🧬 Part 7: Molecular, Biochemical, and Cellular Basis of Genetic Disease English

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Chapter 13: The Molecular Biochemical and Cellular Basis of Genetic Disease

Ch13 · Pt1 chapter 13 The Molecular, Biochemical, and Cellular Basis of Genetic Disease Ada Hamosh In this chapter we extend our examination of the molecular and biochemical basis of genetic disease beyond the hemoglobinopathies to include other diseases and the abnormalities in gene and protein function that cause them. In Chapter 12, we presented an outline of the general mechanisms by which pathogenic variants cause disease (see Fig. 12.1) and reviewed the steps at which they can disrupt the synthesis or function of a protein (see Table 12.1). Those outlines provide a framework for understanding the pathogenesis of all genetic disease. However, pathogenic variants in other classes of proteins often disrupt cell and organ function by processes that differ from those illustrated by the hemoglobinopathies, and we explore them in this chapter. To illustrate these other types of disease mechanisms, we now examine well-known disorders such as phenylketonuria (PKU), cystic fibrosis (CF), familial hypercholesterolemia, Duchenne muscular dystrophy (DMD), and Alzheimer disease (AD). Some less common disorders are included because they best demonstrate a specific principle. The importance of selecting representative disorders becomes apparent now that pathogenic variants in over 4500 genes have been associated with a clinical phenotype. One anticipates that many more of the ~20,000 protein coding genes in the human genome will be associated with both monogenic and genetically complex diseases. DISEASES DUE TO PATHOGENIC VARIANTS IN DIFFERENT CLASSES OF PROTEINS Proteins carry out an astounding number of functions, some of which are presented in Fig. 13.1. Pathogenic variants in virtually every functional class of protein can lead to genetic disorders. In this chapter we describe important genetic diseases that affect representative proteins selected from the groups shown in Fig. 13.1; other proteins listed, as well as the conditions associated with them, are described in the Cases section. Housekeeping Proteins and Specialty Proteins in Genetic Disease Proteins can be separated into two general classes on the basis of their pattern of expression. Housekeeping proteins are present in virtually every cell and have fundamental roles in the maintenance of cell structure and function; tissue-specific specialty proteins are produced in one or few cell types and have unique functions that contribute to the individuality of the cells in which they are expressed. Most cell types in humans express 10,000 to 15,000 protein-coding genes. Knowledge of the tissues in which a protein is expressed, particularly at high levels, may help in understanding the pathogenesis of a disease. Two broad generalizations can be made about the relationship between the site of a protein’s expression and the site of disease. First (and somewhat intuitively), pathogenic variants in a tissue-specific protein most often produce a disease restricted to that tissue. However, there may be secondary effects on other tissues. Pathogenic variants in tissue-specific proteins may cause abnormalities primarily in organs that do not express the protein at all; ironically, the tissue expressing the abnormal protein may be unaffected by the pathologic process. This situation is exemplified by PKU, discussed in depth in the next section. PKU is due to the absence of phenylalanine hydroxylase (PAH) activity in the liver, but it is the brain (which expresses very little of this enzyme), not the liver, that is damaged by the high blood levels of phenylalanine resulting from the lack of hepatic PAH. Second, although housekeeping proteins are expressed in most or all tissues, the clinical effects of pathogenic variants in such proteins are frequently limited to one or few tissues, for at least two reasons. Most often, the housekeeping protein in question is normally expressed abundantly in one or few tissues where it serves a specialty function. This situation is illustrated by Tay-Sachs disease, as discussed later ▶
chapter 13 The Molecular, Biochemical, and Cellular Basis of Genetic Disease Ada Hamosh In this chapter we extend our examination of the molecular and biochemical basis of genetic disease beyond the h...
Ch13 · Pt2 252 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE (and in (Case 43). The enzyme affected in this disorder is hexosaminidase A, which is expressed in virtually all cells, but its absence leads to a fatal neurodegeneration while leaving nonneuronal cell types unscathed. In other instances, another protein with overlapping biologic activity may be expressed in the unaffected tissue, thereby lessening the impact of the loss of function by the variant allele – a situation known as genetic redundancy. Unexpectedly, even pathogenic variants in genes seeming as essential to every cell, such as actin, can result in viable offspring. DISEASES INVOLVING ENZYMES Enzymes are the catalysts that mediate the efficient conversion of a substrate to a product. The diversity of substrates on which enzymes act is huge. Accordingly, the human genome contains more than 3700 genes that encode enzymes, and there are hundreds of human diseases – the enzymopathies – that involve enzyme defects. We first discuss one of the best-known groups of inborn errors of metabolism, the hyperphenylalaninemias. ▶
NUCLEUS Developmental transcription factors Pax 6 - aniridia Genome integrity BRCA1, BRCA2 - breast cancer DNA mismatch repair proteins - Lynch syndrome RNA translation regulation FMRP (RNA binding to...
Ch13 · Pt3 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 253 Aminoacidopathies The Hyperphenylalaninemias The abnormalities that lead to an increase in the blood level of phenylalanine, most notably PAH deficiency or PKU, illustrate almost every principle of biochemical genetics related to enzyme defects. The biochemical causes of hyperphenylalaninemia are illustrated in Fig. 13.2, and the principal features of the diseases associated with the biochemical defect at the six known hyperphenylalaninemia loci are presented in Table 13.1. All the genetic disorders of phenylalanine metabolism are inherited as autosomal recessive conditions and are due to loss-of-function variants, either in the gene encoding PAH or in genes required for the synthesis or reutilization of the PAH cofactor, tetrahydrobiopterin (BH4), or (rarely) in DNAJC12, which encodes a chaperone for PAH. Phenylketonuria. Classic PKU is the epitome of the enzymopathies. It results from pathogenic variants in the gene encoding PAH, which converts phenylalanine to tyrosine (see Fig. 13.2 and Table 13.1). The discovery of PKU in 1934 marked the first demonstration of a TABLE 13.1 Locus Heterogeneity in the Hyperphenylalaninemias Biochemical Defect Incidence/106 Births Enzyme Affected Treatment Variants in the Gene Encoding Phenylalanine Hydroxylase Classic PKU 5–350 (depending on the population) PAH Low-phenylalanine diet* Variant PKU Less than classic PKU PAH Low-phenylalanine diet (less restrictive than that required to treat PKU); BH4 Non-PKU hyperphenylalaninemia 15–75 PAH None, or a much less restrictive low-phenylalanine diet; BH4 in untreated individuals with phenylalanine levels >300 Variants in Genes Encoding Enzymes of Tetrahydrobiopterin Metabolism Impaired BH4 recycling <1 PCD DHPR Low-phenylalanine diet + l-dopa, 5-HT, carbidopa (+ folinic acid for DHPR patients) Impaired BH4 synthesis <1 GTP-CH 6-PTS Low-phenylalanine diet + l-dopa, 5-HT, carbidopa and pharmacological doses of BH4 Variants in the Gene Encoding the PAH chaperone Impaired chaperone and stabilization of PAH <1 DNAJC12 Low-phenylalanine diet + l-dopa, 5-HT, carbidopa and pharmacological doses of BH4 BH4, Tetrahydrobiopterin; DHPR, dihydropteridine reductase; GTP-CH, guanosine triphosphate cyclohydrolase; 5-HT, 5-hydroxytryptophan; PAH, phenylalanine hydroxylase; PCD, pterin 4α-carbinolamine dehydratase; PKU, phenylketonuria; 6-PTS, 6-pyruvoyltetrahydropterin synthase. Protein (diet, endogenous) Phenylalanine Tyrosine Phenylalanine hydroxylase BH4 BH4 DHPR q BH2 PCD 4α OHBH4 GTP DHNP 6-PT phe phe hydroxylase BH4 tyr L-dopa tyr hydroxylase CO2 + H2O dopamine NE E trp 5-OH trp trp hydroxylase serotonin Sepiapterin reductase GTP-cyclohydrolase 6-PT synthase tyr BH4 Figure 13.2 The biochemical pathways affected in the hyperphenylalaninemias. BH4, tetrahydrobiopterin; 4α OHBH4, 4α-hydroxytetrahydrobiopterin; q BH2, quinonoid dihydrobiopterin, the oxidized product of the hydroxylation reactions, which is reduced to BH4 by dihydropteridine reductase (DHPR); PCD, pterin 4α-carbinolamine dehydratase; phe, phenylalanine; tyr, tyrosine; trp, tryptophan; GTP, guanosine triphosphate; DHNP, dihydroneopterin triphosphate; 6-PT, 6-pyruvoyltetrahydropterin; l-dopa, ldihydroxyphenylalanine; NE, norepinephrine; E, epinephrine; 5-OH trp, 5-hydroxytryptophan. *BH4 supplementation may increase the PAH activity of some patients in this group. ▶
CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 253 Aminoacidopathies The Hyperphenylalaninemias The abnormalities that lead to an increase in the blood level of phenyla...
Ch13 · Pt4 254 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE BOX 13.1 MUTANT ENZYMES AND DISEASE: GENERAL CONCEPTS The following concepts are fundamental to the understanding and treatment of enzymopathies. Inheritance patterns Enzymopathies are almost always recessive or X-linked (see Chapter 7). Most enzymes are produced in quantities significantly in excess of minimal biochemical requirements so that heterozygotes (typically with ~50% of residual activity) are clinically normal. In fact, many enzymes may maintain normal substrate and product levels with less than 10% of full activity, a point relevant to the design of therapeutic strategies (e.g., for homocystinuria due to cystathionine synthase deficiency—see Chapter 14). The enzymes of porphyrin synthesis are exceptions (see discussion of acute intermittent porphyria in main text, later). Substrate accumulation or product deficiency Because the function of an enzyme is to convert a substrate to a product, all of the pathophysiologic consequences of enzymopathies can be attributed either to the accumulation of the substrate (as in PKU), to the deficiency of the product (as in glucose-6-phosphate dehydrogenase deficiency (Case 19), or to some combination of the two (Fig. 13.3). Diffusible vs macromolecular substrates An important distinction can be made between enzyme defects in which the substrate is a small molecule (such as phenylalanine, which can be readily distributed throughout body fluids by diffusion or transport) and defects in which the substrate is a macromolecule (such as a mucopolysaccharide or glycosaminoglycan, which remains trapped within its organelle or cell). The pathologic change of the macromolecular diseases, such as Tay-Sachs disease, is confined to the tissues in which the substrate accumulates. In contrast, the site of the disease in the small molecule disorders is often unpredictable because the free-moving unmetabolized substrate or its derivatives can damage cells remote from the affected enzyme, as in PKU. Loss of multiple enzyme activities An individual with a single-gene defect may have loss of function in more than one enzyme. There are several possible mechanisms: the enzymes may use the same cofactor (e.g., BH4 deficiency); the enzymes may share a common subunit or an activating, processing, or stabilizing protein (e.g., the GM2 gangliosidoses); the enzymes may all be processed by a common modifying enzyme, and in its absence, they may be inactive, or their uptake into an organelle may be impaired (e.g., I-cell disease, in which failure to add mannose 6-phosphate to many lysosomal enzymes abrogates the ability of cells to recognize and import the enzymes); or a group of enzymes may be absent or ineffective if the organelle in which they are normally found is not formed or is abnormal (e.g., Zellweger syndrome, a disorder of peroxisome biogenesis). Phenotypic homology The pathologic and clinical features resulting from an enzyme defect are often shared by diseases involving other enzymes that function in the same area of metabolism (e.g., the mucopolysaccharidoses) as well as by the different phenotypes that can result from partial versus complete defects of one enzyme. Partial defects often present with clinical abnormalities that are a subset of those found with the complete deficiency, although the etiologic relationship between the phenotypes may not be immediately obvious. For example, partial deficiency of the purine enzyme, hypoxanthine-guanine phosphoribosyltransferase, causes only hyperuricemia, whereas complete deficiency causes hyperuricemia with a profound neurologic disease, Lesch-Nyhan syndrome, which resembles cerebral palsy and is characterized by severe self-injurious behavior. ▶
genetic defect as a cause of intellectual disability. Because patients with PKU cannot degrade phenylalanine, it accumulates in body fluids and damages the developing central nervous system. A small f...
Ch13 · Pt5 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 255 Variant PKU includes individuals who require only some dietary phenylalanine limitations, less restrictive than for classic PKU, because their blood phenylalanine levels are more moderate and less damaging to the brain. With classic PKU, the plasma phenylalanine levels are more than 1200 μmol/L on a normal diet, whereas non-PKU hyperphenylalaninemia is defined by plasma phenylalanine concentrations above the upper limit of normal (120 μmol/L) but below those seen in classic PKU. If the increase in non-PKU hyperphenylalaninemia is small (<400 μmol/L, termed benign hyperphenylalaninemia), no treatment is required; these individuals come to clinical attention only through newborn screening (see Chapter 19). They are followed to ensure that levels do not rise into the treatment range. Their normal phenotype has been the best indication of the safe target level of plasma phenylalanine in treating classic PKU. The association of these three clinical phenotypes with variants in the PAH gene is a clear example of allelic heterogeneity leading to clinical heterogeneity (see Table 13.1). Allelic and Locus Heterogeneity in the Hyperphenylalaninemias Allelic Heterogeneity in the PAH Gene. A striking degree of allelic heterogeneity at the PAH locus – more than 1200 different variants worldwide – has been identified among individuals with hyperphenylalaninemia associated with classic PKU, variant PKU, or benign hyperphenylalaninemia (see Table 13.1). Seven variants account for a majority of known pathogenic alleles in populations of European descent, whereas six others represent the majority of PAH pathogenic variants in Asian populations. The remaining disease-causing variants are individually rare. To record and make this information publicly available, a PAH variant database has been developed by an international consortium. The allelic heterogeneity at the PAH locus has major clinical consequences. Most important is that most individuals with hyperphenylalaninemia are compound heterozygotes (i.e., they have two different disease-causing alleles) (see Chapter 7). This allelic heterogeneity accounts for much of the enzymatic and phenotypic heterogeneity among affected individuals. Thus pathogenic variants that eliminate or dramatically reduce PAH activity generally cause classic PKU, whereas greater residual enzyme activity is associated with milder phenotypes. However, homozygous patients with certain PAH variants have phenotypes ranging all the way from classic PKU to non-PKU hyperphenylalaninemia. Accordingly, other unidentified biologic variables – undoubtedly including modifier genes – generate variation in the phenotype for a given genotype. This lack of a strict genotype-phenotype correlation, initially somewhat surprising, is now recognized as a feature of most single-gene diseases, highlighting that even monogenic traits like PKU are not genetically simple disorders. Defects in Tetrahydrobiopterin Metabolism. In 1% to 3% of individuals with elevated phenylalanine, the PAH gene is normal, and the hyperphenylalaninemia results from a defect in one of the steps in the biosynthesis or regeneration of BH4 – the cofactor for PAH (see Table 13.1 and Fig. 13.2). The association of a single biochemical phenotype, such as hyperphenylalaninemia, with variants in different genes, is an example of locus heterogeneity (see Table 13.1). The proteins encoded by genes that manifest locus heterogeneity generally act at different steps in a single biochemical pathway: another principle of genetic disease illustrated by hyperphenylalaninemia (see Fig. 13.2). BH4-deficient patients were first recognized because they developed profound neurologic problems in early life, despite the successful administration of a low-phenylalanine diet. This poor outcome is due in part to the requirement for the BH4 cofactor by two other enzymes: tyrosine hydroxylase and tryptophan hydroxylase. These hydroxylases are critical for the synthesis of the monoamine neurotransmitters, dopamine, norepinephrine, epinephrine, and serotonin (see Fig. 13.2). The locus heterogeneity of hyperphenylalaninemia is significant because the treatment of patients with a defect in BH4 metabolism differs markedly from that for subjects with pathogenic variants in PAH, in two ways. First, because the PAH enzyme is itself normal in individuals with BH4 defects, its activity can be restored by large doses of oral BH4, leading to reduction in plasma phenylalanine levels. This practice highlights the principle of product replacement in the treatment of some genetic disorders (see Chapter 14). Consequently, phenylalanine restriction can be significantly relaxed for those with defects in BH4 metabolism, and some actually tolerate an unrestricted diet. Second, one must try to normalize the neurotransmitters in the brains of these patients by administering the products of tyrosine hydroxylase and tryptophan hydroxylase: l-dopa and 5-hydroxytryptophan, respectively (see Fig. 13.2 and Table 13.1). A novel form of hyperphenylalaninemia with movement disorder and sometimes with cognitive impairment is caused by biallelic pathogenic variants in DNAJC12, which codes for a member of the HSP40 family of proteins. It functions as a cochaperone (with members of the HSP70 family of proteins) of the aromatic hydroxylases, including PAH, tyrosine hydroxylase, and tryptophan hydroxylases 1 and 2. So far, more than 20 patients have been described. This condition will be identified by elevated phenylalanine on newborn screening and requires sequencing of the gene for diagnosis. Remarkably, pathogenic variants in sepiapterin reductase, an enzyme in the BH4 synthesis pathway, do not cause hyperphenylalaninemia. Only dopa-­responsive dystonia is seen, due to impaired synthesis of dopamine and serotonin (see Fig. 13.2). Alternative pathways may exist for the final step in BH4 synthesis, bypassing the ▶
CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 255 Variant PKU includes individuals who require only some dietary phenylalanine limitations, less restrictive than for c...
Ch13 · Pt6 256 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE sepiapterin reductase deficiency in peripheral tissues, an example of genetic redundancy. For these reasons, all hyperphenylalaninemic infants must be evaluated to determine whether their hyperphenylalaninemia is the result of an abnormality in PAH, in BH4 metabolism, or in the chaperone. The hyperphenylalaninemias thus illustrate the critical importance of obtaining a specific molecular diagnosis in all patients with a genetic disease phenotype. The underlying genetic defect may not be what one first suspects, and the treatment can vary accordingly. Tetrahydrobiopterin Responsiveness With PAH Variants. Many individuals with variants in the PAH gene (rather than in BH4 metabolism) will also respond to large oral doses of BH4 cofactor, with a substantial decrease in plasma phenylalanine. BH4 supplementation is therefore an important adjunct therapy for PKU patients of this type, allowing a less restricted dietary intake of phenylalanine. The affected individuals most likely to respond are those with significant residual PAH activity (i.e., those with variant PKU and non-PKU hyperphenylalaninemia), but a minority of individuals with classic PKU are also responsive. The presence of residual PAH activity does not, however, guarantee an effect of BH4 administration on plasma phenylalanine levels. Rather, the degree of BH4 responsiveness will depend on the specific properties of each altered PAH protein, reflecting the allelic heterogeneity underlying PAH variants. The provision of increased amounts of a cofactor is a strategy that has been used for the treatment of many inborn errors of enzyme metabolism, as discussed further in Chapter 14. In general, a cofactor comes into contact with the protein component of an enzyme (termed an apoenzyme) to form the active holoenzyme, which consists of both the cofactor and the otherwise inactive apoenzyme. Illustrating this strategy, BH4 supplementation exerts its beneficial effect through one or more mechanisms, all of which result from increased cofactor in contact with the altered PAH apoenzyme. These mechanisms include stabilization of the enzyme, protection of the enzyme from degradation by the cell, and increase in cofactor supply for an altered enzyme with low affinity for BH4. Newborn Screening. PKU is the prototype of genetic diseases for which mass newborn screening is justified (see Chapter 19) because (1) it is relatively common in some populations (up to ~1 in 2900 live births), (2) mass screening is feasible, (3) failure to treat has severe consequences (profound intellectual disability), and (4) treatment is effective if begun early in life. To allow time for the postnatal increase in blood phenylalanine levels, the test is performed after 24 hours of age. Central laboratories assay blood from a heel prick for blood phenylalanine levels and phenylalanine-to-tyrosine ratio. Positive test results must be confirmed quickly because delays in treatment beyond 4 weeks postnatally have profound effects on intellectual outcome. The current recommendation is to initiate treatment within the first week of life. Maternal Hyperphenylalaninemia. Originally, the lowphenyla­lanine diet was discontinued in mid-childhood for most individuals with PKU. It was later found, however, that almost all offspring of women with PKU not on treatment are clinically abnormal; most are severely delayed developmentally, and many have microcephaly, growth impairment, and malformations, particularly of the heart. As predicted by principles of mendelian inheritance, these children are heterozygotes; their neurodevelopmental delay is not due to their own genetic constitution but to the highly teratogenic effect of elevated phenylalanine in the maternal circulation. Accordingly, women with PKU who are planning pregnancies must achieve tight metabolic control with a low-phenylalanine diet and BH4 supplementation (if responsive) prior to conception. Lysosomal Storage Diseases: A Unique Class of Enzymopathies Lysosomes are membrane-bound organelles containing an array of hydrolytic enzymes involved in the degradation of a variety of biologic macromolecules. Pathogenic variants in these hydrolases are unique because they lead to the accumulation of their substrates inside the lysosome, where the substrates remain trapped because their large size prevents their egress from the organelle. Their accumulation and sometimes toxicity interferes with normal cell function, eventually causing cell death. Moreover, the substrate accumulation underlies one uniform clinical feature of these diseases – their unrelenting progression. In most of these conditions, substrate storage increases the mass of the affected tissues and organs. When the brain is affected, the picture is one of neurodegeneration. The clinical phenotypes are very distinct and often make the diagnosis of a storage disease straightforward. More than 50 lysosomal hydrolase or lysosomal membrane transport deficiencies, almost all inherited as autosomal recessive conditions, have been described. Historically these diseases were untreatable. However, bone marrow transplantation and enzyme replacement therapy have dramatically improved the prognosis of these conditions (see Chapter 14). Tay-Sachs Disease Tay-Sachs disease (Case 43) is one of a group of heterogeneous lysosomal storage diseases, the GM2 gangliosidoses, that result from the inability to degrade a sphingolipid, GM2 ganglioside (Fig. 13.4). The biochemical lesion is a marked deficiency of hexosaminidase A (hex A). Although the enzyme is ubiquitous, the disease has its clinical impact almost solely on the brain, the predominant site of GM2 ganglioside synthesis. Catalytically active hex A is the product of a three-gene system (see Fig. 13.4). These genes encode the α and β subunits of the enzyme (the HEXA and HEXB genes, ▶
256 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE sepiapterin reductase deficiency in peripheral tissues, an example of genetic redundancy. For these reasons, all hyperphenylalaninemic infan...
Ch13 · Pt7 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 257 respectively) and an activator protein that must associate with the substrate and the enzyme before the enzyme can cleave the terminal N-acetyl-β-galactosamine residue from the ganglioside. The clinical manifestations of defects in the three genes are indistinguishable, but they can be differentiated by enzymatic analysis. Pathogenic variants in the HEXA gene affect the α subunit and disrupt hex A activity to cause Tay-Sachs disease (or less severe variants of hex A deficiency). Defects in the HEXB gene or in the gene encoding the activator protein impair the activity of both hex A and hex B (see Fig. 13.4) to produce Sandhoff disease or activator protein deficiency (which is very rare), respectively. The clinical course of Tay-Sachs disease is tragic. Affected infants appear normal until ~3 to 6 months of age but then gradually undergo progressive neurologic deterioration until death at 2 to 4 years. The effects of neuronal death can be seen directly in the form of the cherry-red spot in the retina (Case 43). In contrast, HEXA alleles associated with some residual activity lead to later-onset forms of neurologic disease, with manifestations including lower motor neuron dysfunction and ataxia due to spinocerebellar degeneration. In contrast to the infantile disease, vision and intelligence usually remain normal, although psychosis develops in one-third of these patients. Finally, pseudodeficiency alleles (discussed next) cause no disease. Hex A Pseudodeficiency Alleles and Their Clinical Significance. An unexpected consequence of screening for Tay-Sachs carriers in the Ashkenazi Jewish population was the discovery of a unique class of hex A alleles, the pseudodeficiency alleles. Although the two pseudodeficiency alleles are clinically benign, individuals identified as pseudodeficient in screening tests are genetic compounds with a pseudodeficiency allele on one chromosome and a common Tay-Sachs variant on the other chromosome. These individuals have a low level of hex A activity (~20% of controls) that is adequate to prevent GM2 ganglioside accumulation in the brain. The importance of hex A pseudodeficiency alleles is twofold. First, they complicate prenatal diagnosis because a pseudodeficient fetus could be incorrectly diagnosed as affected. More generally, the recognition of the hex A pseudodeficiency alleles indicates that screening programs for other genetic diseases must recognize that comparable alleles may exist at other loci and may confound the correct characterization of individuals in screening or diagnostic tests. Population Genetics. In many monogenic diseases, some alleles are found at higher frequency in some populations than in others (see Chapter 10). This situation is illustrated by Tay-Sachs disease, in which three alleles account for 99% of the variants found in Ashkenazi The GM2 gangliosidoses GM2 ganglioside Disease Tay-Sachs disease and later-onset variants Sandhoff disease and later-onset variants Activator deficiency Affected gene α (chr 15) β (chr 5) activator (chr 5) Polypeptide α subunit β subunit activator Isozyme: subunits Hex A: αβ Hex B: ββ activator αβ Active enzyme complex N-acetylgalactosamine - galactose - glucose - ceramide NANA Cleavage site Figure 13.4 The three-gene system required for hexosaminidase A activity and the diseases that result from defects in each of the genes. The function of the activator protein is to bind the ganglioside substrate and present it to the enzyme. Hex A, Hexosaminidase A; Hex B, hexosaminidase B; NANA, N-acetyl neuraminic acid. (Modified from Sandhoff K, Conzelmann E, Neufeld EF, et al: The GM2 gangliosidoses. In Scriver CR, Beaudet AL, Sly WS, et al, editors: The metabolic bases of inherited disease, ed 6, New York, 1989, Mc Graw-Hill, pp 1807–1839.) ▶
CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 257 respectively) and an activator protein that must associate with the substrate and the enzyme before the enzyme can cl...
Ch13 · Pt8 258 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Jewish patients, the most common of which accounts for 80% of cases (Fig. 13.5). Approximately 1 in 27 Ashkenazi Jews is a carrier of a Tay-Sachs allele, and the incidence of affected infants was 100 times higher than in other populations, prior to screening. A founder effect or heterozygote advantage is the most likely explanation for this high frequency (see Chapter 10). Because most Ashkenazi Jewish carriers will have one of the three common alleles, a practical benefit of the molecular characterization of the disease in this population is the degree to which carrier screening has been simplified. ALTERED PROTEIN FUNCTION DUE TO ABNORMAL POSTTRANSLATIONAL MODIFICATION Congenital Disorders of Glycosylation Approximately 50% of all proteins and 80% of those in blood are glycosylated and need appropriate sugar moieties added to function properly. A broad class of monogenic disease is the congenital disorders of glycosylation (CDGs), involving over 160 different genes (Fig. 13.6): most are autosomal recessive and a few are X-linked. They usually present in infancy with multisystem problems, including failure to thrive, liver disease, hypotonia, intestinal disease (often protein-losing enteropathy), developmental delay, eye and skeletal anomalies, immunologic abnormalities, and may or may not include brain and/or neurodevelopmental abnormalities. CDGs are divided into two main groups: type I CDGs comprise defects in the assembly of the dolichol lipid-linked oligosaccharide (LLO) chain and its transfer to the nascent protein; type II CDGs are due to defects in the processing of the protein-bound glycans either late in the endoplasmic reticulum or in the Golgi apparatus. Screening for N-linked glycosylation disorders can be achieved by isoelectric focusing of transferrin, a heavily glycosylated plasma protein. Decrease in the proportion of tetrasialo-transferrin with increases of asialo- or di- or trisialotransferrin, suggest the diagnosis, which then needs molecular confirmation. Because the multipathway, or type II, CDGs require sequencing and the manifestations are so variable, increasingly the diagnosis is established by direct genome-wide investigation through either exome or genome sequencing. One example is CDG due to pathogenic variants in MPI, called MPI-CDG or CDG1b. This condition spares cognitive development but manifests in infancy with severe failure to thrive, protein-losing enteropathy, hypoglycemia, and coagulation defects. Treatment with high-dose mannose (1 g/kg body weight) results in elimination of hypoglycemia, protein-losing enteropathy, and coagulation defects. Despite treatment, some patients have manifested liver fibrosis in adulthood, so long-term follow-up is essential. A Loss of Glycosylation: Mucolipidosis II or I-Cell Disease Some proteins have information contained in their primary amino acid sequence that directs them to their subcellular residence, whereas others are localized on the basis of posttranslational modifications. This latter mechanism is true of the acid hydrolases found in lysosomes, but this form of cellular trafficking was unrecognized until the discovery of I-cell disease, a severe autosomal recessive lysosomal storage disease. The disorder has a range of phenotypic effects involving facial features, skeletal changes, growth retardation, and intellectual disability and survival of less than 10 years. The cytoplasm of cultured skin fibroblasts from individuals with I-cell disease contains numerous abnormal lysosomes, or inclusions (hence the term inclusion [I] cells). In I-cell disease, the cellular levels of many lysosomal acid hydrolases are greatly diminished, and instead they are found in excess in body fluids, including blood. This unusual situation arises because the hydrolases in these patients have not been properly modified posttranslationally. A typical hydrolase is a glycoprotein, the sugar moiety containing mannose residues, some of which are phosphorylated. The mannose-6-phosphate residues are essential for recognition of the hydrolases by receptors on the cell and lysosomal membrane surface. In I-cell disease there is a defect in the enzyme that transfers a phosphate group to the mannose residues. The fact that many enzymes are affected is consistent with the diversity of clinical abnormalities seen in these patients. Normal HEXA allele Tay-Sachs allele...–Arg–Ile–Ser–Try–Gly–Pro–Asp–......–Arg–Ile–Ser–Ile–Leu–Cys–Pro–Stop... CGT ATA TCC TAT GCC CCT GAC...... CGT ATA TCT ATC CTA TGC CCC TGA C... Altered reading frame Figure 13.5 Four-base insertion (TATC) in the hexosaminidase A (hex A) gene in Tay-Sachs disease, leading to a frameshift. This variant is the major cause of Tay-Sachs disease in Ashkenazi Jews. No detectable hex A protein is made, accounting for the complete enzyme deficiency observed in these infantile-onset patients. ▶
258 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Jewish patients, the most common of which accounts for 80% of cases (Fig. 13.5). Approximately 1 in 27 Ashkenazi Jews is a carrier of a Tay-...
Ch13 · Pt9 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 259 SRD5A3 DOLK CTP CDP DPAGT1 ALG13 ALG14 ALG1 ALG2 ALG2 ALG11 ALG11 RFT1 ALG9 ALG12 ALG9 ALG6 ALG8 ALG10 DOLPP1 DHDDS NUS1 Mevalonate Pathway MOGS PRKCSH GANAB ALG3 Cytoplasm ER Lumen Ribosome 5' 3' mRNA STT3A, STT3B, DDOST, TUSC3 RPN1, RPN2, DAD1, KRTCAP2, DC2 Oligosaccharyltransferase Complex SSR4 SSR1, SSR2, SSR3 Translocon Complex Fructose 6-P MPI 6P PMM2 GTP GMPPA GMPPB GDP 1P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P Asn Asn Asn P OH P P DPM1 DPM2 DPM3 ALG5 P P P P P P MPDU1??? MAN1B1 Asn Polyprenol Dolichol N-acetylglucosamine Mannose Glucose Galactose Fucose Sialic Acid B4GALT1 MGAT1 MGAT2 MAN1A MAN1A2 MAN1C1 MAN2A1 MAN2A2 ST6GAL1 ST6GAL2 FUT8 COG1, COG2, COG4, COG5, COG6, COG7, COG8 COG3 Retrograde Transport Complex SLC35A3 UDP UDP GDP SLC35C1 GDP SLC35A2 UDP UDP SLC35A1 CMP CMP TMEM165 TMEM199 CCDC115 Golgi Homeostasis or p H Maintenance ATP6AP1 ATP6AP2 ATP6V0A2 ATP6AP1 ATP6V1E1 ATP6V1A Asn Asn Asn Asn Asn Asn Asn Asn SLC39A8 Mn+2 Mn+2 OH (13-17) Figure 13.6 A schematic of the N-linked pathway highlighting those genes required for both the initial steps of lipid-linked oligosaccharide synthesis and several key components for glycan processing within the Golgi. These genes highlighted in red represent known loci for glycosylation disorders. The blue arrow indicates MPI. Supplementation with pharmacologic doses of mannose can drive this reaction to create extra mannose-6-phosphate. (Adapted from Ng BG, Freeze HH: Perspectives on glycosylation and its congenital disorders, Trends Genet 34(6):466–476, 2018. https://doi.org/10.1016/j.tig.2018.03.002.) ▶
CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 259 SRD5A3 DOLK CTP CDP DPAGT1 ALG13 ALG14 ALG1 ALG2 ALG2 ALG11 ALG11 RFT1 ALG9 ALG12 ALG9 ALG6 ALG8 ALG10 DOLPP1 DHDDS N...
Ch13 · Pt10 260 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Gains of Glycosylation: Variants That Create New (Abnormal) Glycosylation Sites In contrast to the failure of protein glycosylation exemplified by I-cell disease, it has been shown that an unexpectedly high proportion (~1.5%) of the missense variants that cause human disease may be associated with abnormal gains of N-glycosylation due to pathogenic variants creating new consensus N-glycosylation sites in the mutant proteins. That such novel sites can actually lead to inappropriate glycosylation of the mutant protein, with pathogenic consequences, is highlighted by the rare autosomal recessive disorder, mendelian susceptibility to mycobacterial disease (MSMD). MSMD patients have defects in any one of a number of genes that regulate the defense against some infections. Consequently, they are susceptible to disseminated infections upon exposure to moderately virulent mycobacterial species, such as the bacillus CalmetteGuérin (BCG) used throughout the world as a vaccine against tuberculosis, or to nontuberculous environmental bacteria that do not normally cause illness. Some MSMD patients carry missense variants in the gene for interferon-γ receptor 2 (IFNGR2) that generate novel N-glycosylation sites in the mutant IFNGR2 protein. These novel sites lead to the synthesis of an abnormally large, overly glycosylated receptor. The mutant receptors reach the cell surface but fail to respond to interferon-γ. Variants leading to gains of glycosylation have also been found to lead to a loss of protein function in several other monogenic disorders. The discovery that removal of the abnormal polysaccharides restores function to the mutant IFNGR2 proteins in MSMD offers hope that disorders of this type may be amenable to chemical therapies that reduce the excessive glycosylation. Loss of Protein Function Due to Impaired Binding or Metabolism of Cofactors Some proteins acquire biologic activity only after they associate with cofactors, such as BH4 in the case of PAH, as discussed earlier. Variants that interfere with cofactor synthesis, binding, transport, or removal from a protein (when ligand binding is covalent) are also known. For many of these mutant proteins, an increase in the intracellular concentration of the cofactor is frequently capable of restoring some residual activity to the mutant enzyme, for example, by increasing the stability of the mutant protein. Consequently, enzyme defects of this type are among the most responsive of genetic disorders to specific biochemical therapy because the cofactor or its precursor is often a water-soluble vitamin that can be administered safely in large amounts (see Chapter 14). Impaired Cofactor Binding: Homocystinuria Due to Cystathionine Synthase Deficiency Homocystinuria due to cystathionine synthase deficiency (Fig. 13.7) was one of the first aminoacidopathies to be recognized. The clinical phenotype of this autosomal recessive condition is often dramatic. The most common features include dislocation of the lens, intellectual disability, osteoporosis, long bones, and thromboembolism of both veins and arteries, a phenotype that can be confused with Marfan syndrome, a disorder of connective tissue (Case 30). The accumulation of homocysteine is believed to be central to most, if not all, of the pathology. Homocystinuria was one of the first genetic diseases shown to be vitamin responsive; pyridoxal phosphate is the cofactor of the enzyme, and the administration of large amounts of pyridoxine, the vitamin precursor of the cofactor, often ameliorates the biochemical abnormality and the clinical disease (see Chapter 14). In many patients, the affinity of the mutant enzyme for pyridoxal phosphate is reduced, indicating that altered conformation of the protein impairs cofactor binding. Not all cases of homocystinuria result from pathogenic variants in cystathionine synthase. Pathogenic variants in five different enzymes of cobalamin (vitamin B12) or folate metabolism can also lead to increased levels of homocysteine in body fluids. These variants impair the provision of the vitamin B12 cofactor, methylcobalamin (methyl-B12), or of methyl-H4-folate (see Fig. 13.7) and thus represent another example (like the defects in BH4 Cystathionine synthase Methionine Homocysteine Cystathionine Cysteine Methionine synthase Methyl-B12 H4-folate Methyl-H4-folate Pyridoxal phosphate Vitamin B6 Figure 13.7 Genetic defects in pathways that impinge on cystathionine synthase, or in that enzyme itself, and cause homocystinuria. Classic homocystinuria is due to defective cystathionine synthase. Several different defects in the intracellular metabolism of cobalamins (not shown) lead to a decrease in the synthesis of methylcobalamin (methyl-B12) and thus in the function of methionine synthase. Defects in methylene-H4-folate reductase (not shown) decrease the abundance of methyl-H4-folate, which also impairs the function of methionine synthase. Some patients with cystathionine synthase abnormalities respond to large doses of vitamin B6, increasing the synthesis of pyridoxal phosphate, thereby increasing cystathionine synthase activity and treating the disease (see Chapter 14). ▶
260 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Gains of Glycosylation: Variants That Create New (Abnormal) Glycosylation Sites In contrast to the failure of protein glycosylation exemplif...
Ch13 · Pt11 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 261 synthesis that lead to hyperphenylalaninemia) of genetic diseases due to defects in the biogenesis of enzyme cofactors. The clinical manifestation of these disorders is variable but includes megaloblastic anemia, developmental delay, and failure to thrive. These conditions, all of which are autosomal recessive, are often partially or completely treatable with high doses of vitamin B12. Pathogenic Variants of an Enzyme Inhibitor: α1-Antitrypsin Deficiency α1-Antitrypsin (α1AT) deficiency is an important autosomal recessive condition associated with a substantial risk for chronic obstructive lung disease (emphysema) (Fig. 13.8) and cirrhosis of the liver. The α1AT protein belongs to a major family of protease inhibitors, the serine protease inhibitors or serpins; SERPINA1 is the formal gene name. Notwithstanding the specificity suggested by its name, α1AT actually inhibits a wide spectrum of proteases, particularly elastase released from neutrophils in the lower respiratory tract. In populations of European descent, α1AT deficiency affects ~1 in 6700 persons, and ~4% are carriers. A dozen or so α1AT alleles are associated with an increased risk for lung or liver disease, but only the Z allele (p. Glu 342Lys) is relatively common. The reason for the relatively high frequency of the Z allele in European populations is unknown, but analysis of DNA haplotypes suggests a single origin with subsequent spread throughout northern Europe. Given the increased risk for emphysema, α1AT deficiency is an important public health problem, affecting an estimated 60,000 persons in the United States alone. The α1AT gene is expressed principally in the liver, which normally secretes α1AT into plasma. Approximately 17% of Z/Z homozygotes present with neonatal jaundice, and ~20% of this group subsequently develop cirrhosis. The liver disease associated with the Z allele is thought to result from a novel property of the mutant protein – its tendency to aggregate, trapping it within the rough endoplasmic reticulum (ER) of hepatocytes. The molecular basis of the Z protein aggregation is a consequence of structural changes in the protein that predispose to the formation of long beadlike necklaces of mutant α1AT polymers. Thus, like the sickle cell disease variant in β-globin (see Chapter 12), the Z allele of α1AT is a clear example of a variant that confers a novel property on the protein (in both of these examples, a tendency to aggregate) (see Fig. 12.1). Both sickle cell disease and the α1AT deficiency associated with homozygosity for the Z allele are examples of inherited conformational diseases. These disorders occur when a variant causes the shape or size of a protein to change in a way that predisposes it to self-association and tissue deposition. Notably, some fraction of the mutant protein is invariably correctly folded in these disorders, including α1AT deficiency. Note that not all conformational diseases are single-gene disorders, as illustrated, for example, by nonfamilial AD (discussed later) and prion diseases. The lung disease associated with the Z allele of α1AT deficiency is due to the alteration of the normal balance between elastase and α1AT, which allows progressive degradation of the elastin of alveolar walls (Fig. 13.9). Two mechanisms contribute to the elastase α1AT imbalance. First, the block in the hepatic secretion of the Z protein, although not complete, is severe, and Z/Z patients have only ~15% of the normal plasma concentration of α1AT. Second, the Z protein has only ~20% of the ability of the normal α1AT protein to inhibit neutrophil elastase. The infusion of normal α1AT is used in some patients to augment the level of α1AT in the plasma, to Age (years) 20 30 40 50 60 70 80 90 100 1.0 0.8 0.6 0.4 0.2 0 Cumulative probability of survival Z/Z smokers Z/Z nonsmokers All females (mostly M/M) All males (mostly M/M) Figure 13.8 The effect of smoking on the survival of patients with α1-antitrypsin deficiency. The curves show the cumulative probability of survival to specified ages of smokers, with or without α1-antitrypsin deficiency. (Redrawn from Larson C: Natural history and life expectancy in severe α1-antitrypsin deficiency, Pi Z, Acta Med Scand 204:345–351, 1978.) ▶
CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 261 synthesis that lead to hyperphenylalaninemia) of genetic diseases due to defects in the biogenesis of enzyme cofactor...
Ch13 · Pt12 262 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE rectify the elastase:α1AT imbalance. Although difficult to prove definitively, there is evidence that the progression of the lung disease is slowed by α1AT augmentation. α1-Antitrypsin Deficiency as an Ecogenetic Disease The development of lung or liver disease in subjects with α1AT deficiency is highly variable, and although no modifier genes have yet been identified, a major environmental factor, cigarette smoke, dramatically influences the likelihood of emphysema. The impact of smoking on the progression of the emphysema is a powerful example of the effect that environmental factors may have on the phenotype of a monogenic disease. Thus, for persons with the Z/Z genotype, survival after 60 years of age is ~60% in nonsmokers but only ~10% in smokers (see Fig. 13.8). One molecular explanation for the effect of smoking is that the active site of α1AT, at methionine 358, is oxidized by both cigarette smoke and inflammatory cells, thus reducing its affinity for elastase by 2000-fold. The field of ecogenetics, illustrated by α1AT deficiency, is concerned with the interaction between environmental factors and different human genotypes. This area of medical genetics is one of increasing importance as genotypes are identified that entail an increased risk for disease on exposure to certain environmental agents (e.g., drugs, foods, industrial chemicals, and viruses). At present, the most highly developed area of ecogenetics is that of pharmacogenetics, presented in Chapter 19. Dysregulation of a Biosynthetic Pathway: Acute Intermittent Porphyria Acute intermittent porphyria (AIP) is an autosomal dominant disease associated with intermittent neurologic dysfunction. The primary defect is a deficiency of ­porphobilinogen (PBG) deaminase, an enzyme in the biosynthetic pathway of heme, required for the synthesis of both hemoglobin and hepatic cytochrome p 450 drugmetabolizing enzymes (Fig. 13.10). All individuals with AIP have a ~50% reduction in PBG deaminase enzymatic activity, whether their disease is clinically latent (90% of patients throughout their lifetime) or clinically expressed (~10%). This reduction is consistent with the autosomal dominant inheritance pattern (see Chapter 7). Homozygous deficiency of PBG deaminase, a critical enzyme in heme biosynthesis, would presumably be Clinically latent AIP: No symptoms Hydroxymethylbilane Heme Hydroxymethylbilane Heme Clinically expressed AIP: Postpubertal neurological symptoms Drugs, chemicals, steroids, fasting, etc. Glycine + succinyl Co A ALA PBG ALA synthetase 50% reduction PBG deaminase Glycine + succinyl Co A ALA PBG ALA synthetase 50% reduction PBG deaminase Figure 13.10 The pathogenesis of acute intermittent porphyria (AIP). Patients with AIP who are either clinically latent or clinically affected have approximately half the control levels of porphobilinogen (PBG) deaminase. When the activity of hepatic δ-aminolevulinic acid (ALA) synthase is increased in carriers by exposure to inducing agents (e.g., drugs, chemicals), the synthesis of ALA and PBG is increased to a level that the PBG deaminase can’t handle. The residual PBG deaminase activity (~50% of controls) is overloaded, and the accumulation of ALA and PBG causes clinical disease. Co A, Coenzyme A. (Redrawn from Kappas A, Sassa S, Galbraith RA, et al: The porphyrias. In Scriver CR, Beaudet AL, Sly WS, et al, editors: The metabolic bases of inherited disease, ed 6, New York, 1989, Mc GrawHill, pp 1305–1365.) Figure 13.9 A posteroanterior chest radiograph of an individual carrying two Z alleles of the α1AT gene, showing the hyperinflation and basal hyperlucency characteristic of emphysema. (From Stoller JK, Aboussouan LS: α1-Antitrypsin deficiency, Lancet 365: 2225–2236, 2005.) ▶
262 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE rectify the elastase:α1AT imbalance. Although difficult to prove definitively, there is evidence that the progression of the lung disease is...
Ch13 · Pt13 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 263 incompatible with life. AIP illustrates one molecular mechanism by which an autosomal dominant disease may manifest only episodically. The pathogenesis of the nervous system disease is uncertain but may be mediated directly by the increased levels of δ-aminolevulinic acid (ALA) and PBG that accumulate due to the 50% reduction in PBG deaminase (see Fig. 13.10). The peripheral, autonomic, and central nervous systems are all affected, and the clinical manifestations are diverse. Indeed, this disorder is one of the great mimics in clinical medicine, with manifestations ranging from acute abdominal pain to psychosis. Clinical crises in AIP are elicited by a variety of precipitating factors: drugs (most prominently the barbiturates, and to this extent, AIP is a pharmacogenetic disease; see Chapter 19); some steroid hormones (clinical disease is rare before puberty or after menopause); and catabolic states, including reducing diets, intercurrent illnesses, and surgery. The drugs provoke the clinical manifestations by interacting with drug-sensing nuclear receptors in hepatocytes, which then bind to transcriptional regulatory elements of the ALA synthetase gene, increasing the production of both ALA and PBG. In normal individuals the drug-related increase in ALA synthetase is beneficial because it increases heme synthesis, allowing greater formation of hepatic cytochrome P450 enzymes that metabolize many drugs. In patients with AIP, however, the increase in ALA synthetase causes the accumulation of ALA and PBG because of the 50% reduction in PBG deaminase activity (see Fig. 13.10). The fact that half of the normal activity of PBG deaminase is inadequate to cope with the increased requirement for heme synthesis in some situations accounts for both the dominant inheritance of the condition and the episodic nature of the clinical illness. DEFECTS IN RECEPTOR PROTEINS The recognition of a class of diseases due to defects in receptor molecules began with the identification by Goldstein and Brown of the low-density lipoprotein (LDL) receptor as the polypeptide affected in the most common form of familial hypercholesterolemia. This disorder, which leads to a greatly increased risk for myocardial infarction, is characterized by elevation of plasma cholesterol carried by LDL, the principal cholesterol transport protein in plasma. Goldstein and Brown’s discovery has cast much light on normal cholesterol metabolism and on the biology of cell surface receptors in general. LDL receptor deficiency is representative of a number of disorders now recognized to result from receptor defects. Familial Hypercholesterolemia: A Genetic Hyperlipidemia Familial hypercholesterolemia is one of a group of metabolic disorders called the hyperlipoproteinemias. These diseases are characterized by elevated levels of plasma lipids (cholesterol, triglycerides, or both) carried by apolipoprotein B (apo B)–containing lipoproteins. Other monogenic hyperlipoproteinemias, each with distinct biochemical and clinical phenotypes, have also been recognized. In addition to variants in the LDL receptor gene (Table 13.2), abnormalities in three other genes can lead to familial hypercholesterolemia (Fig. 13.11). Remarkably, all four of the genes associated with familial hypercholesterolemia disrupt the function or abundance either of the LDL receptor at the cell surface or of apo B, the major protein component of LDL and a ligand for the LDL receptor. Because of its importance, we first review familial hypercholesterolemia due to pathogenic variants in the LDL receptor. We also discuss variants in the PCSK9 protease gene; although gain-of-function variants in this gene cause hypercholesterolemia, the greater importance of PCSK9 lies in the fact that several common loss-of-function sequence variants lower plasma LDL cholesterol levels, conferring substantial protection from coronary heart disease. Familial Hypercholesterolemia Due to Pathogenic Variants in the LDL Receptor Pathogenic variants in the LDL receptor gene (LDLR) are the most common cause of familial hypercholesterolemia (Case 16). The receptor is a cell surface protein responsible for binding LDL and delivering it to the cell interior. Elevated plasma concentrations of LDL cholesterol lead to premature atherosclerosis (accumulation of cholesterol by macrophages in the subendothelial space of major arteries) and increased risk for heart attack and stroke in both untreated heterozygote and homozygote TABLE 13.2 Four Genes Associated With Familial Hypercholesterolemia Mutant Gene Product Pattern of Inheritance Effect of DiseaseCausing Variants Typical LDL Cholesterol Level (Normal Adults: ~120 mg/d L) LDL receptor Autosomal dominant Loss of function Heterozygotes: 350 mg/d L Homozygotes: 700 mg/d L Apoprotein B-100 Autosomal dominant* Loss of function Heterozygotes: 270 mg/d L Homozygotes: 320 mg/d L ARH adaptor protein Autosomal recessive† Loss of function Homozygotes: 470 mg/d L PCSK9 protease Autosomal dominant Gain of function Heterozygotes: 225 mg/d L *Principally in individuals of European descent. †Principally in individuals of Italian and Middle Eastern descent. LDL, Low-density lipoprotein. Partly modified from Goldstein JL, Brown MS: The cholesterol quartet, Science 292:1310–1312, 2001. ▶
CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 263 incompatible with life. AIP illustrates one molecular mechanism by which an autosomal dominant disease may manifest o...
Ch13 · Pt14 264 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE carriers of mutant alleles. Physical stigmata of familial hypercholesterolemia include xanthomas (cholesterol deposits in skin and tendons) (Case 16) and premature arcus corneae (deposits of cholesterol around the periphery of the cornea). Few diseases have been as thoroughly characterized; the sequence of pathologic events from the affected locus to its effect on individuals and populations has been meticulously documented. Genetics. Familial hypercholesterolemia due to pathogenic variants in the LDLR gene is inherited as an autosomal semidominant trait. Both homozygous and heterozygous phenotypes are known, and a clear gene dosage effect is evident; the disease manifests earlier and much more severely in homozygotes than in heterozygotes, reflecting the greater reduction in the number of LDL receptors and the greater elevation in plasma LDL cholesterol (Fig. 13.12). Homozygotes may have clinically significant coronary artery disease in childhood and, if untreated, few live beyond the third decade. The heterozygous form of the disease, with a population frequency of ~2 per 1000, is one of the most common single-gene disorders. Heterozygotes have levels of plasma cholesterol that are approximately twice those of controls (see Fig. 13.12). Because of the inherited nature of familial hypercholesterolemia, it is important to make the diagnosis in the ~5% of survivors of premature (<50 years of age) myocardial infarction who are heterozygotes for an LDL receptor defect. It is important to stress, however, that among those in the general population with plasma cholesterol concentrations above the 95th percentile for age and sex, only ~1 in 20 has familial hypercholesterolemia; most such individuals have an uncharacterized hypercholesterolemia due to multiple common genetic variants, as presented in Chapter 9. 1. Mature LDL receptor 2. Apoprotein B-100 surrounding a cholesterol ester core Vesicle Golgi complex Endoplasmic reticulum 3. ARH adaptor protein, required for clustering the LDL receptor in the clathrin-coated pit 4. PCSK9: a protease that targets the LDL receptor for lysosomal degradation Figure 13.11 The four proteins associated with familial hypercholesterolemia. The low-density lipoprotein (LDL) receptor binds apoprotein B-100. Pathogenic variants in the LDL receptor-binding domain of apoprotein B-100 impair LDL binding to its receptor, reducing the removal of LDL cholesterol from the circulation. Clustering of the LDL receptor–apoprotein B-100 complex in clathrincoated pits requires the ARH adaptor protein, which links the receptor to the endocytic machinery of the coated pit. Homozygous variants in the ARH protein impair the internalization of the LDL:LDL receptor complex, thereby impairing LDL clearance. PCSK9 protease activity targets LDL receptors for lysosomal degradation, preventing them from recycling back to the plasma membrane (see text). _ 1000 800 600 400 200 0 Plasma cholesterol (mg/d L) Mean +2 SD Normal Obligate heterozygotes Homozygotes Figure 13.12 Gene dosage in low-density lipoprotein (LDL) deficiency. Shown is the distribution of total plasma cholesterol levels in 49 patients homozygous for deficiency of the LDL receptor, their parents (obligate heterozygotes), and normal controls. (Redrawn from Goldstein JL, Brown MS: Familial hypercholesterolemia. In Scriver CR, Beaudet AL, Sly WS, et al, editors: The metabolic bases of inherited disease, ed 6, New York, 1989, Mc Graw-Hill, pp 1215–1250.) ▶
264 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE carriers of mutant alleles. Physical stigmata of familial hypercholesterolemia include xanthomas (cholesterol deposits in skin and tendons)...
Ch13 · Pt15 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 265 Cholesterol Uptake by the LDL Receptor. Normal cells obtain cholesterol from either de novo synthesis or the uptake from plasma of exogenous cholesterol bound to lipoproteins, especially LDL. The majority of LDL uptake is mediated by the LDL receptor, which recognizes apoprotein B-100, the protein moiety of LDL. LDL receptors on the cell surface are localized to invaginations (coated pits) lined by the protein clathrin (Fig. 13.13). Receptor-bound LDL is brought into the cell by endocytosis of the coated pits, which ultimately evolve into lysosomes in which LDL is hydrolyzed to release free cholesterol. The increase in free intracellular cholesterol reduces endogenous cholesterol formation by suppressing the rate-limiting enzyme of the synthetic pathway, 3-hydroxy-3-methylglutaryl coenzyme A (HMG Co A) reductase. Cholesterol not required for cellular metabolism or membrane synthesis may be reesterified for storage as cholesteryl esters, a process stimulated by the activation of acyl-coenzyme A:cholesterol acyltransferase (ACAT). The increase in intracellular cholesterol also reduces synthesis of the LDL receptor (see Fig. 13.13). Classes of Variants in the LDL Receptor More than 1100 different variants have been identified in the LDLR gene, and these are distributed throughout the gene and protein sequence. Not all of the reported changes are functionally significant, and some disturb receptor function more severely than others. The great majority of alleles are single nucleotide substitutions, small insertions, or deletions; structural rearrangements account for only 2% to 10% of the LDLR alleles in most populations. The mature LDL receptor has five distinct structural domains that for the most part have distinguishable functions that mediate the steps in the life cycle of an LDL receptor, shown in Fig. 13.13. Analysis of the effect on the receptor of variants in each domain has played an important role in defining the function Apoprotein B-100 Cholesteryl ester MUTANT CLASS: EVENT DISRUPTED BY MUTATION: Class 1 Receptor synthesis Class 2 Receptor transport ER Golgi Class 3 LDL binding by receptor Plasma LDL Class 4 Receptor clustering in coated pit Class 6 Defective targeting to the basolateral membrane Mature LDL receptor Vesicle Golgi complex A) B) C) LDL receptor synthesis HMG Co A reductase ACAT Cholesteryl ester droplets Free cholesterol Lysosome Amino acids Coated vesicle Endoplasmic reticulum Endosome Recycling vesicle Class 5 Failure to discharge LDL in endosome (recycling defect) Coated pit H+ Figure 13.13 The cell biology and biochemical role of the low-density lipoprotein (LDL) receptor and the six classes of variants that alter its function. After synthesis in the endoplasmic reticulum (ER), the receptor is transported to the Golgi apparatus and subsequently to the cell surface. Normal receptors are localized to clathrin-coated pits, which invaginate, creating coated vesicles and then endosomes, the precursors of lysosomes. Normally, intracellular accumulation of free cholesterol is prevented because the increase in free cholesterol (A) decreases the formation of LDL receptors, (B) reduces de novo cholesterol synthesis, and (C) increases the storage of cholesteryl esters. The biochemical phenotype of each class of mutant is discussed in the text. ACAT, Acyl-coenzyme A:cholesterol acyltransferase; HMG Co A reductase, 3-hydroxy-3-methylglutaryl coenzyme A reductase. (Modified from Brown MS, Goldstein JL: The LDL receptor and HMG-Co A reductase – Two membrane molecules that regulate cholesterol homeostasis, Curr Top Cell Regul 26:3–15, 1985.) ▶
CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 265 Cholesterol Uptake by the LDL Receptor. Normal cells obtain cholesterol from either de novo synthesis or the uptake f...
Ch13 · Pt16 266 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE of each domain. These studies exemplify the important contribution that genetic analysis can make in determining the structure-function relationships of a protein. Fibroblasts cultured from affected patients have been used to characterize the mutant receptors and the resulting disturbances in cellular cholesterol metabolism. LDLR variants can be grouped into six classes, depending on which step of the normal cellular itinerary of the receptor is disrupted by the variant (see Fig. 13.13). Class 1 variants are null alleles that prevent the synthesis of any detectable receptor; they are the most common type of disease-causing variants at this locus. In the remaining five classes, the receptor is synthesized normally, but its function is impaired. Class 2 variants (like those in classes 4 and 6) define features of the polypeptide critical to its subcellular localization. The relatively common class 2 variants are designated transport deficient because the LDL receptors accumulate at the site of their synthesis, the ER, instead of being transported to the Golgi complex. These alleles are predicted to prevent proper folding of the protein, an apparent requisite for exit from the ER. Class 3 variant receptors reach the cell surface but are incapable of binding LDL. Class 4 variants impair localization of the receptor to the coated pit, and consequently the bound LDL is not internalized. These variants alter or remove the cytoplasmic domain at the carboxyl terminus of the receptor, demonstrating that this region normally targets the receptor to the coated pit. Class 5 variants are recycling-defective alleles. Receptor recycling requires the dissociation of the receptor and the bound LDL in the endosome. Variants in the epidermal growth factor precursor homology domain prevent the release of the LDL ligand. This failure leads to degradation of the receptor, presumably because an occupied receptor cannot return to the cell surface. Class 6 variants lead to defective targeting of the mutant receptor to the basolateral membrane, a process that depends on a sorting signal in the cytoplasmic domain of the receptor. Variants affecting the signal can mistarget the mutant receptor to the apical surface of hepatic cells, thereby impairing the recycling of the receptor to the basolateral membrane and leading to an overall reduction of endocytosis of the LDL receptor. The PCSK9 Protease, a Drug Target for Lowering LDL Cholesterol Rare cases of autosomal dominant familial hypercholesterolemia have been found to result from gain-of-function missense variants in the gene encoding PCSK9 protease (proprotein convertase subtilisin/kexin type 9). The role of PCSK9 is to target the LDL receptor for lysosomal degradation, thereby reducing receptor abundance at the cell surface (see Fig. 13.11). Consequently, the increase in PSCK9 activity associated with gain-of-function variants reduces the levels of the LDL receptor at the cell surface below normal, leading to increased blood levels of LDL cholesterol and coronary heart disease. Conversely, loss-of-function variants in the PCSK9 gene result in an increased number of LDL receptors at the cell surface by decreasing the activity of the protease. More receptors increase cellular uptake of LDL cholesterol, lowering cholesterol and providing protection against coronary artery disease. Notably, the complete absence of PCSK9 activity in the few known individuals with two PCSK9 null alleles appears to have no adverse clinical consequences. Some PCSK9 Sequence Variants Protect Against Coronary Heart Disease. The link between monogenic familial hypercholesterolemia and the PCSK9 gene suggested that common sequence variants in PCSK9 might be linked to very high or very low LDL cholesterol levels in the general population. Importantly, several PCSK9 sequence variants are strongly linked to low levels of plasma LDL cholesterol (Table 13.3). For example, a study that used US census definitions showed that in the Black population one of two PCSK9 nonsense variants is found in 2.6% of all subjects; each variant is associated with a mean reduction in LDL cholesterol of ~40%. This reduction in LDL cholesterol has a powerful protective effect against coronary artery disease, reducing the risk by ~90%; only ~1% of Black subjects carrying one of these two PCSK9 nonsense variants developed coronary artery disease over a 15-year period, compared to almost 10% of individuals without either variant. A missense allele (p. Arg 46Leu) is more common in white US census category populations (3.2% of subjects) but appears to confer only a ~50% reduction in coronary heart disease. These findings have major public health implications because they suggest that modest but lifelong reductions in plasma LDL TABLE 13.3 PCSK9 Variants Associated With Low LDL Cholesterol Levels Sequence Variant Population Frequency LDL Cholesterol Level (Normal ≤~100 mg/d L) Impact on Incidence of Coronary Heart Disease Null or dominant negative alleles Rare genetic compounds, one dominant negative heterozygote 7–16 mg/d L Unknown, but likely to greatly reduce risk Tyr 142Stop or Cys 679Stop Black heterozygotes: 2.6% Mean: 28% (38 mg/d L) 90% reduction Arg 46Leu white heterozygotes: 3.2% Mean: 15% (20 mg/d L) 50% reduction LDL, Low-density lipoprotein. Derived from Cohen JC, Boerwinkle E, Mosley TH, et al: Sequence variants in PCSK9, low LDL, and protection against coronary heart disease, N Engl J Med 354: 1264–1272, 2006. ▶
266 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE of each domain. These studies exemplify the important contribution that genetic analysis can make in determining the structure-function rela...
Ch13 · Pt17 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 267 cholesterol levels of 20 to 40 mg/d L would significantly decrease the incidence of coronary heart disease in the population. The strong protective effect of PCSK9 lossof-function alleles, together with the apparent absence of any clinical sequelae in subjects with a total absence of PCSK9 activity, made PCSK9 a strong candidate target for drugs that inactivate or diminish the activity of the enzyme (see Chapter 14). Finally, these discoveries emphasize how the investigation of rare genetic disorders can lead to important new knowledge about the genetic contribution to common genetically complex diseases. Clinical Implications of the Genetics of Familial Hypercholesterolemia. Early diagnosis of the familial hypercholesterolemias is essential both to permit the prompt application of cholesterol-lowering therapies to prevent coronary artery disease and to initiate genetic screening of first-degree relatives. With appropriate drug therapy, familial hypercholesterolemia heterozygotes have a normal life expectancy. For homozygotes, onset of coronary artery disease can be remarkably delayed by plasma apheresis (which removes the hypercholesterolemic plasma) but will ultimately require liver transplantation. Finally, the elucidation of the biochemical basis of familial hypercholesterolemia has had a profound impact on the treatment of the vastly more common forms of sporadic hypercholesterolemia by leading to the development of the statin class of drugs that inhibit de novo cholesterol biosynthesis (see Chapter 14). Newer therapies include monoclonal antibodies that directly target PCSK9 and lower LDL cholesterol by an additional 60% in clinical trials, prompting approval and use around the world. TRANSPORT DEFECTS Cystic Fibrosis Since the 1960s, CF has been one of the most publicly visible of all human monogenic diseases (Case 12). It is the most common autosomal recessive genetic disorder of children in populations of European ancestry in the United States, with an incidence of ~1 in 2500 births (and thus a carrier frequency of ~1 in 25), whereas it is much less prevalent in other population groups, such as Blacks (1 in 15,000 births) and Asians (1 in 31,000 births). The isolation of the CF gene (called CFTR, for CF transmembrane regulator) (see Chapter 11) more than 30 years ago was one of the first illustrations of the power of molecular genetic and genomic approaches to identify disease genes. Physiologic analyses have shown that the CFTR protein is a regulated chloride channel located in the apical membrane of the epithelial cells affected by the disease. The Phenotypic Features of Cystic Fibrosis. The lungs and exocrine pancreas are the principal organs affected by CF (Case 12), but a major diagnostic feature is increased sweat sodium and chloride concentrations (often first noted when parents kiss their infant). CF is most commonly identified by newborn screening in regions of the world where that is offered. Elsewhere, in most CF patients, the diagnosis is initially based on the clinical pulmonary or pancreatic findings and on an elevated level of sweat chloride. Less than 2% of patients have normal sweat chloride concentration despite an otherwise typical clinical picture; in these cases, molecular analysis can be used to ascertain whether they have pathogenic variants in the CFTR gene. The pancreatic defect in CF is a maldigestion syndrome due to the deficient excretion of pancreatic enzymes (lipase, trypsin, chymotrypsin). Approximately 5% to 15% of patients with CF have enough residual pancreatic exocrine function for normal digestion and are designated “pancreatic sufficient.” Moreover, patients with CF who are pancreatic sufficient have better growth and overall prognosis than the majority, who are “pancreatic insufficient.” The clinical heterogeneity of the pancreatic disease is at least partly due to allelic heterogeneity, as discussed later. Many other features are observed in CF patients. For example, neonatal lower intestinal tract obstruction (meconium ileus) occurs in 10% to 15% of CF newborns. The genital tract is also affected; females with CF have some reduction in fertility, but more than 98% of males with CF are infertile because they lack the vas deferens, a phenotype known as congenital bilateral absence of the vas deferens (CBAVD). In a striking example of allelic heterogeneity giving rise to a partial phenotype, it has been found that some infertile males who are otherwise well (i.e., have no pulmonary or pancreatic disease) have CBAVD associated with specific variants in the CFTR gene. Similarly, some individuals with idiopathic chronic pancreatitis are carriers of variants in CFTR yet lack other clinical signs of CF. The CFTR Gene and Protein. The CFTR gene has 27 exons and spans ~190 kb of DNA. The CFTR protein encodes a large integral membrane protein of ~170 k D (Fig. 13.14). The protein belongs to the ABC (ATP [adenosine triphosphate]–binding cassette) family of transport proteins. At least 27 ABC transporters have been implicated in mendelian disorders and complex trait phenotypes. The CFTR chloride channel has five domains, shown in Fig. 13.14: two membrane-spanning domains, each with six transmembrane sequences; two nucleotide (ATP)–binding domains; and a regulatory domain with multiple phosphorylation sites. The importance of each domain is demonstrated by the identification of CF-causing missense variants in each of them (see Fig. 13.14). The pore of the chloride channel is formed by the 12 transmembrane segments. ATP is bound and hydrolyzed by the nucleotide-binding domains, and the energy released is used to open and close the channel. Regulation of the channel is mediated, at least in part, by phosphorylation of the regulatory domain. ▶
CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 267 cholesterol levels of 20 to 40 mg/d L would significantly decrease the incidence of coronary heart disease in the pop...
Ch13 · Pt18 268 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE The Pathophysiology of Cystic Fibrosis. CF is due to abnormal fluid and electrolyte transport across epithelial apical membranes. This abnormality leads to disease in the lung, pancreas, intestine, hepatobiliary tree, and male genital tract. The physiologic abnormalities have been most clearly elucidated for the sweat gland. The loss of CFTR function means that chloride in the duct of the sweat gland cannot be reabsorbed, leading to a reduction in the electrochemical gradient that normally drives sodium entry across the apical membrane. This defect leads, in turn, to the increased chloride and sodium concentrations in sweat. The effects on electrolyte transport due to the abnormalities in the CFTR protein have also been carefully studied in airway and pancreatic epithelia. In the lung, the hyperabsorption of sodium and reduced chloride secretion result in a depletion of airway surface liquid. Consequently, the mucous layer of the lung may become adherent to cell surfaces, disrupting the cough and cilia-dependent clearance of mucus and providing a niche favorable to Pseudomonas aeruginosa, the major cause of chronic pulmonary infection in CF. The Genetics of Cystic Fibrosis Pathogenic Variants in the Cystic Fibrosis Trans­ membrane Regulator Polypeptide. The most common CF pathogenic variant is a deletion of a phenylalanine residue at position 508 (p. Phe 508del, shortened to F508del) in the first ATP-binding fold (NBD1; see Fig. 13.14), accounting for ~70% of all CF alleles in populations of European ancestry. In these populations, only seven other pathogenic variants are more frequent than 0.5%, and the remainder are each quite rare. Variants of all types have been identified, but the largest single group (nearly half) are missense substitutions. The remainder are point variants of other types, and less than 1% are genomic rearrangements. Although nearly 2000 CFTR gene sequence variants have been associated with disease, the actual number of missense variants that are disease-causing is uncertain because few have been subjected to functional analysis. However, a project called the Clinical and Functional Translation of CFTR (CFTR2 project; cftr 2.org) has succeeded in assigning pathogenicity to more than 466 CFTR variants (including 174 missense variants and in frame deletions), which together account for at least 96% of all CFTR alleles worldwide. Although the specific biochemical abnormalities associated with most CF alleles are not known, six general classes of dysfunction of the CFTR protein have been identified to date. Alleles representative of each class are shown in Fig. 13.14. Class 1 variants are null alleles – no CFTR polypeptide is produced. This class includes alleles with premature stop codons or those that generate highly 23 21 19 15 14b 13 11 9 5 MSD 1 exons NBD1 exons R-domain exon F508del (Phe 508del) is the most common CF allele in those of European ancestry: frequency = 0.68 MSD 2 exons NBD2 exons 3 1 Exon CFTR gene Phe 508del Cell membrane NBD 1 NBD 2 C MSD 2 MSD 1 N CFTR protein Splice mutation intron 4 donor site (G T) Class 1 Absent protein Class 5 Reduced expression of the CFTR gene Arg 117His Class 4 Defective conduction due to alteration of Cl– channel 507 508 509 -Ile Phe Gly- -ATC TTT GGTClass 2 Major block in protein maturation Gly 551Asp Class 3 Defective gating Gln 1412Stop Class 6 Instability at the cell surface R-domain Phe 508del Figure 13.14 The structure of the CFTR gene and a schematic of the CFTR protein. Selected variants are shown. The exons, introns, and domains of the protein are not drawn to scale. Phe 508del results from the deletion of TCT or CTT, replacing the Ile codon with ATT, and deleting the Phe codon. CF, Cystic fibrosis; MSD, membrane-spanning domain; NBD, nucleotide-binding domain; R-domain, regulatory domain. (Based on Zielinski J: Genotype and phenotype in cystic fibrosis, Respiration 67:117–133, 2000.) ▶
268 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE The Pathophysiology of Cystic Fibrosis. CF is due to abnormal fluid and electrolyte transport across epithelial apical membranes. This abnor...
Ch13 · Pt19 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 269 unstable RNAs. Because CFTR is a glycosylated membrane-spanning protein, it must be processed in the endoplasmic reticulum and Golgi apparatus to be glycosylated and secreted. Class 2 variants impair the folding of the CFTR protein, thereby arresting its maturation. The F508del variant typifies this class; this misfolded protein cannot exit from the endoplasmic reticulum. However, the biochemical phenotype of the F508del protein is complex because it also exhibits defects in stability and activation in addition to impaired folding. Class 3 variants allow normal delivery of the CFTR protein to the cell surface but disrupt its function (see Fig. 13.14). The prime example is the p. Gly 551Asp variant that impedes the opening and closing of the CFTR ion channel at the cell surface. Class 4 variants are located in the membrane-spanning domains and, consistent with this localization, have defective chloride ion conduction. Class 5 variants reduce the number of CFTR tran­scripts. Class 6 mutant proteins are synthesized normally but are unstable at the cell surface. A Cystic Fibrosis Genocopy: Pathogenic Variants in the Epithelial Sodium Channel Gene SCNN1. Although CFTR is the only gene that has been associated with classic CF, several families with nonclassic presentations (including CF-like pulmonary infections, less severe intestinal disease, elevated sweat chloride levels) have been found to carry pathogenic variants in the epithelial sodium channel gene SCNN1, a genocopy, that is, a phenotype that, although genetically distinct, has a very closely related phenotype. This finding is consistent with the functional interaction between the CFTR protein and the epithelial sodium channel. Its main clinical significance, at present, is the demonstration that patients with nonclassic CF display locus heterogeneity and that if CFTR pathogenic variants are not identified in a particular case, abnormalities in SCNN1 must be considered. Genotype-Phenotype Correlations in Cystic Fibrosis. Because all patients with the classic form of CF appear to have pathogenic variants in the CFTR gene, clinical heterogeneity in CF must arise from allelic heterogeneity, from the effects of other modifying loci, or from nongenetic factors. Independent of the CFTR alleles that a particular patient may have, a significant genetic contribution from other (modifier) genes to several CF phenotypes has been recognized, with effects on lung function, neonatal intestinal obstruction, and diabetes. Two generalizations have emerged from the genetic and clinical analysis of patients with CF. First, the specific CFTR genotype is a good predictor of exocrine pancreatic function. For example, patients homozygous for the common F508del variant or for predicted null alleles generally have pancreatic insufficiency. On the other hand, alleles that allow the synthesis of a partially functional CFTR protein, such as Arg 117His (see Fig. 13.14), tend to be associated with pancreatic sufficiency. Second, however, the specific CFTR genotype is a poor predictor of the severity of pulmonary disease. For example, among patients homozygous for the F508del variant, the severity of lung disease is variable. One reason for this poor phenotype-genotype correlation is inherited variation in the gene encoding transforming growth factor β1 (TGFβ1), as also discussed in Chapter 9. Overall, the evidence indicates that TGFB1 alleles that increase TGFβ1 expression lead to more severe CF lung disease, perhaps by modulating tissue remodeling and inflammatory responses. Other genetic modifiers of CF lung disease, including alleles of the interferon-related developmental regulator 1 gene (IFRD1) and the interleukin-8 gene (IL8), may act by influencing the ability of the CF lung to tolerate infection. Similarly, a few modifier genes have been identified for other CF-related phenotypes, including diabetes, liver disease, and meconium ileus. The Cystic Fibrosis Gene in Populations. At present, it is not possible to account for the high frequency of disease-causing CFTR alleles (about 1 in 25) among populations of European descent (see Chapter 9). The disease is much less frequent in others, although it has been reported in those of Indigenous, African, and Asian descent (e.g., ~1 in 90,000 Hawaiians of Asian descent). The F508del allele is the only one found to date that is common in virtually all populations of European ancestry, but its frequency among all pathogenic alleles varies significantly in different European populations, from 88% in Denmark to 45% in southern Italy. In populations in which the F508del allele frequency is ~70% of all mutant alleles, ~50% of patients are homozygous for the F508del allele; an additional 40% are genetic compounds for F508del and another mutant allele. In addition, ~70% of CF carriers have the F508del variant. As noted earlier, except for F508del, other variants at the CFTR locus are rare, although in specific populations some alleles are relatively common. Population Screening. Both carrier screening and newborn screening for CF is offered universally in the United States, Canada, Australia, New Zealand, most of western Europe, Russia, Brazil, Argentina, and Chile. Genetic Analysis of Families of Patients and Prenatal Diagnosis. The high frequency of the F508del allele is useful when CF patients without a family history present for DNA diagnosis. The identification of the F508del allele, in combination with a panel of 127 common variants suggested by the American College of Medical Genetics and Genomics, can be used to predict the status of family members for confirmation of disease (e.g., in a newborn or a sibling with an ambiguous ▶
CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 269 unstable RNAs. Because CFTR is a glycosylated membrane-spanning protein, it must be processed in the endoplasmic reti...
Ch13 · Pt20 270 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE presentation), carrier detection, and prenatal diagnosis. Given the vast knowledge of CFTR variants in many populations, direct variant detection is the method of choice for genetic analysis. For couples with a 25% risk, preimplantation genetic testing following in vitro fertilization can be offered; alternatively, for fetuses with a 1 in 4 risk, prenatal diagnosis by DNA analysis at 10 to 12 weeks, with tissue obtained by chorionic villus biopsy, is the method of choice (see Chapter 18). Molecular Genetics and the Treatment of Cystic Fibrosis. Historically, the treatment of CF has been directed toward controlling pulmonary infection and improving nutrition. Increasing knowledge of the molecular pathogenesis has made it possible to design pharmacologic interventions that modulate CFTR function in most patients (see Chapter 14). Alternatively, gene transfer therapy may be possible in the future for CF, but there are many difficulties. DISORDERS OF STRUCTURAL PROTEINS The Dystrophin Glycoprotein Complex: Duchenne, Becker, and Other Muscular Dystrophies Like CF, Duchenne muscular dystrophy (DMD) has long received attention from the general and medical communities as a relatively common, severe, and progressive muscle-wasting disease with relentless clinical deterioration (Case 14). The isolation of the gene affected in this X-linked disorder and the characterization of its protein (named dystrophin because of its association with DMD) have given insight into every aspect of the disease, greatly improved the genetic counseling of affected families, and suggested strategies for treatment. The study of dystrophin led to the identification of a major complex of other muscular dystrophy–associated muscle membrane proteins, the dystrophin glycoprotein complex (DGC), described later in this section. The Clinical Phenotype of Duchenne Muscular Dystrophy. Affected boys are normal for the first 1 to 2 years of life but develop muscle weakness by 3 to 5 years of age, when they begin to have difficulty climbing stairs and rising from a sitting position. The child is typically confined to a wheelchair by the age of 12 years. Although DMD is currently incurable, recent advances in the management of pulmonary and cardiac complications (which were leading causes of death in boys with DMD) have changed the disease from a life-limiting to a life-­threatening disorder. In the preclinical and early stages of the disease, the serum level of creatine kinase is grossly elevated (50–100 times the upper limit of normal) because of its release from diseased muscle. The brain is also affected; on average, there is a moderate decrease in IQ of ~20 points. The Clinical Phenotype of Becker Muscular Dys­ trophy. Becker muscular dystrophy (BMD) is also due to pathogenic variants in the dystrophin gene, but the BMD alleles produce a much milder phenotype and patients often remain ambulant beyond the teenage years. In general, patients with BMD carry ­variant alleles that maintain the reading frame of the protein and thus express some dystrophin, albeit often an altered product at reduced levels. Dystrophin is generally demonstrable in the muscle of patients with BMD (Fig. 13.15). In contrast, patients with DMD have little or no detectable dystrophin. The Genetics of Duchenne Muscular Dystrophy and Becker Muscular Dystrophy Inheritance. DMD has an incidence of ~1 in 3300 live male births, with a calculated mutation rate of 10−4, an order of magnitude higher than the rate observed in genes involved in most other genetic diseases (see Chapter 4). In fact, given a production of ~8 × 107 sperm per day, a normal male produces a sperm with a new mutation in the DMD gene every 10 to 11 seconds! In Chapter 7, DMD was presented as a typical X-linked recessive disorder that is lethal in males, so that one-third of cases are predicted to be due to new mutations and two-thirds of patients have carrier mothers (see also Chapter 17). The great majority of carrier females have no clinical manifestations, although ~70% have slightly elevated levels of serum creatine kinase. In accordance with random inactivation of the X chromosome (see Chapter 6), however, the X chromosome carrying the normal DMD allele appears to be inactivated above a critical threshold of cells in some female heterozygotes. Nearly 20% of adult female carriers have some muscle weakness; whereas in 8%, life-threatening cardiomyopathy and serious proximal muscle disability occur. In rare instances, females have been described with DMD. Some have X;autosome translocations (see Chapter 6), whereas others have only one X chromosome (Turner syndrome) with a DMD pathogenic variant on that chromosome. BMD accounts for ~15% of the variants at the locus. An important genetic distinction between these allelic phenotypes is that whereas DMD is a genetic lethal, the reproductive fitness of males with BMD is high (up to ~70% of normal) so that they can transmit the mutant gene to their daughters. Consequently, and in contrast to DMD, a high proportion of BMD cases are inherited, and relatively few (only ~10%) represent new mutations. The DMD Gene and Its Product. The most remarkable feature of the DMD gene is its size, estimated to be >2000kb, or ~1.5% of the entire X chromosome. This huge gene is among the largest known in any species, by an order of magnitude. The high mutation rate can be at least partly explained by the fact that the locus is a large target for mutation but, as described later, it is also structurally prone to deletion and duplication. The DMD gene is complex, with 79 exons and seven tissue-specific promoters. In muscle, the large (14-kb) ▶
270 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE presentation), carrier detection, and prenatal diagnosis. Given the vast knowledge of CFTR variants in many populations, direct variant dete...
Ch13 · Pt21 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 271 dystrophin transcript encodes a huge 427-k D protein. In accordance with the clinical phenotype, the protein is most abundant in skeletal and cardiac muscle, although many tissues express at least one dystrophin isoform. The Molecular and Physiologic Defects in Becker Muscular Dystrophy and Duchenne Muscular Dystrophy. The most common molecular defects in patients with DMD are deletions (60% of alleles), which are not randomly distributed. Rather, they are clustered in either the 5′ half of the gene or in a central region that encompasses an apparent deletion hot spot. The mechanism of deletion in the central region is unknown, but it appears to involve the tertiary structure of the genome and, in some cases, recombination between Alu repeat sequences (see Chapter 2) in large central introns. Point variants account for approximately one-third of the alleles and are randomly distributed throughout the gene. The absence of dystrophin in DMD destabilizes the myofiber membrane, increasing its fragility and allowing increased Ca++ entry into the cell, with subsequent activation of inflammatory and degenerative pathways. In addition, the chronic degeneration of myofibers eventually exhausts the pool of myogenic stem cells that are normally activated to regenerate muscle. This reduced regenerative capacity eventually leads to the replacement of muscle with fat and fibrotic tissue. The Dystrophin Glycoprotein Complex. Dystrophin is a structural protein that anchors the DGC at the cell membrane. The DGC is a veritable constellation of polypeptides associated with more than a dozen genetically distinct muscular dystrophies (Fig. 13.16). This complex serves several major functions. First, it is thought to be essential for the maintenance of muscle membrane integrity, by linking the actin cytoskeleton to the extracellular matrix. Second, it is required to position the proteins in the complex at the sarcolemma. Although the function of many of the proteins in the complex is unknown, their association with diseases of muscle indicates that they are essential components of the complex. Pathogenic variants in several of these proteins cause autosomal recessive limb girdle muscular dystrophies and other congenital muscular dystrophies (see Fig. 13.16). Normal BMD DMD Figure 13.15 Microscopic visualization of the effect of pathogenic variants in the dystrophin gene in a patient with Becker muscular dystrophy (BMD) and a patient with Duchenne muscular dystrophy (DMD). (Left column) Hematoxylin and eosin staining of muscle. (Right column) Immunofluorescence microscopy staining with an antibody specific to dystrophin. Note the localization of dystrophin to the myocyte membrane in normal muscle, the reduced quantity of dystrophin in BMD muscle, and the complete absence of dystrophin from the myocytes of the DMD muscle. The amount of connective tissue between the myocytes in the DMD muscle is increased. (Courtesy K. Arahata, National Institute of Neuroscience, Tokyo.) ▶
CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 271 dystrophin transcript encodes a huge 427-k D protein. In accordance with the clinical phenotype, the protein is most...
Ch13 · Pt22 272 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE That each component of the DGC is affected by variants that cause other types of muscular dystrophies highlights the principle that no protein functions in isolation but rather is a component of a biologic pathway or a multiprotein complex. Variants in the genes encoding other components of a pathway or a complex often lead to genocopies. Posttranslational Modification of the Dystrophin Glycoprotein Complex. Five of the muscular dystrophies associated with the DGC result from pathogenic variants in glycosyltransferases, leading to hypoglycosylation of α-dystroglycan. That five proteins are required for the posttranslational modification of one other polypeptide testifies to the critical nature of glycosylation to the function of α-dystroglycan in particular but, more generally, to the importance of posttranslational modifications for the normal function of most proteins. Clinical Applications of Gene Testing in Muscular Dystrophy Prenatal Diagnosis and Carrier Detection. With genebased technologies, accurate carrier detection and prenatal diagnosis are available for most families with a history of DMD. In the 60% to 70% of families in whom the allele results from a deletion or duplication, the presence or absence of the defect can be assessed by examination of fetal DNA using methods that assess the gene’s genomic continuity and size (see Fig. 13.17). Cytosol Sarcolemma Extracellular Matrix BMD CMD DMD XDCM Dystrophin-Glycoprotein Complex LGMD MDDGs MDDGs Actin Dp 427 Rod CYS WW C N Na Ch Sarcospan Sarcoglycans Laminin (2 chain) -DG ▶
SYNs Dystrobrevin SYNs n NOS -DG Figure 13.16 In muscle, dystrophin links the extracellular matrix (laminin) to the actin cytoskeleton. Dystrophin interacts with a multimeric complex composed of the d...
Ch13 · Pt23 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 273 In most other families, single nucleotide variants can be identified by sequencing of the coding region and intron-exon boundaries. Because the disease has a very high frequency of new mutations and is not manifested in carrier females, ~80% of Duchenne boys are born into families with no previous history of the disease (see Chapter 7). Thus the incidence of DMD will not decrease substantially until universal prenatal or preconception screening for the disease is possible. Maternal Mosaicism. If a boy with DMD is the first affected member of his family, and if his mother is not found to carry the variant in her lymphocytes, the usual explanation is that he has a new mutation at the DMD locus. However, ~5% to 15% of such cases appear to be due to maternal gonadal mosaicism, in which case the recurrence risk is significant (see Chapter 7). Therapy. At present, only symptomatic treatment is available for DMD. The possibilities for rational therapy for DMD have greatly increased with the understanding of the normal role of dystrophin in the myocyte. Some of the therapeutic considerations are discussed in Chapter 14. Pathogenic Variants in Genes That Encode Collagen or Other Components of Bone Formation: Osteogenesis Imperfecta Osteogenesis imperfecta (OI) is a group of inherited disorders that predispose to skeletal deformity and easy fracturing of bones, even with little trauma (Fig. 13.18). The combined incidence of all forms of the disease is ~1 per 10,000. Approximately 95% of affected individuals have heterozygous pathogenic variants in one of two genes, COL1A1 and COL1A2, that encode the chains of type I collagen, the major protein in bone. A remarkable degree of clinical variation has been recognized, from lethality in the perinatal period to only a mild increase in fracture frequency. The clinical heterogeneity is explained by both locus and allelic heterogeneity; the phenotypes are influenced by which chain of type I procollagen is affected and according to the type and location of the pathogenic variant at the locus. The major phenotypes and genotypes associated with variants in the type I collagen genes are outlined in Table 13.4. Normal Collagen Structure and Its Relationship to Osteogenesis Imperfecta It is important to appreciate the major features of normal type I collagen to understand the pathogenesis of OI. The type I procollagen molecule is formed from two proα1(I) chains (encoded by COL1A1) and one similar but distinct proα2(I) chain (encoded by COL1A2) (Fig. 13.19). Proteins composed of subunits, like collagen, are often subject to variants that prevent subunit association by altering the subunit interfaces. The triple helical (collagen) section is composed of 338 tandemly arranged Gly-X-Y repeats; proline is often in the X position, and hydroxyproline or hydroxylysine is often in the Y position. Glycine, the smallest amino acid, is the only residue compact enough to occupy the axial position of the helix, and consequently, variants that substitute other residues for those glycines are highly disruptive to the helical structure. Several features of procollagen maturation are of special significance to the pathophysiology of OI. First, the assembly of the individual proα chains into the trimer begins at the carboxy terminus, and triple helix formation progresses toward the amino terminus. Consequently, variants that alter residues in the carboxy-terminal part of the triple helical domain are more disruptive because they interfere earlier with the propagation of the triple helix (Fig. 13.20). Second, the Figure 13.18 Radiograph of a premature (26 weeks of gestation) infant with the perinatal lethal form (type II) of osteogenesis imperfecta. The skull is relatively large and unmineralized and was soft to palpation. The thoracic cavity is small, the long bones of the arms and legs are short and deformed, and the vertebral bodies are flattened. All the bones are undermineralized. (Courtesy T. Costa, The Hospital for Sick Children, Toronto.) ▶
CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 273 In most other families, single nucleotide variants can be identified by sequencing of the coding region and intron-ex...
Ch13 · Pt24 274 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE posttranslational modification (e.g., proline or lysine hydroxylation; hydroxylysyl glycosylation) of procollagen continues on any part of a chain not assembled into the triple helix. Thus, when triple helix assembly is slowed by a change, the unassembled sections of the chains amino-terminal to the defect are modified excessively, which slows their secretion into the extracellular space. Overmodification may also interfere with the formation of collagen fibrils. As a result of all these abnormalities, the number of secreted collagen molecules is reduced, and many of them are abnormal. In bone, the abnormal chains and their reduced number lead to defective mineralization of collagen fibrils (see Fig. 13.18). Molecular Abnormalities of Collagen in Osteogenesis Imperfecta More than 2000 different pathogenic variants affecting the synthesis or structure of type I collagen have been found in individuals with OI. The clinical heterogeneity of this disease reflects even greater heterogeneity at the molecular level (see Table 13.4). For the type I collagen TABLE 13.4 Summary of the Genetic, Biochemical, and Molecular Features of the Types of Osteogenesis Imperfecta Due to Variants in Type 1 Collagen Genes Type Phenotype Inheritance Biochemical Defect Gene Defect Defective Production of Type I Collagen* I Mild: blue sclerae, brittle bones but no bone deformity Autosomal dominant All the collagen made is normal (i.e., solely from the normal allele), but the quantity is reduced by half Largely null alleles that impair the production of proα1(I) chains, such as defects that interfere with mRNA synthesis Structural Defects in Type I Collagen II Perinatal lethal: severe skeletal abnormalities, dark sclerae, death within 1 mo (see Fig. 13.18) Autosomal dominant (new mutation) Production of abnormal collagen molecules due to substitution of the glycine in Gly-X-Y of the triple helical domain located, in general, throughout the protein Missense variants in the glycine codons of the genes for the α1 and α2 chains III Progressive deforming: with blue sclerae; fractures, often at birth; progressive bone deformity, limited growth Autosomal dominant† IV Normal sclerae, deforming: mildmoderate bone deformity, short stature, fractures Autosomal dominant mRNA, Messenger RNA. Modified from Byers PH: Disorders of collagen biosynthesis and structure. In Scriver CR, Beaudet AL, Sly WS, et al, eds: The metabolic basis of inherited disease, ed 6, New York, 1989, Mc Graw-Hill, pp 2805–2842; Byers PH: Brittle bones – Fragile molecules: disorders of collagen structure and expression, Trends Genet 6:293–300, 1990. Type I procollagen Triple helix Type I collagen Collagen fibrils Mineralization (in bone) Protease cleavage site Protease cleavage site Aminoterminal peptide Carboxylterminal peptide proα1(I) proα1(I) proα2(I) Figure 13.19 The structure of type I procollagen. Each collagen chain is made as a procollagen triple helix that is secreted into the extracellular space. The amino- and carboxyl-terminal domains are cleaved extracellularly to form collagen; mature collagen fibrils are then assembled and, in bone, mineralized. Note that type I procollagen is composed of two proα1(I) chains and one proα2(I) chain. (Redrawn from Byers PH: Disorders of collagen biosynthesis and structure. In Scriver CR, Beaudet AL, Sly WS, et al, editors: The metabolic bases of inherited disease, ed 6, New York, 1989, Mc Graw-Hill, pp 2805–2842.) *A few patients with type I disease have substitutions of glycine in one of the type I collagen chains. †Rare cases are autosomal recessive. ▶
274 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE posttranslational modification (e.g., proline or lysine hydroxylation; hydroxylysyl glycosylation) of procollagen continues on any part of a...
Ch13 · Pt25 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 275 genes, the variants fall into two general classes, those that reduce the amount of type I procollagen made and those that alter the structure of the molecules assembled. Type I: Diminished Collagen Production. Most individuals with OI type I have variants that result in production by cells of approximately half the normal amount of type I procollagen. Most of these variants result in Normal type I collagen Abnormal type I collagen Biochemical abnormalities similar to the above but may be less severe Type II, III, or IV OI (phenotype depends on the substitution) Normal type I collagen Abnormal type I collagen Rate of triple helix formation Secretion and degradation Defective collagen fibrils Poor mineralization (in bone) Posttranslational modification NH2-terminal to mutation Type I, II, III, or IV OI (phenotype depends on the substitution) Consequences of mutation Normal type I collagen 1/2 normal amount Reduced number of type I fibrils Type I OI Available types of unassembled chains Proα10 stoichiometric effect: Proα1 Proα10 null allele Proα2 Proα2 Assembled chains α1 α1 α2 1/2 number of normal molecules Proα1M stoichiometric effect: Proα1M Proα1 Proα2 Proα2 Proα2M stoichiometric effect: Proα1 Proα1 Proα2 Proα2M Ratio of normal: mutant molecules = 1:1 α1 α1 α2 α1 α1 α2M IM I I IM IM IMM α1 α1 α2 α1M α1 α2 α1 α1M α2 α1M α1M α2 Ratio of normal: mutant molecules = 1:3 I Figure 13.20 The pathogenesis of the major classes of type I procollagen mutants. (Column 1) The types of procollagen chains available for assembly into a triple helix. Although there are two α1 and two α2 collagen genes/genome, as implied in the left column, twice as many α1 collagen molecules are produced, compared to α2 collagen molecules, as shown in the central column. (Column 2) The effect of type I procollagen stoichiometry on the ratio of normal to defective collagen molecules formed in mutants with proα1 chain versus proα2 chain variants. The small vertical bars on each procollagen chain indicate posttranslational modifications (see text). (Column 3) The effect of variants on the biochemical processing of collagen. OI, Osteogenesis imperfecta; Proα1M, a proα1 chain with a missense variant; Proα2M, a proα2 chain with a missense variant; Proα10, a proα1 chain null allele. ▶
CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 275 genes, the variants fall into two general classes, those that reduce the amount of type I procollagen made and those...
Ch13 · Pt26 276 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE premature termination codons in one COL1A1 allele that render the mRNA from that allele untranslatable. Because type I procollagen molecules must have two proα1(I) chains to assemble into a triple helix, loss of half the mRNA leads to production of half the normal quantity of type I procollagen molecules, although these molecules are normal (see Fig. 13.20). Missense variants can also give rise to this milder form of OI when the amino acid change is located in the amino terminus. This is because amino terminal substitutions tend to be less disruptive of collagen chain assembly, which can still initiate as usual at the carboxy terminus. Types II, III, and IV: Structurally Defective Collagens. The type II, III, and IV phenotypes of OI usually result from variants that produce structurally abnormal proα1(I) or proα2(I) chains (see Fig. 13.20 and Table 13.4). Most of these patients have substitutions in the triple helix that replace a glycine with a bulkier residue that disrupts formation of the triple helix. The specific collagen affected, the location of the substitution, and the nature of the substituting residue are all important phenotypic determinants, but some generalizations about the phenotype likely to result from a specific substitution are nevertheless possible. Thus substitutions in the proα1(I) chain are more prevalent in patients with OI types III and IV and are more often lethal. In either chain, replacement of glycine (a neutral residue) with a charged residue (aspartic acid, glutamic acid, arginine) or large residue (tryptophan) is usually very disruptive and often associated with a severe (type II) phenotype (see Fig. 13.20). Sometimes, a specific substitution is associated with more than one phenotype, an outcome that is likely to reflect the influence of powerful modifier genes. Novel Forms of Osteogenesis Imperfecta That Do Not Result From Collagen Variants Seventeen additional forms of clinically defined OI (types V–XXII, or 5–22) do not result from pathogenic variants in type I collagen genes but involve defects in other genes. These 5% of OI subjects with normal collagen genes have either dominant variants in the IFITM5 gene (encoding interferon-induced transmembrane protein 5) or biallelic variants in any of more than a dozen other genes that encode proteins that regulate osteoblast development and facilitate bone formation or that mediate collagen assembly by interacting with collagens during synthesis and secretion. These genes include, for example, WNT1, which encodes a secreted signaling protein, and BMP1, which encodes bone morphogenetic protein 1, an inducer of cartilage formation. The Genetics of Osteogenesis Imperfecta As just discussed, most of the variants in type I collagen genes that cause OI act in a dominant manner. This group of disorders illustrates the genetic complexities that result when variants alter structural proteins, particularly those composed of multiple different subunits, or alter proteins that are involved in the folding and transport of collagens to their place of action. The relatively mild phenotype and dominant inheritance of OI type I are consistent with the fact that although only half the normal number of molecules is made, they are of normal quality (see Fig. 13.20). The more severe consequences of producing structurally defective proα1(I) chains from one allele (compared with producing no chains) partly reflect the stoichiometry of type I collagen, which contains two proα1(I) chains and one proα2(I) chain (see Fig. 13.20). Accordingly, if half the proα1(I) chains are abnormal, three of four type I molecules have at least one abnormal chain; in contrast, if half the proα2(I) chains are defective, only one in two molecules is affected. Variants such as the proα1(I) missense allele (proα1M) shown in Fig. 13.20 are thus dominant negative alleles because they impair the contribution of both the normal proα1(I) chains and the normal proα2(I) chains. In other words, the effect of the mutant allele is amplified because of the trimeric nature of the collagen molecule. Consequently, in dominantly inherited diseases such as OI, it is actually better to have a variant that generates no gene product than one that produces an abnormal gene product. The biochemical mechanism in OI by which the dominant negative effect of dominant negative alleles of the COL1A1 genes is exerted is one of the best understood in all of human genetics (see Cases 8 and 30 for other examples of dominant negative alleles). Although variants that produce structurally abnormal proα2(I) chains reduce the number of normal type I collagen molecules by half, this reduction is nevertheless sufficient, in the case of some variants, to cause the severe perinatal lethal phenotype (see Table 13.4). Most infants with OI type II, the perinatal lethal form, have a de novo dominant mutation, and consequently there is a very low likelihood of recurrence in the family. In occasional families, however, more than one sibling is affected with OI type II. Such recurrences are usually due to parental germline or gonadal mosaicism, as described in Chapter 7. Clinical Management. If a patient’s molecular defect can be determined, increasing knowledge of the correlation between OI genotypes and phenotypes has made it possible to predict, at least to some extent, the natural history of the disease. The treatment of children with the more clinically significant forms of OI is based on physical medicine approaches to increase ambulation and mobility, often in the context of treatment with parenteral bisphosphonates, a class of drugs that act by decreasing bone resorption, to increase bone density and reduce fracture rate. These drugs appear to be less effective in individuals with the recessive forms of OI. The development of better and targeted drugs is a critical issue to improve care. ▶
276 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE premature termination codons in one COL1A1 allele that render the mRNA from that allele untranslatable. Because type I procollagen molecules...
Ch13 · Pt27 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 277 THE EFFECT OF GENE DUPLICATION AND RETAINED FUNCTION ON PHENOTYPE: SPINAL MUSCULAR ATROPHY The Phenotypes of Spinal Muscular Atrophy Spinal muscular atrophy (SMA), an autosomal recessive disease, is the most frequent genetic cause of infant mortality, affecting 1 in 10,000 live births and a carrier rate of 1 in 40 to 50. The disorder leads to progressive loss of the alpha motor neurons of the ventral spinal cord and motor nuclei of the lower brainstem causing hypotonia, muscle weakness, and atrophy of variable severity depending on the underlying genotype. SMA has traditionally been classified into four different phenotypes. Patients with SMA type 1, which is the most common form (~45% of patients), present with symptoms around 0 to 6 months of age. Clinical presentation includes predominant proximal limb weakness, respiratory insufficiency, and poor feeding. Patients also show signs of intercostal muscle weakness, relative to preserved diaphragm strength, and over time develop a bell-shaped chest deformity with signs of paradoxic breathing. Tongue fasciculations are present, while facial and ocular muscle strength are unaffected. Interestingly, cognitive function is normal to above average. Patients usually do not achieve the ability to sit independently (nonsitters) and have a limited life expectancy. Type 2 SMA, comprising 30% of cases, presents with muscle weakness by the age of 6 to 18 months. Most patients are able to achieve the ability to sit unsupported (sitters), although they may later lose this ability and are almost never able to stand or walk without support. Patients demonstrate proximal muscle weakness, often more pronounced in the lower extremities, tongue atrophy, and fasciculations. Respiratory insufficiency and dysphagia are common, particularly in more severe phenotypes. Given the significant axial muscle weakness, many patients develop significant scoliosis, which in turn often leads to restrictive lung disease and respiratory insufficiency. Aggressive supportive treatments prior to the onset of disease-modifying therapy led to increased life span, with 68.5% of this historic cohort surviving to age 25. SMA type 3 (15% of cases) usually shows an onset of symptoms that can present from 18 months to adulthood. Patients are generally able to stand or walk without support (walkers), although some lose this ability with ongoing disease progression. Patients may present with symptoms of proximal weakness such as falls, abnormal gait, and difficulty climbing stairs. In contrast to SMA types 1 and 2, type 3 patients generally have a normal life expectancy and do not develop significant respiratory muscle weakness. The recent development of disease-modifying therapies has changed the phenotypic landscape significantly, and clinical presentations and disease progression have become more diverse. This has changed previous clinical classifications to focus on the functional status of patients (nonsitters, sitters, walkers) or the response treatment (decline, no change, improvement). Therapeutic approaches are discussed in Chapter 14. Genetics of Spinal Muscular Atrophy The SMN1 gene exhibits a duplication event with subsequent SNV in exon 7 of SMN2 that affects splicing (Fig. 13.21). The SMN2 gene retains about 10% wildtype transcript and is present in zero to four copies in the general population. With a carrier frequency of 1 in 40 to 50 and an estimated incidence of 1 in 10,000 live births, SMA is the second most common autosomal recessive disorder. On chromosome 5q13, the survival motor neuron protein SMN1 is reduced in function; however, the modifying SMN2 gene is maintained. The absence of the SMN1 gene accounts for most SMA cases. Ninety-five percent of SMA-affected individuals have a homozygous deletion of SMN1 exon 7 or gene conversion from SMN1 to SMN2, and most of the remaining 5% are compound heterozygotes for an SMN1 exon 7 deletion and an SMN1 SNV (see Fig. 13.21: normal in A, variants in B and C). A number of intragenic variants can be detected in the compound heterozygous state with an SMN1 deletion and can include missense, nonsense, splice site variants, insertions, deletions, and duplications (see Fig. 13.21C). Recurrent variants have been discovered in exons 3 and 6, representing hot spots for small and missense changes, respectively (see Fig. 13.21D). Exon 6 codes for a domain in the protein, which plays a role in protein oligomerization, and individuals with exon 6 missense variants have decreased SMN protein self-oligomerization capacity. The exon 6 p. Tyr 272Cys missense variant is the most frequently reported SNV in the SMN1 gene. Because both copies of SMN1 exon 7 are lost in the majority of patients, no phenotype-genotype correlation was initially observed in SMA. The copy number of SMN2 has now been shown to be a significant modifier of SMA severity. All individuals with SMA have at least one copy of SMN2, which generates low amounts of SMN protein but does not fully compensate for the loss of SMN1. The SMN2 gene is unable to create a full transcript because of the presence of the splice variant in exon 7. The copy number varies from zero to three copies in the general population, with around 10% of individuals having no SMN2. A fetus that lacks SMN1 function and has no copies of SMN2 would presumably be nonviable. The majority of individuals who have type 1 SMA have one to two copies of SMN2. The typical number of SMN2 copies in persons with type 2 SMA is three. Patients with milder type 3 and 4 individuals generally exhibit four or more copies of SMN2. These phenotypegenotype correlations have paved the way for developing disease-modifying therapies for SMA (see Chapter 14). Molecular Testing for Spinal Muscular Atrophy Screening for a missing or deficient exon 7 is the first step in diagnostic testing in patients, with 95% having a ▶
CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 277 THE EFFECT OF GENE DUPLICATION AND RETAINED FUNCTION ON PHENOTYPE: SPINAL MUSCULAR ATROPHY The Phenotypes of Spinal M...
Ch13 · Pt28 278 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE homozygous lack of SMN1 exon 7. Several methodologies can detect the absence of SMN1 exon 7, all based on the c.840C>T variation. One of the most popular techniques as a first deletion test in laboratories is multiplex ligation-dependent probe amplification (MLPA). It is simple to use and highly sensitive and can determine both SMN1 and SMN2 copy numbers. Preimplantation genetic diagnosis and prenatal testing for SMA is available for at-risk couples or due to the presence of abnormal findings on fetal ultrasound, such as decreased fetal movements, contractures in utero, or increased nuchal translucency. The presence of maternal cell contamination of the fetal specimen may result in a false-negative test result and therefore must be tested and shown to be absent prior to reporting the prenatal test result. Newborn Screening The SMN1 exon 7 deletion test can be used as a reliable confirmatory test for the majority of patients suspected to have SMA and can be reported within 24 hours. The test is highly sensitive (~95%) and nearly 100% specific. Newborn screening for SMA has now been developed and is standard of care in many jurisdictions and is primarily based on real-time polymerase chain reaction that detects the common SMN1 deletion and may also detect SMN2 copy number on dried blood spots. Follow-up molecular genetic testing confirmation of a positive newborn screening result is always strongly recommended. NEURODEGENERATIVE DISORDERS Until recently, the biochemical and molecular mechanisms underlying almost all neurodegenerative diseases were completely obscure. In this section, we discuss three different conditions, each with a different genetic and genomic basis and illustrating different mechanisms of pathogenesis: Alzheimer disease Disorders of mitochondrial DNA Diseases due to the expansion of unstable repeat sequences Alzheimer Disease One of the most common adult-onset neurodegenerative conditions is Alzheimer disease (AD) (Case 4), introduced in Chapter 9 in the context of complex genetic disorders. AD generally manifests in the sixth to ninth decades, but there are monogenic forms that often present earlier, sometimes as soon as the third decade. The clinical picture of AD is characterized by a progressive deterioration of memory and of higher cognitive functions, such as reasoning, in addition to behavioral changes. These abnormalities reflect degeneration of neurons in specific regions of the cerebral cortex and hippocampus. AD affects ~1.4% of persons in developed countries and is responsible for over 120,000 deaths per year in the United States alone. The Genetics of Alzheimer Disease The lifetime risk for AD in the general population is 12.1% in men and 20.3% in women by age 85. Most of A B C D Figure 13.21 (A) A chromosome carrying a normal copy of SMN1 and SMN2. (B) The blank box indicates a deleted gene. A deletion can remove part or all of the SMN1 gene. (C) The curved arrow represents a conversion. With the C>T transition in SMN1, the SMN1 copy now closely resembles SMN2 and is considered SMN2-like. (D) Point mutations occurring in any of the SMN1 exons prior to the last exon can affect the SMN protein. (From Keinath MC, Prior DE, Prior TW: Spinal muscular atrophy: Mutations, testing, and clinical relevance, Appl Clin Genet 14:11–25, 2021. https://doi.org/10.2147/TACG. S239603) ▶
278 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE homozygous lack of SMN1 exon 7. Several methodologies can detect the absence of SMN1 exon 7, all based on the c.840C>T variation. One of the...
Ch13 · Pt29 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 279 the increased risk in relatives of affected individuals is not due to mendelian inheritance; rather, as described in Chapter 9, this familial aggregation results from a complex genetic contribution involving one or more incompletely penetrant genes that act independently, from multiple interacting genes, or from some combination of genetic and environmental factors. Approximately 7% to 10% of patients, however, have a monogenic highly penetrant form of AD that is inherited in an autosomal dominant manner. In the 1990s, four genes associated with AD were identified (Table 13.5). Pathogenic variants in three of these genes – encoding the β-amyloid precursor protein (β APP), presenilin 1, and presenilin 2 – lead to autosomal dominant AD. The fourth gene, APOE, encodes apolipoprotein E (apo E), the protein component of several plasma lipoproteins. Variants in APOE are not associated with monogenic AD. Rather, as we saw in Chapter 9, the ε4 allele of APOE modestly increases susceptibility to nonfamilial AD and influences the age at onset of at least some of the monogenic forms (see later). The identification of the four genes associated with AD has provided great insight not only into the pathogenesis of monogenic AD but also, as is commonly the case in medical genetics, into the mechanisms that underlie the more common form, nonfamilial or sporadic AD. Indeed, overproduction of one proteolytic product of β APP, called the Aβ peptide, appears to be at the center of AD pathogenesis, and the currently available experimental evidence suggests that the β APP, presenilin 1, and presenilin 2 proteins all play a direct role in the pathogenesis of AD. The Pathogenesis of Alzheimer Disease: β-Amyloid Peptide and Tau Protein Deposits The most important pathologic abnormalities of AD are the deposition in the brain of two fibrillary proteins, β-amyloid peptide (Aβ) and tau protein. The Aβ peptide is generated from the larger β APP protein (see Table 13.5), as discussed in the next section, and is found in extracellular amyloid or senile plaques in the extracellular space of AD brains. Amyloid plaques contain other proteins besides the Aβ peptide, notably apo E (see Table 13.5). Tau is a microtubule-associated protein expressed abundantly in neurons of the brain. Hyperphosphorylated forms of tau compose the neurofibrillary tangles that, in contrast to the extracellular amyloid plaques, are found within AD neurons. The tau protein normally promotes the assembly and stability of microtubules, functions that are diminished by phosphorylation. Although the formation of tau neurofibrillary tangles appears to be one cause of the neuronal degeneration in AD, variants in the tau gene are associated not with AD but with another autosomal dominant dementia, frontotemporal dementia. The Amyloid Precursor Protein Gives Rise to the β-Amyloid Peptide The major features of the β APP and its corresponding gene are summarized in Table 13.5. β APP is a singlepass intracellular transmembrane protein found in TABLE 13.5 Genes and Proteins Associated With Inherited Susceptibility to Alzheimer Disease Gene Inheritance % of FAD Protein Normal Function Role in FAD PSEN1 AD 50% Presenilin 1 (PS1): A 5–10 membrane-spanning domain protein found in cell types both inside and outside the brain Unknown, but required for γ-secretase cleavage of β APP May participate in the abnormal cleavage at position 42 of β APP and its derivative proteins; >100 variants identified in Alzheimer disease PSEN2 AD 1–2% Presenilin 2 (PS2): structure similar to PS1, maximal expression outside the brain Unknown, likely to be similar to PS1 At least 5 missense variants identified APP AD 1–2% Amyloid precursor protein (β APP): an intracellular transmembrane protein. Normally, β APP is cleaved endoproteolytically within the transmembrane domain (see Fig. 13.24), so that little of the β-amyloid peptide (Aβ) is formed. Unknown β-amyloid peptide (Aβ) is the principal component of senile plaques. Increased Aβ production, especially of the Aβ42 form, is a key pathogenic event. Approximately 10 variants have been identified in FAD. APOE See Table 13.6 NA Apolipoprotein E (apo E): a protein component of several plasma lipoproteins. The apo E protein is imported into the cytoplasm of neurons from the extracellular space. Normal function in neurons is unknown. Outside the brain, apo E participates in lipid transport between tissues and cells. Loss of function causes one form (type III) of hyperlipoproteinemia. An Alzheimer disease susceptibility gene (see Table 13.6). Apo E is a component of senile plaques. AD, Autosomal dominant; FAD, familial Alzheimer disease; NA, not applicable. Data derived from St. George-Hyslop PH, Farrer LA: Alzheimer’s disease and the fronto-temporal dementias: diseases with cerebral deposition of fibrillar proteins. In Scriver CR, Beaudet AL, Sly WS, et al, editors: The molecular and metabolic bases of inherited disease, ed 8, New York, 2000, Mc Graw-Hill; Martin JB: Molecular basis of the neurodegenerative disorders, N Engl J Med 340:1970–1980, 1999. Updated in 2022 from Clin Var. ▶
CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 279 the increased risk in relatives of affected individuals is not due to mendelian inheritance; rather, as described in...
Ch13 · Pt30 280 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE endosomes, lysosomes, the ER, and the Golgi apparatus. It is subject to three distinct proteolytic fates, depending on the relative activity of three different proteases: α-secretase and β-secretase, which are cell surface proteases, and γ-secretase, which is an atypical protease that cleaves membrane proteins within their transmembrane domains. The predominant fate of ~90% of β APP is cleavage by the α-secretase (Fig. 13.22), an event that precludes the formation of the Aβ peptide because α-secretase cleaves within the Aβ peptide domain. The other ~10% of β APP is cleaved by the β- and γ-secretases to form either the nontoxic Aβ40 ­peptide or the Aβ42 peptide. The Aβ42 peptide is thought to be neurotoxic because it is more prone to aggregation than its Aβ40 counterpart, a feature that makes AD a conformational disease like α1AT deficiency (described previously in this chapter). Normally, little Aβ42 peptide is produced, and the factors that determine whether γ-secretase cleavage will produce the Aβ40 or Aβ42 peptide are not well defined. In monogenic AD due to missense substitutions in the gene encoding β APP (APP), however, several variants lead to the relative overproduction of the Aβ42 peptide. This increase leads to accumulation of the neurotoxic Aβ42, an occurrence that appears to be the central pathogenic event of all forms of AD, monogenic or sporadic. Consistent with this model is the fact that patients with Down syndrome, who possess three copies of the APP gene (which is on chromosome 21), typically develop the neuropathologic changes of AD by 40 years of age. Moreover, pathogenic variants in the AD genes presenilin 1 and presenilin 2 (see Table 13.5) also lead to increased production of Aβ42. Notably, the amount of the neurotoxic Aβ42 peptide is increased in the serum of individuals with pathogenic variants in the β APP, presenilin 1, and presenilin 2 genes; furthermore, in cultured cell systems, the expression of mutant β APP, presenilin 1, and presenilin 2 increases the relative production of Aβ42 peptide by two- to tenfold. The central role of the Aβ42 peptide in AD is highlighted by the discovery of a coding variant (p. Ala 673Thr) in the APP gene (Fig. 13.23) that protects against both AD and cognitive decline in older adults. The protective effect is likely due to reduced formation of the Aβ42 peptide, reflecting the proximity of Thr 673 to the β-secretase cleavage site (see Fig. 13.23). The Presenilin 1 and 2 Genes The genes encoding presenilin 1 and presenilin 2 (see Table 13.5) were identified in families with autosomal Effect of an Ala 692Gly mutation on processing α-secretase β-secretase α-secretase β-secretase γ-secretase Increased production of Aβ40 and Aβ42 3 k D γ-secretase Effect of Val 717Gly, Val 717Ile, and Val 717Phe mutations on processing α-secretase β-secretase α-secretase β-secretase γ-secretase Increased production of Aβ42 3 k D γ-secretase Normal processing of β-amyloid precursor protein α-secretase β-secretase α-secretase β-secretase γ-secretase Cell membrane Aβ40 3 k D γ-secretase Figure 13.22 The normal processing of β-amyloid precursor protein (β APP) and the effect on processing of missense variants in the β APP gene associated with familial Alzheimer disease. The ovals show the locations of the missense changes. (Reproduced with permission from Nussbaum RL, Ellis CE: Alzheimer’s disease and Parkinson’s disease, N Engl J Med 348:1356–1364, 2003.) ▶
280 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE endosomes, lysosomes, the ER, and the Golgi apparatus. It is subject to three distinct proteolytic fates, depending on the relative activity...
Ch13 · Pt31 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 281 dominant AD. Presenilin 1 is required for γ-secretase cleavage of β APP derivatives. Indeed, some evidence suggests that presenilin 1 is a critical cofactor protein of γ-secretase. The pathogenic variants in presenilin 1 associated with AD, through an unclear mechanism, increase production of the Aβ42 peptide. A major difference between presenilin 1 and presenilin 2 pathogenic variants is that the age at onset with the latter is much more variable (presenilin 1, 35–60 years; presenilin 2, 40–85 years); indeed, in one family, an asymptomatic octogenarian carrying a presenilin 2 pathogenic variant transmitted the disease to his offspring. The basis of this variation is partly dependent on the number of APOE ε4 alleles (see Table 13.5 and later discussion) carried by individuals with a presenilin 2 pathogenic variant; two ε4 alleles are associated with an earlier age at onset than one allele, and one confers an earlier onset than other APOE alleles. The APOE Gene Is an Alzheimer Disease Susceptibility Locus As presented in Chapter 9, the ε4 allele of the APOE gene is a major risk factor for the development of AD. The role for APOE as a major AD susceptibility locus 670 671 692 715 716 723 693 Cleavage by α-secretase Ala 673Thr (Protective) Cleavage by β-secretase Cleavage by γ-secretase Aβ40 or Aβ42 717 COOH M I T K K V V P V V I L M I N V E D H R F A D NH2 Y H H V E G S K I F D K Q A E Q K S V F V G K N G A I G L G I G G V V V M T K M G I F A I Figure 13.23 The topology of the amyloid precursor protein (β APP), its nonamyloidogenic cleavage by α-secretase, and its alternative cleavage by putative β-secretase and γ-secretase to generate the amyloidogenic β amyloid peptide (Aβ). Letters are the single-letter code for amino acids in β-amyloid precursor protein, and numbers show the position of the affected amino acid. Normal residues involved in missense variants are shown in highlighted circles, whereas the amino acid residues representing various missense pathogenic variants are shown in boxes. The mutated amino acid residues are near the sites of β-, α-, and γ-secretase cleavage (black arrowheads). The pathogenic variants lead to the accumulation of toxic peptide Aβ42 rather than the wild-type Aβ40 peptide. The location of the protective allele Ala 673Thr is indicated by the dashed arrow. (Reproduced with permission from Nussbaum RL, Ellis CE: Alzheimer’s disease and Parkinson’s disease, N Engl J Med 348:1356–1364, 2003.) ▶
CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 281 dominant AD. Presenilin 1 is required for γ-secretase cleavage of β APP derivatives. Indeed, some evidence suggests t...
Ch13 · Pt32 282 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE was suggested by multiple lines of evidence, including linkage to AD in late-onset families, increased association of the ε4 allele with AD patients compared with controls, and the finding that apo E binds to the Aβ peptide. The APOE protein has three common forms encoded by corresponding APOE alleles (Table 13.6). The ε4 allele is significantly overrepresented in patients with AD (~40% vs ~15% in the general population) and is associated with an early onset of AD (for ε4/ε4 homozygotes, the age at onset of AD is ~10–15 years earlier than in the general population; see Chapter 9). Moreover, the relationship between the ε4 allele and the disease is dose dependent; two copies of ε4 are associated with an earlier age at onset (mean onset before 70 years) than with one copy (mean onset after 70 years). In contrast, the ε2 allele has a protective effect and correspondingly is more common in elderly subjects who are unaffected by AD (see Table 13.6). The mechanisms underlying these effects are not known, but apo E polymorphisms may influence the processing of β APP and the density of amyloid plaques in AD brains. It is also important to note that the APOE ε4 allele is not only associated with an increased risk for AD; carriers of ε4 alleles can also have poorer neurologic outcomes after head injury, stroke, and other neuronal insults. Although carriers of the APOE ε4 allele have a clearly increased risk for development of AD, there is currently no role for screening for the presence of this allele in healthy individuals; such testing has poor positive and negative predictive values and would therefore generate highly uncertain estimates of future risk for AD (see Chapter 19). Other Genes Associated With Alzheimer Disease One significant modifier of AD risk, the TREM2 gene (which encodes the triggering receptor expressed on myeloid cells 2), was identified by whole exome and whole genome sequencing in families with multiple individuals affected with AD. Several moderately rare missense coding variants in this gene are associated with a fivefold increase in risk for late-onset AD, making TREM2 variants the second most common contributor to classic lateonset AD after APOE ε4. Statistical analyses suggest that an additional four to eight genes may significantly modify the risk for AD, but their identity remains obscure. Although case-control association studies (see Chapter 11) of candidate genes with hypothetical functional links to the known biology of AD have suggested more than 100 genes in AD, only one such candidate gene, SORL1 (sortilin-related receptor 1), has been robustly implicated. Single nucleotide polymorphisms (SNPs) in the SORL1 gene confer a moderately increased relative risk for AD of less than 1.5. The SORL1-encoded protein affects the processing of APP and favors the production of the neurotoxic Aβ42 peptide from β APP. Genome-wide association study analyses (see Chapter 11), on the other hand, have greatly expanded the number of genes believed to be associated with AD, identifying many novel SNPs associated with a predisposition to nonfamilial late-onset forms of AD. The genes implicated by these SNPs and their causal role(s) in AD are presently uncertain. Overall, it is becoming clear that genetic variants alter the risk for AD in at least two general ways: first, by modulating the production of Aβ, and second, through their impact on other processes, including the regulation of innate immunity, inflammation, and the resecretion of protein aggregates. These latter variants likely modulate AD risk by altering the flux through downstream pathways in response to a given load of Aβ. DISEASES OF MITOCHONDRIAL DNA (mtDNA) Neal Sondheimer The Genetics of mtDNA Disease The general characteristics of the mtDNA genome and the features of the inheritance of disorders caused by pathogenic variants in this genome were first described in Chapters 2 and 7 but are reviewed briefly here. The small circular mtDNA chromosome is located inside mitochondria and contains only 37 genes (Fig. 13.24). Unlike nuclear chromosomes, different cell types have a wide range in the copy number of mtDNA. The oocyte at fertilization has ~106 copies, fibroblasts may have thousands of copies, and red blood cells have none. In addition to encoding two ribosomal RNAs (rRNAs) and 22 transfer RNAs (tRNAs), mtDNA encodes 13 proteins that are subunits of oxidative phosphorylation. TABLE 13.6 Amino Acid Substitutions Underlying the Three Common Apolipoprotein E Polymorphisms Allele ε2 ε3 ε4 Residue 112 Cys Cys Arg Residue 158 Cys Arg Arg Frequency in US populations of European ancestry 10% 65% 25% Frequency in patients with Alzheimer disease 2% 58% 40% Effect on Alzheimer disease Protective None known 30–50% of the genetic risk for Alzheimer disease These figures are estimates, with differences in allele frequencies that vary with ancestry in control populations, and with age, gender, and ancestry in Alzheimer disease subjects. Data derived from St. George Hyslop PH, Farrer LA, Goedert M: Alzheimer disease and the frontotemporal dementias: Diseases with cerebral deposition of fibrillar proteins. In Valle D, Beaudet AL, Vogelstein B, et al, editors: The online metabolic & molecular bases of inherited disease (OMMBID). http://www. ommbid.com/ ▶
282 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE was suggested by multiple lines of evidence, including linkage to AD in late-onset families, increased association of the ε4 allele with AD...
Ch13 · Pt33 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 283 Pathogenic variants in mtDNA, and the associated disorders, can be inherited or acquired as somatic mutations. These diseases show distinctive patterns of inheritance due to three features of mtDNA: Maternal inheritance Homoplasmy and heteroplasmy Replicative segregation The maternal inheritance of mtDNA (discussed in greater detail in Chapter 7; see Fig. 7.22) reflects the fact that sperm mtDNA are generally eliminated from the embryo so that mtDNA is inherited entirely from the mother. Paternal inheritance has been well documented in only one instance, and this case may represent a unique failure in the clearance of paternal mtDNA. Replicative segregation refers to the fact that the multiple copies of mtDNA in each mitochondrion replicate and assort randomly among newly synthesized mitochondria, which in turn are distributed randomly between the daughter cells (see Fig. 7.23). This occurs in both mitotic and meiotic divisions and impacts the transmission of heteroplasmy between generations (a phenomenon known as a germline bottleneck), which is described in greater detail later. The 74 polypeptides of the oxidative phosphorylation complex not encoded in the mtDNA are encoded by the nuclear genome, which contains the genes for most of the estimated 1500 mitochondrial proteins. To date, more than 300 nuclear genes are associated with disorders of the respiratory chain. Thus diseases of oxidative phosphorylation arise not only from pathogenic variants in the mitochondrial genome but also from variants in nuclear genes that encode oxidative phosphorylation MELAS tRNALeu (A3243G) Aminoglycoside-Induced Deafness 12S tRNA (A1555G) OH LHON ND4 (G11778A) Leigh disease MELAS ND5 (G13513A) NARP and Leigh Disease ATPase 6 (T8993C) (T8993G) MERRF tRNALys (A8344G) F P T Cyt b E ND6 CO I W ND2 I ND1 L 16S 12S V C P E O ▶
a n d K S S co m m o n 5 k b de le tio n ND5 L S2 H ND4 ND4L R ND3 G CO III S OL A N Q M A6 A8 Y C K CO II D Figure 13.24 Representative disease-causing variants and deletions in the human mtDNA ge...
Ch13 · Pt34 284 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE components. Furthermore, the nuclear genome encodes up to 200 proteins required for the maintenance and expression of mtDNA genes or for the assembly of oxidative phosphorylation protein complexes. Defects in many of these nuclear genes can also lead to disorders with the phenotypic characteristics of mtDNA diseases, but of course the patterns of inheritance in these cases are those typically seen with other mendelian disorders (see Chapter 7). Diseases Caused by Pathogenic Variants in mtDNA The sequence of the mtDNA genome and the presence of pathogenic variants in mtDNA have been known for over 4 decades. The disorders are not uncommon, and the prevalence has been shown, in at least one population, to be ~1 per 5000. The range of clinical disease resulting from mtDNA is diverse (Fig. 13.25), although neuromuscular disease predominates. Nearly 100 different disease-related variants have been identified in mtDNA, in addition to more than 100 rearrangements that cause disease. Representative pathogenic variants and the diseases associated are presented in Fig. 13.24 and Table 13.7. In general, as illustrated in the sections to follow, three types of variants have been identified in mtDNA: rearrangements that generate deletions or duplications of the mtDNA molecule, point variants in tRNA or rRNA genes that impair mitochondrial translation, and missense variants in the coding regions of genes that alter the activity of an oxidative phosphorylation protein. Deletions of mtDNA and Disease. In many cases, mtDNA deletions that cause disease, such as KearnsSayre syndrome (see Table 13.7), are inherited from an unaffected mother who carries the deletion in her oocytes but generally not elsewhere, an example of gonadal mosaicism. Under these circumstances, disorders caused by mtDNA deletions appear to be sporadic because oocytes carrying the deletion are relatively rare. In ~5% of cases, the mother may be affected and transmit the deletion. The reason for the low frequency of transmission is uncertain, but it may simply reflect the fact that women with a high proportion of the deleted mtDNA in their germ cells have such a severe phenotype that they rarely reproduce. Eye External ophthalmoplegia Ptosis Cataract Pigmentary retinopathy Optic atrophy Hearing Bilateral sensorineural deafness Gastrointestinal Dysphagia Pseudo-obstruction Constipation Hepatic failure Peripheral nervous system Myopathy Axonal sensorimotor neuropathy Central nervous system Encephalopathy Strokelike episodes Seizures and dementia Psychosis and depression Ataxia Migraine Cardiac Hypertrophic cardiomyopathy Dilated cardiomyopathy Heart block Pre-excitation syndrome Endocrine and diabetes Diabetes mellitus Hypoparathyroidism Hypothyroidism Gonadal failure Renal Renal tubular defects De Toni-Fanconi-Debré syndrome Figure 13.25 The range of affected tissues and clinical phenotypes associated with variants in mitochondrial DNA (mtDNA). (Modified from Chinnery PF, Turnbull DM: Mitochondrial DNA and disease, Lancet 354:SI17–SI21, 1999.) ▶
284 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE components. Furthermore, the nuclear genome encodes up to 200 proteins required for the maintenance and expression of mtDNA genes or for the...
Ch13 · Pt35 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 285 Mitochondrial tRNA and rRNA Are Associated With Disease. Pathogenic variants in the tRNA and rRNA genes of mtDNA are significant because they illustrate that not all disease-causing variants in humans occur in genes that encode proteins (Case 33). Unlike nuclear tRNA and rRNA, which are present at high copy number in the chromosomes, the mitochondrial genes encoding these RNAs exist at unique locations in the genome (see Fig. 13.24) so that single nucleotide variants can disrupt their function. Fifty pathogenic variants have been identified in 15 of the 22 tRNA genes of the mtDNA, and they are the most common cause of oxidative phosphorylation abnormalities in humans (see Fig. 13.24 and Table 13.7). Pathogenic tRNA variants include 11 different substitutions in the tRNAleu(UUR) gene, some of which, like the m.3243A>G variant, cause a phenotype referred to as MELAS, an acronym for mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (see Fig. 13.24 and Table 13.7); others are associated predominantly with myopathy. The m.3243A>G variant, for reasons that are not entirely clear, is the most commonly observed mitochondrial pathogenic variant in clinical practice. It is only found in a heteroplasmic state, and the homoplasmic state is presumed to be lethal. An example of an rRNA variant causing disease is the m.1555A>G variant in the 12S ribosomal RNA. This variant causes sensorineural prelingual deafness after exposure to aminoglycoside antibiotics and illustrates an important rule for homoplasmic variants, which is that they must either be incompletely penetrant or must allow females to survive to reproduction; otherwise, they would be eliminated from the population. The Phenotypes of Mitochondrial Disorders Oxidative Phosphorylation and mtDNA Diseases. Mitochondrial diseases generally affect tissues that depend on intact oxidative phosphorylation to satisfy high demands for metabolic energy. This phenotypic focus reflects the central role of the oxidative phosphorylation complex in the production of ATP. The evidence that mechanisms other than decreased energy production contribute to the pathogenesis of mtDNA diseases is either indirect or weak, but the generation of reactive oxygen species as a byproduct of faulty oxidative phosphorylation may also contribute to the pathology of mtDNA disorders. A substantial body of evidence indicates that there is a phenotypic threshold effect associated with mtDNA heteroplasmy (see Fig. 7.23); a critical threshold in the proportion of mtDNA molecules carrying the detrimental variant must be exceeded in cells from the affected tissue before clinical disease becomes apparent. The threshold for the appearance of disease is dependent upon the nature of each variant and its impact on the underlying gene. The neuromuscular system is most commonly affected by mitochondrial diseases; the consequences can include encephalopathy, myopathy, ataxia, retinal degeneration, and loss of function of the external ocular muscles. Mitochondrial myopathy is characterized by ragged-red (muscle) fibers, a histologic phenotype due to the proliferation of structurally and biochemically abnormal mitochondria in muscle fibers. The spectrum of mitochondrial disease is broad and, as illustrated in Fig. 13.25, may include liver dysfunction, bone marrow failure, pancreatic islet cell deficiency and diabetes, ­deafness, and other disorders. TABLE 13.7 Representative Examples of Disorders due to Variants in Mitochondrial DNA and Their Inheritance Disease Phenotypes Typical Variant in mtDNA Homoplasmy vs Heteroplasmy Inheritance Leber hereditary optic neuropathy (LHON) Rapid onset of blindness in young adult life due to optic nerve atrophy; some recovery of vision, depending on the variant. Strong sex bias: ~50% of male carriers have visual loss vs. ~10% of females. m.1178A>G in the complex I gene ND4 Largely homoplasmic Maternal Leigh syndrome Early-onset progressive neurodegeneration with characteristic necrosis of basal ganglia m.8993T>G in the complex V gene ATP6 Heteroplasmic Maternal MELAS Myopathy, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; may present only as diabetes mellitus and deafness m.3243A>G in MTTL1, encoding the tRNAleu(UUR) Heteroplasmic Maternal MERRF (Case 33) Myoclonic epilepsy with ragged-red muscle fibers, myopathy, ataxia, sensorineural deafness, dementia m.8344A>G in MTTK, encoding the tRNAlys Heteroplasmic Maternal Deafness Aminoglycoside-induced sensorineural deafness m.1555A>G in MTRNR1, encoding the 12S rRNA Homoplasmic Maternal Kearns-Sayre syndrome (KSS) Progressive myopathy, progressive external ophthalmoplegia of early onset, cardiomyopathy, heart block, ptosis, retinal pigmentation, ataxia, diabetes 5-kb large deletion (see Fig. 13.24) Heteroplasmic Generally sporadic, likely due to maternal gonadal mosaicism mtDNA, Mitochondrial DNA; rRNA, ribosomal RNA; tRNA, transfer RNA. ▶
CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 285 Mitochondrial tRNA and rRNA Are Associated With Disease. Pathogenic variants in the tRNA and rRNA genes of mtDNA are...
Ch13 · Pt36 286 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Unexplained and Unexpected Phenotypic Variation in mtDNA Diseases. As seen in Table 13.7, heteroplasmy is the rule for many mtDNA diseases. Heteroplasmy leads to an unpredictable and variable fraction of diseaseassociated mtDNA being present in any particular tissue, undoubtedly accounting for much of the pleiotropy and variable expressivity of mtDNA mutations (see Box 13.2). An example is provided by the m.3243A>G substitution in the tRNAleu(UUR) gene, previously mentioned in the context of the MELAS phenotype. It also leads to maternally inherited diabetes and deafness in some families, whereas in others it causes a disease called chronic progressive external ophthalmoplegia. Moreover, a very small fraction (<1%) of diabetes mellitus in the general population has been attributed to the m.3243A>G substitution. Disorders of mtDNA Replication Because both the nuclear and mitochondrial genomes contribute polypeptides to oxidative phosphorylation, it is not surprising that the phenotypes associated with defects in nuclear genes can be clinically indistinguishable from those due to mitochondrial genes. One additional concept of importance to disease is that the mtDNA itself depends on nuclear genome–encoded proteins for its replication and the maintenance of its integrity. The medical consequence of this dependency is diseases with mendelian inheritance patterns (dominant or recessive), due to variants in nuclear genes, that have their impact on the mtDNA. An example of this class of disorders is the mtDNA depletion syndromes, which result from pathogenic variants in any of 17 nuclear genes that lead to a reduction in the number of copies of mtDNA (both per mitochondrion and per cell) in various tissues. Some of the affected genes encode proteins required to maintain nucleotide pools or to metabolize nucleotides appropriately in the mitochondrion. One example of a mitochondrial depletion syndrome is Alpers syndrome, which is a recessive disorder caused by pathogenic variants in DNA polymerase γ (POLG). POLG is the DNA-dependent DNA polymerase that replicates mtDNA, and variants in this gene can cause loss of mtDNA but may also lead to excess mutations or rearrangements in mtDNA. Environmental Influences Modify the Phenotype of mtDNA Diseases. Although heteroplasmy is a major source of phenotypic variability in mtDNA diseases (see Box), additional factors, including environmental stressors, also play a role. Strong evidence for environmental influence is provided by families carrying variants associated with Leber hereditary optic neuropathy (LHON; see Table 13.7), which is generally homoplasmic (thus ruling out heteroplasmy as the explanation for the observed phenotypic variation). LHON is expressed phenotypically as rapid, painless bilateral loss of central vision due to optic nerve atrophy in young adults (see Table 13.7 and Fig. 13.24). There is a striking increase in the penetrance of the disease in males; ~50% of male carriers but only ~10% of female carriers of an LHON variant develop symptoms. Studies of large numbers of individuals have strongly implicated that cigarette smoking, and possibly alcohol consumption, drives this discrepancy in risk. This suggests that environmental stressors causing oxidative injury may play an important and synergistic role in determining the penetrance of a mitochondrial variant. It has additionally been suggested that interactions with nuclear-encoded variants may also alter the risk of developing LHON symptoms. Problems in Therapy for mtDNA-Associated Dis-­ orders. Mitochondrial disorders lag other diseases in the development of new therapies based on genetic manipulation. For diseases due to mtDNA there are two critical drivers of this problem. The first is that mitochondrial disease tends to affect many tissues simultaneously, so approaches to correction have to be applied across the whole patient rather than to a single organ or tissue. The second, and more remarkable challenge, is that genetic manipulation of human mtDNA is largely impossible with current technologies. Mitochondria are impermeable to nucleic acids (DNA and RNA), so techniques of mutagenesis that rely on recombination are not effective, even when used on isolated cells BOX 13.2 HETEROPLASMY AND MITOCHONDRIAL DISEASE Heteroplasmy accounts for three general characteristics of genetic disorders of mtDNA that are of importance to their pathogenesis. These features of the inheritance of heteroplasmic variants greatly complicate the counseling of families affected by mitochondrial disease, and the risk of recurrence of disease cannot be precisely estimated as it is with dominant or recessive disorders. First, female carriers of disease-causing heteroplasmic mtDNA variants usually transmit some variant mtDNAs to their offspring. Second, the fraction of variant mtDNA molecules inherited by each child of a carrier mother is not identical in each of her children. This is because the number of mtDNA molecules within each developing oocyte is reduced before being subsequently amplified to the huge total seen in mature oocytes. This restriction and subsequent amplification of mtDNA during oogenesis is termed the mitochondrial genetic bottleneck. Consequently, the variability in the percentage of variant-bearing mtDNA molecules seen in the offspring of a mother carrying a mtDNA mutation arises, at least in part, from the sampling of only a subset of the mtDNAs during oogenesis. Third, despite the variability in the degree of heteroplasmy arising from the bottleneck, mothers with a high proportion of disease-associated mtDNA molecules are more likely to have clinically affected offspring than are mothers with a lower proportion, as one would predict from the distribution of mtDNA ratios through the ­bottleneck. Nevertheless, even women carrying low proportions of pathogenic mtDNA molecules have some risk for having an affected child because the bottleneck can lead to the sampling and subsequent expansion, by chance, of even a rare mtDNA species. ▶
286 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Unexplained and Unexpected Phenotypic Variation in mtDNA Diseases. As seen in Table 13.7, heteroplasmy is the rule for many mtDNA diseases....
Ch13 · Pt37 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 287 in a laboratory. A related challenge is that there are no viruses that infect the mitochondrion, so genetic delivery systems will need to be developed rather than adapted from existing viruses. DISEASES DUE TO THE EXPANSION OF UNSTABLE REPEAT SEQUENCES Ryan Yuen The inheritance pattern of diseases due to unstable repeat expansions was presented in Chapter 7, with emphasis on the unusual genetics of this unique group of almost 60 disorders. These features include the unstable and dynamic nature of the variants, which arise from expansion of repeated sequences within the transcribed region of the affected gene. Examples include the codon for glutamine (CAG) in Huntington disease (Case 24) and in most of a group of neurodegenerative disorders called the spinocerebellar ataxias, or the codon for alanine (GCG) in diseases such as oculopharyngeal muscular dystrophy. The expansion can also be of trinucleotides in noncoding regions of RNAs, including CGG in fragile X syndrome (Case 17), GAA in Friedreich ataxia, CUG in myotonic dystrophy 1 (Fig. 13.26), or GCA in glutaminase deficiency. Although the nucleotide repeat diseases initially described are all due to the expansion of trinucleotide repeats, with the help of advanced genomic technologies, other disorders have been found to result from the expansion of longer repeats; these include a tetranucleotide (CCTG) in myotonic dystrophy 2 (a close genocopy of myotonic dystrophy 1), a pentanucleotide (ATTCT) in spinocerebellar atrophy 10, an inserted pentanucleotide (TTTCA) in a group of familial adult myoclonic epilepsies, and a hexanucleotide (GGGGCC) in amyotrophic lateral sclerosis. Because the affected gene is passed from generation to generation, the number of repeats may expand to a degree that is pathogenic, ultimately interfering with normal gene expression and function. The intergenerational expansion of the repeats accounts for the phenomenon of anticipation: the appearance of the disease at an earlier age or with more severe form as it is transmitted through a family. The biochemical mechanism most proposed to underlie the expansion of unstable repeat sequences is slipped mispairing (Fig. 13.27). Remarkably, the repeat expansions appear to occur both in proliferating cells, such as spermatogonia (during meiosis), and in nonproliferating somatic cells, such as neurons. Consequently, expansion can occur – depending on the disease – ­during both DNA replication (as shown in Fig. 13.27) and genome maintenance (i.e., DNA repair). The clinical phenotypes of Huntington disease and fragile X syndrome are presented in Chapter 7 and in Cases 24 and 17, respectively. It has become apparent, particularly with fragile X syndrome, that diseases due to the expansion of unstable repeats are primarily neurologic; the clinical presentations include ataxia, cognitive exon intron intron 5' AUG pre-mRNA (CGG)n>200 Fragile X syndrome (CGG)n Transcriptional silencing = loss-offunction mutation Loss of RNA binding = impaired translational repression of target RNAs (GAA)n200 Friedreich ataxia (GAA)n Impaired transcriptional elongation = loss of frataxin function Increased Fe in mitochondria, reduced heme synthesis, reduced activity of Fe-S complex containing proteins (CTG)n50 Myotonic dystrophy 1 (CUG)n Expanded CUG repeats in the RNA confer novel properties on the RNA Expanded CUG repeats bind increased amounts of RNA-binding proteins  impaired RNA splicing of key proteins (CCTG)n75 Myotonic dystrophy 2 (CCUG)n (CAG)n40 Huntington disease (CAG)n Expanded polyglutamine tracts in the huntingtin protein confer novel properties on the protein Increased and/or promiscuous protein:protein interactions with transcription factors  loss of their function (CGG)n>60 to 200 Fragile X tremor/ataxia syndrome 2- to 5-fold increase in FMR1 mRNA =?gain-ofRNA function Neuronal intranuclear inclusions stop 3' 3' UTR 5' UTR Figure 13.26 The locations of the nucleotide repeat expansions and the sequence of each nucleotide in five representative nucleotide repeat diseases, shown on a schematic of a generic pre–messenger RNA (mRNA). The minimal number of repeats in the DNA sequence of the affected gene associated with the disease is also indicated. The effect of the expansion on the mutant RNA or protein is also indicated. (Based partly on an unpublished figure courtesy John A. Phillips III, Vanderbilt University Nashville.) ▶
CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 287 in a laboratory. A related challenge is that there are no viruses that infect the mitochondrion, so genetic delivery...
Ch13 · Pt38 288 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE defects, dementia, nystagmus, parkinsonism, and spasticity. Nevertheless, other systems are sometimes involved, as illustrated by some of the diseases discussed here. The Pathogenesis of Diseases Due to Unstable Repeat Expansions Diseases of unstable repeat expansion are diverse in their pathogenic mechanisms. They can be divided into three main classes, considered in turn in the sections to follow. Class 1: diseases due to the expansion of noncoding repeats that cause a loss of protein expression Class 2: disorders resulting from expansions of noncoding repeats that confer novel properties on the RNA Class 3: diseases due to repeat expansion of a codon such as CAG (for glutamine) that confers novel properties on the affected protein Class 1: Diseases Due to the Expansion of Noncoding Repeats That Cause a Loss of Protein Expression Fragile X Syndrome. In the X-linked fragile X syndrome, expansion of the CGG repeat in the 5′ untranslated region (UTR) of the FMR1 gene to more than 200 copies leads to excessive methylation of cytosines in the promoter; this epigenetic modification of the DNA silences transcription of the gene (see Figs. 7.21 and 13.26). Remarkably, the epigenetic silencing appears to be mediated by the variant FMR1 mRNA itself. The initial step in the silencing of FMR1 results from the FMR1 mRNA (containing the transcribed CGG repeat) hybridizing with the complementary CGG-repeat sequence of the FMR1 gene, to form an RNA:DNA duplex. The mechanisms that subsequently maintain the silencing of the FMR1 gene are unknown. The loss of the related protein (FMRP) is the cause of the intellectual disability, learning deficits, and nonneurologic features of the clinical phenotype, including postpubertal macroorchidism and connective tissue dysplasia (Case 17). FMRP is an RNA-binding protein that associates with polyribosomes to suppress the translation of proteins from its RNA targets. These targets appear to be involved in cytoskeletal structure, synaptic transmission, and neuronal maturation; disruption of these processes is likely to underlie the intellectual disability and learning abnormalities seen in individuals with fragile X. For example, FMRP appears to regulate the translation of proteins required for the formation of synapses because the brains of individuals with the fragile X syndrome have increased density of abnormally long, immature dendritic spines. Moreover, FMRP localizes to dendritic spines, where at least one of its roles is to regulate synaptic plasticity – the capacity to alter the strength of a synaptic connection – a process critical to learning and memory. Fragile X Tremor/Ataxia Syndrome. Remarkably, in individuals with less pronounced CGG repeat expansion (60–200 repeats) in the FMR1 gene, causing the clinically distinct fragile X tremor/ataxia syndrome (FXTAS), the pathogenesis is entirely different from that of the fragile X syndrome itself. Although decreased translational efficiency impairs the expression of FMRP in FXTAS, this reduction cannot be responsible for the disease because males with full expansions and virtually complete loss of function of the FMR1 gene never develop FXTAS. Rather, FXTAS seems to result from the two- to fivefold increase of FMR1 mRNA in these patients, representing a gain-of-function variant. This pathogenic RNA leads to the formation of intranuclear neuronal inclusions, the cellular signature of the disease. Class 2: Disorders Resulting From Expansions of Noncoding Repeats That Confer Novel Properties on the RNA Myotonic Dystrophy. Myotonic dystrophy 1 (DM1) is an autosomal dominant condition with the most pleiotropic phenotype of all the unstable repeat expansion disorders. In addition to myotonia, it is characterized by muscle weakness and wasting, cardiac conduction defects, testicular atrophy, insulin resistance, and cataracts; there is also a congenital form with intellectual disability. The disease results from a CTG expansion in the 3′ UTR of the DMPK gene, which encodes a protein kinase (see Fig. 13.26). Myotonic dystrophy 2 (DM2) is Starting (template) strand of genomic DNA Replicating strand detaches inappropriately from template during replication. Replicating strand slips from its proper alignment with the template strand, by one repeat (R) length. Mismatched R2 repeat loops out. Newly synthesized strand contains an extra repeat. R2 R3 R1 R3 R2 R3 R1 R3 R3 R1 R2 R3 R1 R2 R3 R1 R2 R3 R1 R2 Figure 13.27 The slipped mispairing mechanism thought to underlie the expansion of unstable repeats, such as the (CAG)n repeat found in Huntington disease and the spinocerebellar ataxias. An insertion occurs when the newly synthesized strand aberrantly dissociates from the template strand during replication synthesis. When the new strand reassociates with the template strand, the new strand may slip back to align out of register with an incorrect repeat copy. Once DNA synthesis is resumed, the misaligned molecule will contain one or more extra copies of the repeat (depending on the number of repeat copies that slipped out in the misalignment event). ▶
288 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE defects, dementia, nystagmus, parkinsonism, and spasticity. Nevertheless, other systems are sometimes involved, as illustrated by some of th...
Ch13 · Pt39 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 289 also an autosomal dominant trait and shares most of the clinical features of DM1, except that there is no associated congenital presentation. DM2 is due to the expansion of a CCTG tetranucleotide in the first intron of the gene encoding zinc finger protein 9 (see Fig. 13.26). The strikingly similar phenotypes of DM1 and DM2 suggest that they have a common pathogenesis. Because the unstable expansions occur within the noncoding regions of two different genes that encode unrelated proteins, the CTG trinucleotide expansion itself (and the resulting expansion of CUG in the mRNA) is thought to underlie an RNA-mediated pathogenesis. What is the mechanism by which large tracts of the CUG trinucleotide, in the noncoding regions of genes, lead to the DM1 and DM2 phenotypes? The pathogenesis appears to result from the binding of the CUG repeats to RNA-binding proteins. Consequently, the pleiotropy that typifies the disease may reflect the broad array of RNA-binding proteins to which the CUG repeats bind. Many of the RNA-binding proteins sequestered by the excessive number of CUG repeats are regulators of splicing. Indeed, more than a dozen distinct pre-mRNAs have splicing alterations in patients with DM1, including cardiac troponin T (which might account for the cardiac abnormalities) and the insulin receptor (which may explain the insulin resistance). Thus the myotonic dystrophies are referred to as spliceopathies. Knowledge of the abnormal processes underlying DM1 and DM2 is incomplete, but these molecular insights offer hope that a rational small molecule therapy might be developed. Class 3: Diseases Due to Repeat Expansion of a Codon That Confers Novel Properties on the Affected Protein Huntington Disease. Huntington disease is an autosomal dominant neurodegenerative disorder associated with chorea, athetosis (uncontrolled writhing movements of the extremities), loss of cognition, and psychiatric abnormalities (Case 24). The pathologic process is caused by the expansion – to more than 40 repeats – of a CAG codon in the HTT gene, resulting in long polyglutamine tracts in the protein, huntingtin (see Figs. 7.19 and 7.20). Evidence suggests that the proteins with expanded polyglutamine sequences have novel properties: the expanded tract confers novel features on the protein that damage specific populations of neurons and produce neurodegeneration by unique toxic mechanisms. The most striking cellular hallmark of the disease is the presence of insoluble aggregates of the abnormal protein (as well as other polypeptides) clustered in nuclear inclusions in neurons. The aggregates are thought to result from normal cellular responses to the misfolding of huntingtin that results from the polyglutamine expansion. Dramatic as these inclusions are, however, their formation may be protective rather than pathogenic. There is no unifying model of the neuronal death mediated by polyglutamine expansion in huntingtin. Many cellular processes are disrupted by expanded huntingtin in its soluble or aggregated form, including transcription, vesicular transport, mitochondrial fission, and synaptic transmission and plasticity. Ultimately, the most critical and primary events in the pathogenesis will be identified, perhaps guided by genetic analyses, leading to correction of the phenotype. For example, expanded huntingtin associates abnormally with a mitochondrial fission protein, GTPase dynamin-related protein 1 (DRP1), leading to multiple mitochondrial abnormalities in individuals with Huntington disease. Remarkably, in mice, these defects are rescued by reducing DRP1 GTPase activity, suggesting both that DRP1 may be a therapeutic target for the disorder and that mitochondrial abnormalities play important roles in Huntington disease. Despite the substantial progress in identifying novel repeat expansions and our understanding of the molecular events that underlie the pathology of the unstable repeat expansion diseases, we are only beginning to dissect the genetic and pathogenic complexity of these important conditions. It is clear that the use of new genomic technologies and study of animal models are providing critical insights into these disorders. Such insights should soon lead to interventions to prevent or to reverse the pathogenesis of these slowly developing disorders. We begin to explore the concepts relevant to the treatment of disease in the next chapter. GENERAL REFERENCES Adam MP, editor: Molecular genetics. In Gene Reviews, Seattle, 1993– 2022, University of Washington. http://www.ncbi.nlm.nih.gov/ books/NBK1116/ Hamosh A: Online mendelian inheritance in man, OMIM. Mc KusickNathans Department of Genetic Medicine, Baltimore, MD, Johns Hopkins University. http://omim.org/ Strachan T, Read AP: Human molecular genetics, ed 5, 2019, Garland Science. Valle DL, Antonarakis S, Ballabio A, et al, editors: The online metabolic and molecular bases of inherited disease, New York, 2019, Mc Graw Hill. https://ommbid.mhmedical.com REFERENCES TO SPECIFIC TOPICS Bettens K, Sleegers K, Van Broeckhoven C: Genetic insights in Alzheimer’s disease, Lancet Neurol 12:92–104, 2013. Bird TD: Alzheimer disease overview. In Gene Reviews, Seattle, 1998, University of Washington. https://www.ncbi.nlm.nih.gov/books/ NBK1161/ Blau N, Hennermann JB, Langenbeck U, et al: Diagnosis, classification, and genetics of phenylketonuria and tetrahydrobiopterin (BH4) deficiencies, Mol Genet Metab 104:S2–S9, 2011. Brais B, Bouchard J-P, Xie Y-G, et al: Short GCG expansions in the PABP2 gene cause oculopharyngeal muscular dystrophy, Nat Genet 18:164–167, 1998. Byers PH, Pyott SM: Recessively inherited forms of osteogenesis imperfecta, Ann Rev Genet 46:475–497, 2012. Chamberlin JS: Duchenne muscular dystrophy models show their age, Cell 143:1040–1042, 2010. ▶
CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 289 also an autosomal dominant trait and shares most of the clinical features of DM1, except that there is no associated...
Ch13 · Pt40 290 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Chillon M, Casals T, Mercier B, et al: Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens, N Engl J Med 332:1475–1480, 1995. Colak D, Zaninovic N, Cohen MS, et al: Promoter-bound trinucleotide repeat mRNA drives epigenetic silencing in fragile X syndrome, Science 343:1002–1005, 2014. Cutting GR: Modifier genes in mendelian disorders: The example of cystic fibrosis, Ann N Y Acad Sci 1214:57–69, 2010. De Jesus-Hernandez M, Mackenzie IR, Boeve BF, et al: Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS, Neuron 72:245–256, 2011. Duan D, Goemans N, Takeda S, et al: Duchene muscular dystrophy, Nat Rev Dis Primers 7:13, 2021. Flanigan KM: The muscular dystrophies, Semin Neurol 32:255–263, 2012. Fong LG, Young SG: PCSK9 function and physiology, J Lipid Res 49: 1152–1156, 2008. 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Jonsson T, Atwal JK, Steinberg S, et al: A mutation in APP protects against Alzheimer’s disease and age-related cognitive decline, Nature 488:96–99, 2012. Kathiresan S, Melander O, Guiducci C, et al: Six new loci associated with blood low-density lipoprotein cholesterol, high-density lipoprotein cholesterol or triglycerides in humans, Nat Genet 40: 189–197, 2008. Keinath MC, Prior DE, Prior TW: Spinal muscular atrophy: mutations, testing, and clinical relevance, Appl Clin Genet 14:11–25, 2021. https://doi.org/10.2147/TACG. S239603 Kirkman MA, Yu-Wai-Man P, Korsten A, et al: Gene-environment interactions in Leber hereditary optic neuropathy, Brain 132(Pt 9): 2317–2326, 2009. https://doi.org/10.1093/brain/awp 158 Koopman WJ, Willems PH, Smeitink JA: Monogenic mitochondrial disorders, N Engl J Med 366:1132–1141, 2012. Laine CM, Joeng KS, Campeau PM, et al: WNT1 mutations in earlyonset osteoporosis and osteogenesis imperfecta, N Engl J Med 368: 1809–1816, 2013. 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Rahaghi FF, Miravitlles M: Long-term clinical outcomes following treatment with alpha 1-proteinase inhibitor for COPD associated with alpha-1 antitrypsin deficiency: A look at the evidence, Respir Res 18(1):105, 2017. https://doi.org/10.1186/s 12931-017-0574-1 Ramsey BW, Banks-Schlegel S, Accurso FJ, et al: Future directions in early cystic fibrosis lung disease research: An NHLBI workshop report, Am J Respir Crit Care Med 185:887–892, 2012. Renton AE, Majounie E, Waite A, et al: A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD, Neuron 72:257–268, 2011. Schon EA, Di Mauro S, Hirano M: Human mitochondrial DNA: Roles of inherited and somatic mutations, Nat Rev Genet 13:878–890, 2012. Selkoe DJ: Alzheimer’s disease, Cold Spring Harb Perspect Biol 3:a 00 4457, 2011. Sosnay PR, Siklosi KR, Van Goor F, et al: Defining the disease liability of mutations in the cystic fibrosis transmembrane conductance regulator gene, Nature Genet 45:1160–1167, 2013. 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USEFUL WEBSITES Variant Databases Clin Var (an annotated aggregation of variants by gene submitted from clinical labs and researchers): https://www.ncbi.nlm.nih.gov/clinvar Global Variome Shared LOVD: https://databases.lovd.nl/shared/genes Clin Gen Evidence Repository: https://erepo.clinicalgenome.org/evrepo/ Clinical and functional translation of CFTR (CFTR2 project): http:// www.cftr 2.org/ Collagen variant database (the osteogenesis imperfecta and Ehlers-Danlos syndrome variant database: http://www.le.ac.uk/genetics/collagen/ Human mitochondrial genome database: https://www.mitomap.org/ MITOMAP Phenylalanine hydroxylase variant database: http://www.biopku.org/ home/pah.asp The Human Gene Mutation Database: http://www.hgmd.cf.ac.uk/ac/ index.php PROBLEMS 1. One variant allele at the LDL receptor locus (leading to familial hypercholesterolemia) encodes an elongated protein that is ~50,000 Da larger than the normal 120,000Da receptor. Indicate at least three mechanisms that could account for this abnormality. Approximately how many extra nucleotides would need to be translated to add 50,000 Da to the protein? 2. Comparing autosomal dominant PSCK9 gain-of-function variants to autosomal dominant variants in the LDL receptor gene, are these phenocopies or genocopies? Explain your answer. 3. In discussing the nucleotide changes in the coding region of the CFTR gene, we stated that some of the changes (the missense changes) so far are only “putative” diseasecausing variants. What criteria would one need to fulfill before knowing that a nucleotide change is pathogenic and not benign? continued ▶
290 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Chillon M, Casals T, Mercier B, et al: Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens, N Engl...
Ch13 · Pt41 CHAPTER 13 — The Molecular, Biochemical, and Cellular Basis of Genetic Disease 291 4. Johnny, 2 years of age, is failing to thrive. Investigations show that although he has clinical findings of CF, his sweat chloride concentration is normal. The sweat chloride concentration is normal in less than 2% of patients with CF. His pediatrician and parents want to know whether DNA analysis can determine whether he indeed has CF. a. Would DNA analysis be useful in this case? Briefly outline the steps involved in obtaining a DNA diagnosis for CF. b. If he has CF, what is the probability that he is homozygous for the c.1521_1523del CTT (p. Phe 508del) variant? (Assume that 95% of CFTR variants could be detected at the time you are consulted and that his parents are from northern Europe, where the Phe 508del allele has a frequency of 0.70.) c. If he does not have the common variant, does this disprove the diagnosis? Explain. 5. James is the only person in his kindred affected by DMD. He has one unaffected brother, Joe. DNA analysis shows that James has a deletion in the DMD gene and that Joe has received the same maternal X chromosome, but one without a deletion. What genetic counseling would you give the parents regarding the recurrence risk for DMD in a future pregnancy? 6. DMD has a high mutation rate but shows no ancestral variation in frequency. Use your knowledge of the gene and the genetics of DMD to suggest why this disorder is equally common in all populations. 7. A 3 12-year-old girl, T. N., has increasing difficulty standing up after sitting on the floor. Her serum level of creatine kinase is grossly elevated. Although a female, the presumptive clinical diagnosis is Duchenne muscular dystrophy. Identify three mechanisms that could account for the rare occurrence of DMD in a female. 8. In patients with osteogenesis imperfecta, explain why the missense variants at glycine positions in the triple helix of type I collagen are confined to a limited ­number of replacement amino acid residues (Ala, Ser, Cys, Arg, Val, Asp). 9. A 2-year-old infant, the child of first-cousin parents, has unexplained developmental delay. A survey of various biochemical parameters indicates that he has a deficiency of four lysosomal enzyme activities. Explain how a single autosomal recessive pathogenic variant might cause the loss of function of four enzyme activities. Why is it most likely that the child has an autosomal recessive condition, if he has a genetic condition at all? 10. The effect of a dominant negative allele illustrates one general mechanism by which changes in a protein cause dominantly inherited disease. What mechanisms are commonly associated with dominance in genes encoding the subunits of multimeric proteins? 11. The clinical effects of pathogenic variants in a housekeeping protein are frequently limited to one or a few tissues, often tissues in which the protein is abundant and serves a specialty function. Identify and discuss examples that illustrate this generalization, and explain why they fit it. 12. The relationship between the site at which a protein is physically present/active and the site of pathological change in a genetic disease may be unpredictable. Give examples of this phenomenon and discuss them. 13. The two pseudodeficiency alleles of hex A are p. Arg 247Trp and p. Arg 249Trp. What is the probable reason that the missense substitutions of these alleles are so close together in the protein? 14. Why are gain-of-function variants in proteins, as seen with the autosomal dominant PCSK9 variants that cause hypercholesterolemia, almost always missense variants? 15. What are the possible explanations for the presence of three predominant alleles for Tay-Sachs disease (as well as other lysosomal storage disorders) in Ashkenazi Jews? Does the presence of three alleles, and the relatively high frequency of Tay-Sachs disease in this population, necessarily accord with a heterozygote advantage hypothesis or a founder effect hypothesis? 16. The known loci associated with Alzheimer disease fail to account for the implied genetic contribution to risk. Identify at least three other sources of genetic variation that may account for the genetic contribution to this disorder. 17. The two forms of myotonic dystrophy are characterized by an expansion of a CUG trinucleotide in the RNA, which is thought to lead to an RNA-mediated pathogenesis. Propose a molecular therapy that might counteract the effect of the CUG expansions in the RNAs and that would reduce the binding of RNA-binding proteins to the CUG repeats. Anticipate some possible undesirable effects of your proposed therapy. ▶
PROBLEMS—CONT’D .
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