Chromosome Disorders

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Part 3: Genes, the Environment, and Disease



Principles of Human Genetics

J. Larry Jameson, Peter Kopp



The prevalence of genetic diseases, combined with their potential severity and chronic nature, imposes great human, social, and financial burdens on society. Human genetics refers to the study of individual genes, their role and function in disease, and their mode of inheritance. Genomics refers to an organism’s entire genetic information, the genome, and the function and interaction of DNA within the genome, as well as with environmental or nongenetic factors, such as a person’s lifestyle. With the characterization of the human genome, genomics complements traditional genetics in our efforts to elucidate the etiology and pathogenesis of disease and to improve therapeutic interventions and outcomes. Following impressive advances in genetics, genomics, and health care information technology, the consequences of this wealth of knowledge for the practice of medicine are profound and play an increasingly prominent role in the diagnosis, prevention, and treatment of disease (Chap. 84).

Personalized medicine, the customization of medical decisions to an individual patient, relies heavily on genetic information. For example, a patient’s genetic characteristics (genotype) can be used to optimize drug therapy and predict efficacy, adverse events, and drug dosing of selected medications (pharmacogenetics) (Chap. 5). The mutational profile of a malignancy allows the selection of therapies that target mutated or overexpressed signaling molecules. Although still investigational, genomic risk prediction models for common diseases are beginning to emerge.

Genetics has traditionally been viewed through the window of relatively rare single-gene diseases. These disorders account for ~10% of pediatric admissions and childhood mortality. Historically, genetics has focused predominantly on chromosomal and metabolic disorders, reflecting the long-standing availability of techniques to diagnose these conditions. For example, conditions such as trisomy 21 (Down’s syndrome) or monosomy × (Turner’s syndrome) can be diagnosed using cytogenetics (Chap. 83e). Likewise, many metabolic disorders (e.g., phenylketonuria, familial hypercholesterolemia) are diagnosed using biochemical analyses. The advances in DNA diagnostics have extended the field of genetics to include virtually all medical specialties and have led to the elucidation of the pathogenesis of numerous monogenic disorders. In addition, it is apparent that virtually every medical condition has a genetic component. As is often evident from a patient’s family history, many common disorders such as hypertension, heart disease, asthma, diabetes mellitus, and mental illnesses are significantly influenced by the genetic background. These polygenic or multifactorial (complex) disorders involve the contributions of many different genes, as well as environmental factors that can modify disease risk (Chap. 84). Genome-wide association studies (GWAS) have elucidated numerous disease-associated loci and are providing novel insights into the allelic architecture of complex traits. These studies have been facilitated by the availability of comprehensive catalogues of human single-nucleotide polymorphism (SNP) haplotypes generated through the HapMap Project. The sequencing of whole genomes or exomes (the exons within the genome) is increasingly used in the clinical realm in order to characterize individuals with complex undiagnosed conditions or to characterize the mutational profile of advanced malignancies in order to select better targeted therapies.

Cancer has a genetic basis because it results from acquired somatic mutations in genes controlling growth, apoptosis, and cellular differentiation (Chap. 101e). In addition, the development of many cancers is associated with a hereditary predisposition. Characterization of the genome (and epigenome) in various malignancies has led to fundamental new insights into cancer biology and reveals that the genomic profile of mutations is in many cases more important in determining the appropriate chemotherapy than the organ in which the tumor originates. Hence, comprehensive mutational profiling of malignancies has increasing impact on cancer taxonomy, the choice of targeted therapies, and improved outcomes.

Genetic and genomic approaches have proven invaluable for the detection of infectious pathogens and are used clinically to identify agents that are difficult to culture such as mycobacteria, viruses, and parasites, or to track infectious agents locally or globally. In many cases, molecular genetics has improved the feasibility and accuracy of diagnostic testing and is beginning to open new avenues for therapy, including gene and cellular therapy (Chaps. 90e and 91e). Molecular genetics has also provided the opportunity to characterize the microbiome, a new field that characterizes the population dynamics of bacteria, viruses, and parasites that coexist with humans and other animals (Chap. 86e). Emerging data indicate that the microbiome has significant effects on normal physiology as well as various disease states.

Molecular biology has significantly changed the treatment of human disease. Peptide hormones, growth factors, cytokines, and vaccines can now be produced in large amounts using recombinant DNA technology. Targeted modifications of these peptides provide the practitioner with improved therapeutic tools, as illustrated by genetically modified insulin analogues with more favorable kinetics. Lastly, there is reason to believe that a better understanding of the genetic basis of human disease will also have an increasing impact on disease prevention.

The astounding rate at which new genetic information is being generated creates a major challenge for physicians, health care providers, and basic investigators. Although many functional aspects of the genome remain unknown, there are many clinical situations where sufficient evidence exits for the use of genetic and genomic information to optimize patient care and treatment. Much genetic information resides in databases or is being published in basic science journals. Databases provide easy access to the expanding information about the human genome, genetic disease, and genetic testing (Table 82-1). For example, several thousand monogenic disorders are summarized in a large, continuously evolving compendium, referred to as the Online Mendelian Inheritance in Man (OMIM) catalogue (Table 82-1). The ongoing refinement of bioinformatics is simplifying the analysis and access to this daunting amount of new information.

TABLE 82-1




Structure of the Human GenomeHUMAN GENOME PROJECT The Human Genome Project was initiated in the mid-1980s as an ambitious effort to characterize the entire human genome. 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 2000 and the completion of the DNA sequence for the last of the human chromosomes in May 2006. Currently, facilitated by rapidly decreasing costs for comprehensive sequence analyses and improvement of bioinformatics pipelines for data analysis, the sequencing of whole genomes and exomes is used with increasing frequency in the clinical setting. The scope of a whole genome sequence analysis can be illustrated by the following analogy. Human DNA consists of ~3 billion base pairs (bp) of DNA per haploid genome, which is nearly 1000-fold greater than that of the Escherichia coli genome. If the human DNA sequence were printed out, it would correspond to about 120 volumes of Harrison’s Principles of Internal Medicine.

In addition to the human genome, the genomes of numerous organisms have been sequenced completely (~4000) or partially (~10,000) (Genomes Online Database [GOLD]; Table 82-1). They include, among others, eukaryotes such as the mouse (Mus musculus), Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster; bacteria (e.g., E. coli); and Archaea, viruses, organelles (mitochondria, chloroplasts), and plants (e.g., Arabidopsis thaliana). Genomic information of infectious agents has significant impact for the characterization of infectious outbreaks and epidemics. Other ramifications arising from the availability of genomic data 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) 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 (4) the identification of genes that play critical roles in the development of polygenic and multifactorial disorders.

CHROMOSOMES    The human genome is divided into 23 different chromosomes, including 22 autosomes (numbered 1–22) and the × and Y sex chromosomes (Fig. 82-1). Adult cells are diploid, meaning they contain two homologous sets of 22 autosomes and a pair of sex chromosomes. Females have two × chromosomes (XX), whereas males have one × 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.


FIGURE 82-1   Structure of chromatin and chromosomes. Chromatin is composed of double-strand DNA that is wrapped around histone and nonhistone 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.

STRUCTURE 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 (Fig. 82-1). DNA has several remarkable features that make it ideal for the transmission of genetic information. It is relatively stable, and the double-stranded nature of DNA and its feature of strict base-pair complementarity permit faithful replication during cell division. Complementarity also allows the transmission of genetic information from DNA → RNA → protein (Fig. 82-2). mRNA is encoded by the so-called sense or coding strand of the DNA double helix and is translated into proteins by ribosomes.


FIGURE 82-2   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. CBP, CREB-binding protein; CoA, co-activator; COOH, carboxyterminus; CRE, cyclic AMP responsive element; CREB, cyclic AMP response element–binding protein; GTF, general transcription factors; HAT, histone acetyl transferase; NH2, aminoterminus; RE, response element; TAF, TBP-associated factors; TATA, TATA box; TBP, TATA-binding protein.

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.

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 sequences, often of highly repetitive nature, the function of which is poorly understood. These repetitive DNA regions, along with nonrepetitive sequences that do not encode genes, serve, in part, a structural role in the packaging of DNA into chromatin (i.e., DNA bound to histone proteins, and chromosomes) and exert regulatory functions (Fig. 82-1).

GENES 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 (Fig. 82-3). 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 size of genes is quite broad; some genes are only a few hundred base pairs, whereas others are extraordinarily large (2 Mb). The number of genes greatly underestimates the complexity of genetic expression, because single genes can generate multiple spliced messenger RNA (mRNA) products (isoforms), which are translated into proteins that are subject to complex posttranslational modification such as phosphorylation. Exons refer to the portion of genes that are eventually spliced together to form mRNA. Introns refer to the spacing regions between the exons that are spliced out of precursor RNAs during RNA processing. The gene locus also includes regions that are necessary to control its expression (Fig. 82-2). Current estimates predict 20,687 protein-coding genes in the human genome with an average of about four different coding transcripts per gene. Remarkably, the exome only constitutes 1.14% of the genome. In addition, thousands of noncoding transcripts (RNAs of various length such as microRNAs and long noncoding RNAs), which function, at least in part, as transcriptional and posttranscriptional regulators of gene expression, have been identified. Aberrant expression of microRNAs has been found to play a pathogenic role in numerous diseases.


FIGURE 82-3   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 1900 mutations in this gene have been found in patients with cystic fibrosis. A 20-kb region encompassing exons 4–9 is shown further amplified to illustrate the SNPs in this region.

SINGLE-NUCLEOTIDE POLYMORPHISMS An SNP is a variation of a single base pair in the DNA. 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. 82-3). SNPs are the most common type of sequence variation and account for ~90% of all sequence variation. They occur on average every 100 to 300 bases and are the major source of genetic heterogeneity. Remarkably, however, the primary DNA sequence of humans has ~99.9% similarity compared to that of any other human. SNPs that are in close proximity are inherited together (e.g., they are linked) and are referred to as haplotypes (Fig. 82-4). The HapMap describes the nature and location of these SNP haplotypes and how they are distributed among individuals within and among populations. The haplotype map information, referred to as HapMap, is greatly facilitating GWAS designed to elucidate the complex interactions among multiple genes and lifestyle factors in multifactorial disorders (see below). Moreover, haplotype analyses are useful to assess variations in responses to medications (pharmacogenomics) and environmental factors, as well as the prediction of disease predisposition.


FIGURE 82-4   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 (SNPs), an approach that facilitates performing genome-wide association studies (GWAS).

COPY NUMBER VARIATIONS Copy number variations (CNVs) are relatively large genomic regions (1 kb to several Mb) that have been duplicated or deleted on certain chromosomes (Fig. 82-5). It has been estimated that as many as 1500 CNVs, scattered throughout the genome, are present in an individual. When comparing the genomes of two individuals, approximately 0.4–0.8% of their genomes differ in terms of CNVs. Of note, de novo CNVs have been observed between monozygotic twins, who otherwise have identical genomes. Some CNVs have been associated with susceptibility or resistance to disease, and CNVs can be elevated in cancer cells.


FIGURE 82-5   Copy number variations (CNV) encompass relatively large regions of the genome that have been duplicated or deleted. Chromosome 8 is shown with CNV detected by genomic hybridization. An increase in the signal strength indicates a duplication, a decrease reflects a deletion of the covered chromosomal regions.

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).

Prior to mitosis, cells exit the resting, or G0 state, and enter the cell cycle (Chap. 101e). 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. 101e). 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. 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.

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. 82-6). 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).


FIGURE 82-6   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.

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.


Regulation by Transcription Factors The expression of genes is regulated by DNA-binding proteins that activate or repress transcription. The number of DNA sequences and transcription factors that regulate transcription is much greater than originally anticipated. Most genes contain at least 15–20 discrete regulatory elements within 300 bp of the transcription start site. This densely packed promoter region often contains binding sites for ubiquitous transcription factors such as CAAT box/enhancer binding protein (C/EBP), cyclic AMP response element–binding (CREB) protein, selective promoter factor 1 (Sp-1), or activator protein 1 (AP-1). However, factors involved in cell-specific expression may also bind to these sequences. Key regulatory elements may also reside at a large distance from the proximal promoter. The globin and the immunoglobulin genes, for example, contain locus control regions that are several kilobases away from the structural sequences of the gene. Specific groups of transcription factors that bind to these promoter and enhancer sequences provide a combinatorial code for regulating transcription. In this manner, relatively ubiquitous factors interact with more restricted factors to allow each gene to be expressed and regulated in a unique manner that is dependent on developmental state, cell type, and numerous extracellular stimuli. Regulatory factors also bind within the gene itself, particularly in the intronic regions. The transcription factors that bind to DNA actually represent only the first level of regulatory control. Other proteins—co-activators and co-repressors—interact with the DNA-binding transcription factors to generate large regulatory complexes. These complexes are subject to control by numerous cell-signaling pathways and enzymes, leading to phosphorylation, acetylation, sumoylation, and ubiquitination. Ultimately, the recruited transcription factors interact with, and stabilize, components of the basal transcription complex that assembles at the site of the TATA box and initiator region. This basal transcription factor complex consists of >30 different proteins. Gene transcription occurs when RNA polymerase begins to synthesize RNA from the DNA template. A large number of identified genetic diseases involve transcription factors (Table 82-2).

TABLE 82-2



The field of functional genomics is based on the concept that understanding alterations of gene expression under various physiologic and pathologic conditions provides insight into the underlying functional role of the gene. By revealing specific gene expression profiles, this knowledge may be of diagnostic and therapeutic relevance. The large-scale study of expression profiles, which takes advantage of microarray and bead array technologies, is also referred to as transcriptomics because the complement of mRNAs transcribed by the cellular genome is called the transcriptome.

Most studies of gene expression have focused on the regulatory DNA elements of genes that control transcription. However, it should be emphasized that gene expression requires a series of steps, including mRNA processing, protein translation, and posttranslational modifications, all of which are actively regulated (Fig. 82-2).

Epigenetic Regulation of Gene Expression Epigenetics describes mechanisms and phenotypic changes that are not a result of variation in the primary DNA nucleotide sequence, but are caused by secondary modifications of DNA or histones. These modifications include heritable changes such as X-inactivation and imprinting, but they can also result from dynamic posttranslational protein modifications in response to environmental influences such as diet, age, or drugs. The epigenetic modifications result in altered expression of individual genes or chromosomal loci encompassing multiple genes. The term epigenome describes the constellation of covalent modifications of DNA and histones that impact chromatin structure, as well as noncoding transcripts that modulate the transcriptional activity of DNA. Although the primary DNA sequence is usually identical in all cells of an organism, tissue-specific changes in the epigenome contribute to determining the transcriptional signature of a cell (transcriptome) and hence the protein expression profile (proteome).

Mechanistically, DNA and histone modifications can result in the activation or silencing of gene expression (Fig. 82-7). DNA methylation involves the addition of a methyl group to cytosine residues. This is usually restricted to cytosines of CpG dinucleotides, which are abundant throughout the genome. Methylation of these dinucleotides is thought to represent a defense mechanism that minimizes the expression of sequences that have been incorporated into the genome such as retroviral sequences. CpG dinucleotides also exist in so-called CpG islands, stretches of DNA characterized by a high CG content, which are found in the majority of human gene promoters. CpG islands in promoter regions are typically unmethylated, and the lack of methylation facilitates transcription.


FIGURE 82-7   Epigenetic modifications of DNA and histones. Methylation of cytosine residues is associated with gene silencing. Methylation of certain genomic regions is inherited (imprinting), and it is involved in the silencing of one of the two × chromosomes in females (X-inactivation). Alterations in methylation can also be acquired, e.g., in cancer cells. Covalent posttranslational modifications of histones play an important role in altering DNA accessibility and chromatin structure and hence in regulating transcription. Histones can be reversibly modified in their amino-terminal tails, which protrude from the nucleosome core particle, by acetylation of lysine, phosphorylation of serine, methylation of lysine and arginine residues, and sumoylation. Acetylation of histones by histone acetylases (HATs), e.g., leads to unwinding of chromatin and accessibility to transcription factors. Conversely, deacetylation by histone deacetylases (HDACs) results in a compact chromatin structure and silencing of transcription.

Histone methylation involves the addition of a methyl group to lysine residues in histone proteins (Fig. 82-7). Depending on the specific lysine residue being methylated, this alters chromatin configuration, either making it more open or tightly packed. Acetylation of histone proteins is another well-characterized mechanism that results in an open chromatin configuration, which favors active transcription. Acetylation is generally more dynamic than methylation, and many transcriptional activation complexes have histone acetylase activity, whereas repressor complexes often contain deacetylases and remove acetyl groups from histones. Other histone modifications, whose effects are incompletely characterized, include phosphorylation and sumoylation. Lastly, noncoding RNAs that bind to DNA can have a significant impact on transcriptional activity.

Physiologically, epigenetic mechanisms play an important role in several instances. For example, X-inactivation refers to the relative silencing of one of the two × chromosome copies present in females. The inactivation process is a form of dosage compensation such that females (XX) do not generally express twice as many X-chromosomal gene products as males (XY). In a given cell, the choice of which chromosome is inactivated occurs randomly in humans. But once the maternal or paternal × chromosome is inactivated, it will remain inactive, and this information is transmitted with each cell division. The X-inactive specific transcript (Xist) gene encodes a large noncoding RNA that mediates the silencing of the × chromosome from which it is transcribed by coating it with Xist RNA. The inactive × chromosome is highly methylated and has low levels of histone acetylation.

Epigenetic gene inactivation also occurs on selected chromosomal regions of autosomes, a phenomenon referred to as genomic imprinting. Through this mechanism, a small subset of genes is only expressed in a monoallelic fashion. Imprinting is heritable and leads to the preferential expression of one of the parental alleles, which deviates from the usual biallelic expression seen for the majority of genes. Remarkably, imprinting can be limited to a subset of tissues. Imprinting is mediated through DNA methylation of one of the alleles. The epigenetic marks on imprinted genes are maintained throughout life, but during zygote formation, they are activated or inactivated in a sex-specific manner (imprint reset) (Fig. 82-8), which allows a differential expression pattern in the fertilized egg and the subsequent mitotic divisions. Appropriate expression of imprinted genes is important for normal development and cellular functions. Imprinting defects and uniparental disomy, which is the inheritance of two chromosomes or chromosomal regions from the same parent, are the cause of several developmental disorders such as Beckwith-Wiedemann syndrome, Silver-Russell syndrome, Angelman’s syndrome, and Prader-Willi syndrome (see below). Monoallelic loss-of-function mutations in the GNAS1 gene lead to Albright’s hereditary osteodystrophy (AHO). Paternal transmission of GNAS1 mutations leads to an isolated AHO phenotype (pseudopseudohypoparathyroidism), whereas maternal transmission leads to AHO in combination with hormone resistance to parathyroid hormone, thyrotropin, and gonadotropins (pseudohypoparathyroidism type IA). These phenotypic differences are explained by tissue-specific imprinting of the GNAS1 gene, which is expressed primarily from the maternal allele in the thyroid, gonadotropes, and the proximal renal tubule. In most other tissues, the GNAS1 gene is expressed biallelically. In patients with isolated renal resistance to parathyroid hormone (pseudohypoparathyroidism type IB), defective imprinting of the GNAS1 gene results in decreased Gsα expression in the proximal renal tubules. Rett’s syndrome is an X-linked dominant disorder resulting in developmental regression and stereotypic hand movements in affected girls. It is caused by mutations in the MECP2 gene, which encodes a methyl-binding protein. The ensuing aberrant methylation results in abnormal gene expression in neurons, which are otherwise normally developed.


FIGURE 82-8   A few genomic regions are imprinted in a parent-specific fashion. The unmethylated chromosomal regions are actively expressed, whereas the methylated regions are silenced. In the germline, the imprint is reset in a parent-specific fashion: both chromosomes are unmethylated in the maternal (mat) germline and methylated in the paternal (pat) germline. In the zygote, the resulting imprinting pattern is identical with the pattern in the somatic cells of the parents.

Remarkably, epigenetic differences also occur among monozygotic twins. Although twins are epigenetically indistinguishable during the early years of life, older monozygotic twins exhibit differences in the overall content and genomic distribution of DNA methylation and histone acetylation, which would be expected to alter gene expression in various tissues.

In cancer, the epigenome is characterized by simultaneous losses and gains of DNA methylation in different genomic regions, as well as repressive histone modifications. Hyper- and hypomethylation are associated with mutations in genes that control DNA methylation. Hypomethylation is thought to remove normal control mechanisms that prevent expression of repressed DNA regions. It is also associated with genomic instability. Hypermethylation, in contrast, results in the silencing of CpG islands in promoter regions of genes, including tumor-suppressor genes. Epigenetic alterations are considered to be more easily reversible compared to genetic changes, and modification of the epigenome with demethylating agents and histone deacetylases is being explored in clinical trials.


Several organisms have been studied extensively as genetic models, including M. musculus (mouse), D. melanogaster (fruit fly), C. elegans (nematode), S. cerevisiae (baker’s yeast), and E. coli (colonic bacterium). The ability to use these evolutionarily distant organisms as genetic models that are relevant to human physiology reflects a surprising conservation of genetic pathways and gene function. Transgenic mouse models have been particularly valuable, because many human and mouse genes exhibit similar structure and function and because manipulation of the mouse genome is relatively straightforward compared to that of other mammalian species. Transgenic strategies in mice can be divided into two main approaches: (1) expression of a gene by random insertion into the genome, and (2) deletion or targeted mutagenesis of a gene by homologous recombination with the native endogenous gene (knock-out, knock-in). Previous versions of this chapter provide more detail about the technical principles underlying the development of genetically modified animals. Several databases provide comprehensive information about natural and transgenic animal models, the associated phenotypes, and integrated genetic, genomic, and biologic data (Table 82-1).


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 may also 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. 83e). 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. 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.

Mutations can occur in all domains of a gene (Fig. 82-9). A point mutation occurring within the coding region leads to an amino acid substitution if the codon is altered (Fig. 82-10). Point mutations that introduce a premature stop codon result in a truncated protein. Large deletions may affect a portion of a gene or an entire gene, whereas small deletions and insertions alter the reading frame if they do not represent a multiple of three bases. These “frameshift” mutations lead to an entirely altered carboxy terminus. Mutations in intronic sequences or in exon junctions may destroy or create splice donor or splice acceptor sites. Mutations may also be found in the regulatory sequences of genes, resulting in reduced or enhanced gene transcription.


FIGURE 82-9   Point mutations causing β thalassemia as example of allelic heterogeneity. The β-globin gene is located in the globin gene cluster. Point mutations can be located in the promoter, the CAP site, the 5’-untranslated region, the initiation codon, each of the three exons, the introns, or the polyadenylation signal. Many mutations introduce missense or nonsense mutations, whereas others cause defective RNA splicing. Not shown here are deletion mutations of the β-globin gene or larger deletions of the globin locus that can also result in thalassemia. image, promoter mutations; *, CAP site; •, 5’UTR;images, initiation codon; ♦, defective RNA processing; images, missense and nonsense mutations; images, Poly A signal.


FIGURE 82-10   A. Examples of mutations. The coding strand is shown with the encoded amino acid sequence. B. Chromatograms of sequence analyses after amplification of genomic DNA by polymerase chain reaction.

Certain DNA sequences are particularly susceptible to mutagenesis. Successive pyrimidine residues (e.g., T-T or C-C) are subject to the formation of ultraviolet light–induced photoadducts. If these pyrimidine dimers are not repaired by the nucleotide excision repair pathway, mutations will be introduced after DNA synthesis. The dinucleotide C-G, or CpG, is also a hot spot for a specific type of mutation. In this case, methylation of the cytosine is associated with an enhanced rate of deamination to uracil, which is then replaced with thymine. This C → T transition (or G → A on the opposite strand) accounts for at least one-third of point mutations associated with polymorphisms and mutations. In addition to the fact that certain types of mutations (C → T or G → A) are relatively common, the nature of the genetic code also results in overrepresentation of certain amino acid substitutions.

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. 82-10). The detection of sequence variants poses a practical problem because it is often unclear whether it creates a mutation with functional consequences or a benign polymorphism. In this situation, the sequence alteration is described as variant of unknown significance (VUS).

MUTATION RATES 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. 83e). Thus, the incidence of new point mutations in spermatogonia increases with paternal age (e.g., achondrodysplasia, Marfan’s syndrome, neurofibromatosis). It is estimated that about 1 in 10 sperm carries a new deleterious mutation. The rates for new mutations are calculated most readily for autosomal dominant and X-linked disorders and are ~10–5–10–6/locus per generation. Because most monogenic diseases are relatively rare, new mutations account for a significant fraction of cases. This is important in the context of genetic counseling, because a new mutation can be transmitted to the affected individual but does not necessarily imply that the parents are at risk to transmit the disease to other children. An exception to this is when the new mutation occurs early in germline development, leading to gonadal mosaicism.

UNEQUAL CROSSING-OVER Normally, DNA recombination in germ cells occurs with remarkable fidelity to maintain the precise junction sites for the exchanged DNA sequences (Fig. 82-6). However, mispairing of homologous sequences leads to unequal crossover, with gene duplication on one of the chromosomes and gene deletion on the other chromosome. A significant fraction of growth hormone (GH) gene deletions, for example, involve unequal crossing-over (Chap. 402). 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. 459).

Glucocorticoid-remediable aldosteronism (GRA) is caused by a gene fusion or rearrangement involving the genes that encode aldosterone synthase (CYP11B2) and steroid 11β-hydroxylase (CYP11B1), normally arranged in tandem on chromosome 8q. These two genes are 95% identical, predisposing to gene duplication and deletion by unequal crossing-over. The rearranged gene product contains the regulatory regions of 11β-hydroxylase fused to the coding sequence of aldosterone synthetase. Consequently, the latter enzyme is expressed in the adrenocorticotropic hormone (ACTH)–dependent zona fasciculata of the adrenal gland, resulting in overproduction of mineralocorticoids and hypertension (Chap. 406).

Gene conversion refers to a nonreciprocal exchange of homologous genetic information. It has been used to explain how an internal portion of a gene is replaced by a homologous segment copied from another allele or locus; these genetic alterations may range from a few nucleotides to a few thousand nucleotides. As a result of gene conversion, it is possible for short DNA segments of two chromosomes to be identical, even though these sequences are distinct in the parents. A practical consequence of this phenomenon is that nucleotide substitutions can occur during gene conversion between related genes, often altering the function of the gene. In disease states, gene conversion often involves intergenic exchange of DNA between a gene and a related pseudogene. For example, the 21-hydroxylase gene (CYP21A2) is adjacent to a nonfunctional pseudogene (CYP21A1P). Many of the nucleotide substitutions that are found in the CYP21A2 gene in patients with congenital adrenal hyperplasia correspond to sequences that are present in the CYP21A1P pseudogene, suggesting gene conversion as one cause of mutagenesis. In addition, mitotic gene conversion has been suggested as a mechanism to explain revertant mosaicism in which an inherited mutation is “corrected” in certain cells. For example, patients with autosomal recessive generalized atrophic benign epidermolysis bullosa have acquired reverse mutations in one of the two mutated COL17A1 alleles, leading to clinically unaffected patches of skin.

INSERTIONS AND DELETIONS Although many instances of insertions and deletions occur as a consequence of unequal crossing-over, there is also evidence for internal duplication, inversion, or deletion of DNA sequences. The fact that certain deletions or insertions appear to occur repeatedly as independent events indicates that specific regions within the DNA sequence predispose to these errors. For example, certain regions of the DMD gene, which encodes dystrophin, appear to be hot spots for deletions and result in muscular dystrophy (Chap. 462e). Some regions within the human genome are rearrangement hot spots and lead to CNVs.

ERRORS IN DNA REPAIR 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. 102e). 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. 105). 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. 450). The discovery of the ataxia telangiectasia mutated (ATM) gene reveals that it is homologous to genes involved in DNA repair and control of cell cycle checkpoints. Mutations in the ATM gene give rise to defects in meiosis as well as increasing susceptibility to damage from ionizing radiation. Fanconi’s anemia is also associated with an increased risk of multiple acquired genetic abnormalities. It is characterized by diverse congenital anomalies and a strong predisposition to develop aplastic anemia and acute myelogenous leukemia (Chap. 132). Cells from these patients are susceptible to chromosomal breaks caused by a defect in genetic recombination. At least 13 different complementation groups have been identified, and the loci and genes associated with Fanconi’s anemia have been cloned. HNPCC (Lynch’s syndrome) is characterized by autosomal dominant transmission of colon cancer, young age (<50 years) of presentation, predisposition to lesions in the proximal large bowel, and associated malignancies such as uterine cancer and ovarian cancer. HNPCC is predominantly caused by mutations in one of several different mismatch repair (MMR) genes including MutS homologue 2 (MSH2), MutL homologue 1 and 6 (MLH1, MLH6), MSH6, PMS1, and PMS2 (Chap. 110). These proteins are involved in the detection of nucleotide mismatches and in the recognition of slipped-strand trinucleotide repeats. Germline mutations in these genes lead to microsatellite instability and a high mutation rate in colon cancer. Genetic screening tests for this disorder are now being used for families considered to be at risk (Chap. 84). Recognition of HNPCC allows early screening with colonoscopy and the implementation of prevention strategies using nonsteroidal anti-inflammatory drugs.

UNSTABLE DNA SEQUENCES Trinucleotide repeats may be unstable and expand beyond a critical number. Mechanistically, the expansion is thought to be caused by unequal recombination and slipped mispairing. A premutation represents a small increase in trinucleotide copy number. In subsequent generations, the expanded repeat may increase further in length and result in an increasingly severe phenotype, a process called dynamic mutation (see below for discussion of anticipation). Trinucleotide expansion was first recognized as a cause of the fragile × syndrome, one of the most common causes of intellectual disability. Other disorders arising from a similar mechanism include Huntington’s disease (Chap. 448), X-linked spinobulbar muscular atrophy (Chap. 452), and myotonic dystrophy (Chap. 462e). Malignant cells are also characterized by genetic instability, indicating a breakdown in mechanisms that regulate DNA repair and the cell cycle.

Functional Consequences of Mutations Functionally, mutations can be broadly classified as gain-of-function and loss-of-function mutations. Gain-of-function mutations are typically dominant (e.g., they result in phenotypic alterations when a single allele is affected). Inactivating mutations are usually recessive, and an affected individual is homozygous or compound heterozygous (e.g., carrying two different mutant alleles of the same gene) for the disease-causing mutations. Alternatively, mutation in a single allele can result in haploinsufficiency, a situation in which one normal allele is not sufficient to maintain a normal phenotype. Haploinsufficiency is a commonly observed mechanism in diseases associated with mutations in transcription factors (Table 82-2). Remarkably, the clinical features among patients with an identical mutation in a transcription factor often vary significantly. One mechanism underlying this variability consists in the influence of modifying genes. Haploinsufficiency can also affect the expression of rate-limiting enzymes. For example, haploinsufficiency in enzymes involved in heme synthesis can cause porphyrias (Chap. 430).

An increase in dosage of a gene product may also result in disease, as illustrated by the duplication of the DAX1 gene in dosage-sensitive sex reversal (Chap. 410). Mutation in a single allele can also result in loss of function due to a dominant-negative effect. In this case, the mutated allele interferes with the function of the normal gene product by one of several different mechanisms: (1) a mutant protein may interfere with the function of a multimeric protein complex, as illustrated by mutations in type 1 collagen (COL1A1, COL1A2) genes in osteogenesis imperfecta (Chap. 427); (2) a mutant protein may occupy binding sites on proteins or promoter response elements, as illustrated by thyroid hormone resistance, a disorder in which inactivated thyroid hormone receptor β binds to target genes and functions as an antagonist of normal receptors (Chap. 405); or (3) a mutant protein can be cytotoxic as in α1 antitrypsin deficiency (Chap. 314)

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