell lines in the tissues of an individual. It results from a mutation that occurs during embryonic, fetal, or extrauterine development. The developmental stage at which the mutation arises will determine whether germ cells and/or somatic cells are involved. Chromosomal mosaicism results from nondisjunction at an early embryonic mitotic division, leading to the persistence of more than one cell line, as exemplified by some patients with Turner's syndrome (Chap. 349). 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. 353). 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. 347).
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 also occurs on selected chromosomal regions of autosomes. This phenomenon, referred to as genomic imprinting, 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. 62). 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).
Imprinting and the related phenomenon of allelic exclusion may be more common than currently documented, as 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. 62). 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. 83). These children show somatic overgrowth with organomegalies and hemihypertrophy, and they have an increased risk of embryonal malignancies such as Wilms' tumor. Normally, only the paternally derived copy of the IGF-II gene is active and the maternal copy is inactive. Imprinting of the IGF-II gene is regulated by H19, which encodes an RNA transcript that is not translated into protein. Disruption or lack of H19 methylation leads to a relaxation of IGF-II imprinting and expression of both alleles.
Meiotically and mitotically heritable changes in gene expression not associated with DNA sequence alterations are referred to as epigenetic effects. These changes involve DNA methylation, histone modifications, and RNA-mediated silencing, resulting in gene repression without a change in the coding sequence. Epigenetic alterations are increasingly recognized to play a role in human diseases such as cancer, mental retardation, hematologic disorders, and possibly in aging. For example, de novo methylation of CpG islands, regions of >500 bp in size with a GC content >55% in promoter regions that are normally unmethylated, is a hallmark of human cancers. Inhibitors of enzymes controlling epigenetic modifications such as histone deacetylases and DNA methyltransferases reverse gene silencing and represent a promising new group of antineoplastic agents.
Cancer can be defined as a genetic disease at the cellular level (Chap. 83). Cancers are monoclonal in origin, indicating that they have arisen from a single precursor cell with one or several mutations in genes controlling growth (proliferation or apoptosis) and/or differentiation. These acquired somatic mutations are restricted to the tumor and its metastases and are not found in the surrounding normal tissue. The molecular alterations include dominant gain-of-function mutations in oncogenes, recessive loss-of-function mutations in tumor-suppressor genes and DNA repair genes, gene amplification, and chromosome rearrangements. Rarely, a single mutation in certain genes may be sufficient to transform a normal cell into a malignant cell. In most cancers, however, the development of a malignant phenotype requires several genetic alterations for the gradual progression from a normal cell to a cancerous cell, a phenomenon termed multistep carcinogenesis (Chaps. 83 and 84). Genomewide analyses of cancers using deep sequencing often reveal somatic rearrangements and mutations in multiple genes. 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 may provide a novel strategy for treating advanced human cancers.
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, though tumorigenesis results from a biallelic loss of the tumor-suppressor gene in an affected tissue. The classic example to illustrate this phenomenon is retinoblastoma, which can occur as a sporadic or hereditary tumor. In sporadic retinoblastoma, both copies of the retinoblastoma (RB) gene are inactivated through two somatic events. In hereditary retinoblastoma, one mutated or deleted RB allele is inherited in an autosomal dominant manner and the second allele is inactivated by a subsequent somatic mutation. This two-hit model applies to other inherited cancer syndromes such as MEN-1 (Chap. 351) and neurofibromatosis type 2 (Chap. 379).
Nucleotide Repeat Expansion Disorders
Several diseases are associated with an increase in the number of nucleotide repeats above a certain threshold (Table 61-6). 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. 366). Anticipation has also been documented in other diseases caused by dynamic mutations in trinucleotide repeats (Table 61-6). 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. 387).
Table 61-6 Selected Trinucleotide Repeat Disorders |Favorite Table|Download (.pdf)
Table 61-6 Selected Trinucleotide Repeat Disorders
|Disease||Locus||Repeat||Triplet Length (Normal/Disease)||Inheritance||Gene Product|
|X-chromosomal spinobulbar muscular atrophy (SBMA)||Xq11-q12||CAG||11–34/40–62||XR||Androgen receptor|
|Fragile X-syndrome (FRAXA)||Xq27.3||CGG||6–50/200–300||XR||FMR-1 protein|
|Fragile X-syndrome (FRAXE)||Xq28||GCC||6–25/>200||XR||FMR-2 protein|
|Dystrophia myotonica (DM)||19q13.2-q13.3||CTG||5–30/200–1000||AD, variable penetrance||Myotonin protein kinase|
|Huntington's disease (HD)||4p16.3||CAG||6–34/37–180||AD||Huntingtin|
|Spinocerebellar ataxia type 1 (SCA1)||6p21.3-21.2||CAG||6–39/40–88||AD||Ataxin 1|
|Spinocerebellar ataxia type 2 (SCA2)||12q24.1||CAG||15–31/34–400||AD||Ataxin 2|
|Spinocerebellar ataxia type 3 (SCA3); Machado-Joseph disease (MD)||14q21||CAG||13–36/55–86||AD||Ataxin 3|
|Spinocerebellar ataxia type 6 (SCA6, CACNAIA)||19p13.1-13.2||CAG||4–16/20–33||AD||Alpha 1A voltage-dependent L-type calcium chan-nel|
|Spinocerebellar ataxia type 7 (SCA7)||3p21.1-p12||CAG||4–19/37 to >300||AD||Ataxin 7|
|Spinocerebellar ataxia type 12 (SCA12)||5q31||CAG||6–26/66–78||AD||Protein phosphatase 2A|
|Dentorubral pallidoluysian atrophy (DRPLA)||12p||CAG||7–23/49–75||AD||Atrophin 1|
|Friedreich ataxia (FRDA1)||9q13-21||GAA||7–22/200–900||AR||Frataxin|
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 modifying 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.
Type 2 diabetes mellitus provides a paradigm for considering a multifactorial disorder, as genetic, nutritional, and lifestyle factors are intimately interrelated in disease pathogenesis (Table 61-7) (Chap. 344). 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 genetic and molecular mechanisms important in the pathogenesis of complex diseases. For example, the identification of the hepatocyte nuclear factor 1α (HNF1α) in maturity-onset of diabetes type 4 defined it as a candidate gene in the pathogenesis of diabetes mellitus type 2 (Tables 61-2 and 61-7). Genome scans have identified various 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 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.
Table 61-7 Genes and Loci Involved in Mono- and Polygenic Forms of Diabetes |Favorite Table|Download (.pdf)
Table 61-7 Genes and Loci Involved in Mono- and Polygenic Forms of Diabetes
|Disorder||Genes or Susceptibility Locus||Chromosomal Location||Other Factors|
|Monogenic forms of diabetes|
|MODY 1||HNF4α (hepatocyte nuclear factor 4α)||20q12-q13.1||AD inheritance|
|MODY 2||GCK (glucokinase)||7p15-p13|
|MODY 3||HNF1α (hepatocyte nuclear factor 1α)||12q24.2|
|MODY 4||IPF1 (insulin receptor substrate)||13q12.1|
|MODY 5 (renal cysts, diabetes)||HNF1β (hepatocyte nuclear factor 1β)||17cen-q21.3|
|MODY 6||NeuroD1 (neurogenic differention factor 1)||2q32|
|Diabetes mellitus type 2; loci and genes linked and/or associated with susceptibility for diabetes mellitus type 2||Genes and loci identified by linkage/association studies|
|HNF4α (hepatocyte nuclear factor 4α)||20q12-q13.1||Energy expenditure|
|PTPN1 (protein-tyrosine phosphatase)||20q13.1-q13.2||Obesity|
|PKLR (liver pyruvate kinase)||1q21|
|CASQ1 (calsequestrin 1)||1q21|
|TCF7L2 (transcription factor 7-like 2)||10q25.3|
|Selected candidate genes with possible contribution|
|PPARγ (Peroxisome proliferator receptor γ)||3p25|
|KCNJ11(ATP-sensitive K channel Kir6.2)||11p15.1|
|ABCC8 (ATP-binding cassette, subfamily c, member 8)||11p15.1|
|IRS-1 (insulin receptor substrate)||2q36|
|PGC1α (PPAR γ coactivator α)||4p15.1|
|ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1)||6q22-23|
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 61-8).
Table 61-8 Genetic Approaches for Identifying Disease Genes |Favorite Table|Download (.pdf)
Table 61-8 Genetic Approaches for Identifying Disease Genes
|Method||Indications and Advantages||Limitations|
|Classical linkage analysis (parametric methods)|
Analysis of monogenic traits
Suitable for genome scan
Control population not required
Useful for multifactorial disorders in isolated populations
Difficult to collect large informative pedigrees
Difficult to obtain sufficient statistical power for complex traits
Allele-sharing methods (nonparametric methods)
Affected sib and relative pair analyses
Sib pair analysis
Suitable for identification of susceptibility genes in polygenic and multifactorial disorders
Suitable for genome scan
Control population not required if allele frequencies are known
Statistical power can be increased by including parents and relatives
Difficult to collect sufficient number of subjects
Difficult to obtain sufficient statistical power for complex traits
Reduced power compared to classical linkage, but not sensitive to specification of genetic mode
Transmission disequilibrium test (TDT)
Whole-genome association studies
Suitable for identification of susceptibility genes in polygenic and multifactorial disorders
Suitable for testing specific allelic variants of known candidate loci
Facilitated by HapMap data, making GWAS more feasible
Does not necessarily need relatives
Requires large sample size and matched control population
False-positive results in the absence of suitable control population
Candidate gene approach does not permit to detect novel genes and pathways
Whole-genome association studies very expensive
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. 61-3 and 61-7); 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.
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 measured readily using PCR (Fig. 61-12). Characterization of SNPs, using DNA chips or beads, permit comprehensive analyses of genetic variation, linkage, and association studies. Although these sequence variations usually have no apparent functional consequences, they provide much of the basis for variation in genetic traits.
CAG repeat length and linkage analysis in multiple endocrine neoplasia (MEN) type 1. Upper panel. Detection of different alleles using polymorphic microsatellite markers. The example depicts a CAG trinucleotide repeat. PCR with primers flanking the polymorphic region results in products of variable length, depending on the number of CAG repeats. After characterization of the alleles in the parents, transmission of the paternal and maternal alleles can be determined. Lower panel. Genotype analysis using microsatellite markers in a family with MEN-1. Two microsatellite markers, A and B, are located in close proximity to the MEN1 gene on chromosome 11q13. For each individual, the A and B alleles have been determined. Based on this analysis, the genotype A3,B4 is linked to the disease because it occurs in the two affected individuals I-1 and II-2 but not in unaffected siblings. Because the disease allele is linked to A3,B4 within the affected family, it is likely that the individual III-1 is a carrier of the mutated MEN1 gene. Although III-5 also has the A3,B4 genotype, she has inherited the allele from her unaffected father (II-4), who is not related to the original family. The A3,B4 genotype is only associated with MEN-1 in the original family, but not in the general population. Therefore, individual III-5 is not at risk for developing the disease.
In order to identify a chromosomal locus that segregates with a disease, it is necessary to characterize polymorphic DNA markers from affected and unaffected individuals of one or several pedigrees. One can then assess whether certain marker alleles cosegregate with the disease. Markers that are closest to the disease gene are less likely to undergo recombination events and therefore receive a higher linkage score. Linkage is expressed as a lod (logarithm of odds) score'the ratio of the probability that the disease and marker loci are linked rather than unlinked. Lod scores of +3 (1000:1) are generally accepted as supporting linkage, whereas a score of –2 is consistent with the absence of linkage.
An example of the use of linkage analysis is shown in Fig. 61-12. In this case, the gene for the autosomal dominant disorder MEN-1 is known to be located on chromosome 11q13. Using positional cloning, the MEN1 gene was identified and shown to encode menin, a tumor suppressor. Affected individuals inherit a mutant form of the MEN1 gene, predisposing them to certain types of tumors (parathyroid, pituitary, pancreatic islet) (Chap. 351). In the tissues that develop a tumor, a "second hit" occurs in the normal copy of the MEN1 gene. This somatic mutation may be a point mutation, a microdeletion, or loss of a chromosomal fragment (detected as loss of heterozygosity, LOH). Within a given family, linkage to the MEN1 gene locus can be assessed without necessarily knowing the specific mutation in the MEN1 gene. Using polymorphic STRs that are close to the MEN1 gene, one can assess transmission of the different MEN1 alleles and compare this pattern to development of the disorder to determine which allele is associated with risk of MEN-1. In the pedigree shown, the affected grandfather in generation I carries alleles 3 and 4 on the chromosome with the mutated MEN1 gene and alleles 2 and 2 on his other chromosome 11. Consistent with linkage of the 3/4 genotype to the MEN1 locus, his son in generation II is affected, whereas his daughter (who inherits the 2/2 genotype from her father) is unaffected. In the third generation, transmission of the 3/4 genotype indicates risk of developing MEN-1, assuming that no genetic recombination between the 3/4 alleles and the MEN1 gene has occurred. After a specific mutation in the MEN1 gene is identified within a family, it is possible to track transmission of the mutation itself, thereby eliminating uncertainty caused by recombination.
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.
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.
Detecting the genetic factors contributing to the pathogenesis of common complex disorders remains a great challenge. In many instances, these are low-penetrancealleles (i.e., variations that individually only have a subtle effect on disease development, and they can only be identified by unbiased GWAS). Most variants are 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 selected marker SNPs, so-called Tag SNPs, thereby reducing cost and workload (Fig. 61-8). 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 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.
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.
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. Though homozygosity for the sickle cell gene is associated with severe anemia and sickle crises (Chap. 104), 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.
Approach to the Patient: Inherited Disorders
For the practicing clinician, the family history remains an essential step in recognizing the possibility of a hereditary component. When taking the history, it is useful to draw a detailed pedigree of the first-degree relatives (e.g., parents, siblings, and children), since they share 50% of genes with the patient. Standard symbols for pedigrees are depicted in Fig. 61-9. The family history should include information about ethnic background, age, health status, and (infant) deaths. 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 will have to assess whether some of the patient's relatives may be at risk of carrying or transmitting the disease. In this circumstance, it is useful to confirm and extend the pedigree based on input from several family members. This information may form the basis for carrier detection, genetic counseling, early intervention, and prevention of a disease in relatives of the index patient (Chap. 63).
In instances where a diagnosis at the molecular level may be relevant, the physician will have to identify an appropriate laboratory that can perform the 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 on-line resources (GeneTests; Table 61-1). If genetic testing is considered, the patient and the family should be informed about the potential implications of positive results, including psychological distress and the possibility of discrimination. The patient or caretakers should be informed about the meaning of a negative result, technical limitations, and the possibility of false-negative and inconclusive results. For these reasons, genetic testing should only be performed after obtaining informed consent. Published ethical guidelines address the specific aspects that should be considered when testing children and adolescents. Genetic testing should usually be limited to situations in which the results may have an impact on the medical management.
Identifying the Disease-Causing Gene
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.
DNA testing is performed by mutational analysis or linkage studies in individuals at risk for a genetic disorder known to be present in a family. Mass screening programs require tests of high sensitivity and specificity to be cost effective. Prerequisites for the success of genetic screening programs include the following: that the disorder is potentially serious; that it can be influenced at a presymptomatic stage by changes in behavior, diet, and/or pharmaceutical manipulations; and that the screening does not result in any harm or discrimination. Screening in Jewish populations for the autosomal recessive neurodegenerative storage disease Tay-Sachs has reduced the number of affected individuals. In contrast, screening for sickle cell trait/disease in African Americans has led to unanticipated problems of discrimination by health insurers and employers. Mass screening programs harbor additional potential problems. For example, screening for the most common genetic alteration in cystic fibrosis, the ΔF508 mutation with a frequency of ∼70% in northern Europe is feasible and seems to be effective. One has to keep in mind, however, that there is pronounced allelic heterogeneity and that the disease can be caused by >1700 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 will permit 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 deficiency and smoke or dust exposure).
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. 63). Numerous techniques are available for the detection of mutations (Table 61-9). In a very broad sense, one can distinguish between techniques that allow for screening for the absence or presence 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. 62), as well as more sensitive novel techniques that search for multiple single exon deletions or duplications.
Table 61-9 Techniques Commonly Used for Mutation Detection |Favorite Table|Download (.pdf)
Table 61-9 Techniques Commonly Used for Mutation Detection
|Method||Principle||Type of Mutation Detected|
|Cytogenetic analysis||Unique visual appearance of various chromosomes||Numerical or structural abnormalities in chromosomes|
|Fluorescent in situ hybridization (FISH)||Hybridization to chromosomes with fluorescently labeled probes||Numerical or structural abnormalities in chromosomes|
|Southern blot||Hybridization with genomic probe or cDNA probe after digestion of high-molecular-weight DNA||Large deletion, insertion, rearrangement, expansions of triplet repeat, amplification|
|Polymerase chain reaction (PCR)||Amplification of DNA segment||Expansion of triplet repeats, variable number of tandem repeats (VNTR), gene rearrangements, translocations; prepare DNA for other mutation methods|
|Reverse transcriptase PCR (RT-PCR)||Reverse transcription, amplification of DNA segment → absence or reduction of mRNA transcription||Analyze expressed mRNA (cDNA) sequence; detect loss of expression|
|Traditional DNA sequencing|
Direct sequencing of PCR products
Sequencing of DNA segments cloned into plasmid vectors
|Point mutations, small deletions and insertions|
|Next-generation DNA sequencing||Sequencing of large contiguous genomic regions, exome of single or all chromosomes.||Deep sequencing for detection of mutations and SNPs. Sequencing of whole genomes of microorganisms|
|Microarrays||Hybridization of PCR products to wild-type or mutated oligonucleotides|
Point mutations, small deletions and insertions
Genotyping of SNPs
More discrete sequence alterations rely heavily on the use of the 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.
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 currently 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 of an individual is expected to cost about $1000 within this decade. 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 for data handling.
The huge amount of data generated by massive parallel sequencing underscores the need for ongoing attention to ethical and legal concerns. They include, among others, issues related to consent, sequence interpretation, the discovery of incidental findings that are predictors of serious disorders, the ability to link the information to an individual despite the absence of any conventional personal identifier, and data storage.
A general algorithm for the approach to mutational analysis is outlined in Fig. 61-13. 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.
Approach to genetic disease.
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 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. In combination with in vitro fertilization (IVF) techniques, it is even possible to perform genetic diagnoses in a single cell removed from the four- to eight-cell embryo or to analyze the first polar body from an oocyte. Preconceptual diagnosis thereby avoids therapeutic abortions but is extremely costly and labor intensive. Lastly, it has to 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 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 1/300 individuals. Patients with intermediate or deficient TPMT activity are at risk for excessive toxicity, including fatal myelosuppression. Characterization of these polymorphisms allows mercaptopurine doses to be modified based on TPMT genotype. Pharmacogenomics may increasingly permit individualized drug therapy, improve drug effectiveness, reduce adverse side effects, and provide cost-effective pharmaceutical care.