Thompson & Thompson Genetics in Medicine, 8th Edition

CHAPTER 12. The Molecular, Biochemical, and Cellular Basis of Genetic Disease

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 11, we presented an outline of the general mechanisms by which mutations cause disease (see Fig. 11-1) and reviewed the steps at which mutations can disrupt the synthesis or function of a protein (see Table 11-2). Those outlines provide a framework for understanding the pathogenesis of all genetic disease. However, mutations 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 examine here well-known disorders such as phenylketonuria, cystic fibrosis, familial hypercholesterolemia, Duchenne muscular dystrophy, and Alzheimer disease. In some instances, less common disorders are included because they best demonstrate a specific principle. The importance of selecting representative disorders becomes apparent when one considers that to date, mutations in almost 3000 genes have been associated with a clinical phenotype. In the coming decade, one anticipates that many more of the approximately 20,000 to 25,000 coding genes in the human genome will be shown to be associated with both monogenic and genetically complex diseases.

Diseases Due to Mutations in Different Classes of Proteins

Proteins carry out an astounding number of different functions, some of which are presented in Figure 12-1. Mutations 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 Figure 12-1; many other of the proteins listed, as well as the diseases associated with them, are described in the Cases section.


FIGURE 12-1 Examples of the classes of proteins associated with diseases with a strong genetic component (most are monogenic), and the part of the cell in which those proteins normally function. CFTR, Cystic fibrosis transmembrane regulator; FMRP, fragile X mental retardation protein; HLA, human leukocyte antigen; LDL, low-density lipoprotein; MELAS, mitochondrial encephalomyopathy with lactic acidosis and strokelike episodes; PKU, phenylketonuria.

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, which are present in virtually every cell and have fundamental roles in the maintenance of cell structure and function; and tissue-specific specialty proteins, which are produced in only one or a limited number of 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, is often useful 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), mutation in a tissue-specific protein most often produces a disease restricted to that tissue. However, there may be secondary effects on other tissues, and in some cases mutations in tissue-specific proteins may cause abnormalities primarily in organs that do not express the protein at all; ironically, the tissue expressing the mutant protein may be left entirely unaffected by the pathological process. This situation is exemplified by phenylketonuria, discussed in depth in the next section. Phenylketonuria 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), and not the liver, that is damaged by the high blood levels of phenylalanine resulting from the lack of hepatic PAH. Consequently, one cannot necessarily infer that disease in an organ results from mutation in a gene expressed principally or only in that organ, or in that organ at all.

• Second, although housekeeping proteins are expressed in most or all tissues, the clinical effects of mutations in housekeeping proteins are frequently limited to one or just a few tissues, for at least two reasons. In most such instances, a single or a few tissue(s) may be affected because the housekeeping protein in question is normally expressed abundantly there and serves a specialty function in that tissue. This situation is illustrated by Tay-Sachs disease, as discussed later; the mutant enzyme in this disorder is hexosaminidase A, which is expressed in virtually all cells, but its absence leads to a fatal neurodegeneration, leaving non-neuronal cell types unscathed. In other instances, another protein with overlapping biological activity may also be expressed in the unaffected tissue, thereby lessening the impact of the loss of function of the mutant gene, a situation known as genetic redundancy. Unexpectedly, even mutations in genes that one might consider 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 5000 genes that encode enzymes, and there are hundreds of human diseases—the so-called enzymopathies—that involve enzyme defects. We first discuss one of the best-known groups of inborn errors of metabolism, the hyperphenylalaninemias.


The Hyperphenylalaninemias

The abnormalities that lead to an increase in the blood level of phenylalanine, most notably PAH deficiency or phenylketonuria (PKU), illustrate almost every principle of biochemical genetics related to enzyme defects. The biochemical causes of hyperphenylalaninemia are illustrated in Figure 12-2, and the principal features of the diseases associated with the biochemical defect at the five known hyperphenylalaninemia loci are presented in Table 12-1. All the genetic disorders of phenylalanine metabolism are inherited as autosomal recessive conditions and are due to loss-of-function mutations in the gene encoding PAH or in genes required for the synthesis or reutilization of its cofactor, tetrahydrobiopterin (BH4).


FIGURE 12-2 The biochemical pathways affected in the hyperphenylalaninemias. BH4, tetrahydrobiopterin; 4αOHBH4, 4α-hydroxytetrahydrobiopterin; qBH2, 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, L-dihydroxyphenylalanine; NE, norepinephrine; E, epinephrine; 5-OH trp, 5-hydroxytryptophan.

TABLE 12-1

Locus Heterogeneity in the Hyperphenylalaninemias


*BH4 supplementation may increase the PAH activity of some patients in each of these three groups.

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.


Classic PKU is the epitome of the enzymopathies. It results from mutations in the gene encoding PAH, which converts phenylalanine to tyrosine (see Fig. 12-2 and Table 12-1). The discovery of PKU in 1934 marked the first demonstration of a 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 in early childhood. A small fraction of phenylalanine is metabolized to produce increased amounts of phenylpyruvic acid, the keto acid responsible for the name of the disease. Ironically, although the enzymatic defect has been known for many decades, the precise pathogenetic mechanism(s) by which increased phenylalanine damages the brain is still uncertain. Importantly, the neurological damage is largely avoided by reducing the dietary intake of phenylalanine. The management of PKU is a paradigm of the treatment of the many metabolic diseases whose outcome can be improved by preventing accumulation of an enzyme substrate and its derivatives; this therapeutic principle is described further in Chapter 13.

Variant Phenylketonuria and Nonphenylketonuria Hyperphenylalaninemia.

Whereas PKU results from a virtual absence of PAH activity (less than 1% of that in controls), less severe phenotypes, designated non-PKU hyperphenylalaninemia and variant PKU (see Table 12-1), result when the mutant PAH enzyme has some residual activity. The fact that a very small amount of residual enzyme activity can have a large impact on phenotype is another general principle of the enzymopathies (see Box).

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 approximately 50% of residual activity) are clinically normal. In fact, many enzymes may maintain normal substrate and product levels with activities of less than 10%, a point relevant to the design of therapeutic strategies (e.g., homocystinuria due to cystathionine synthase deficiency—see Chapter 13). 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 pathophysiological consequences of enzymopathies can be attributed 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. 12-3).


FIGURE 12-3 A model metabolic pathway showing that the potential effects of an enzyme deficiency include accumulation of the substrate (S) or derivatives of it (S1, S2, S3) and deficiency of the product (P) or compounds made from it (P1, P2). In some cases, the substrate derivatives are normally only minor metabolites that may be formed at increased rates when the substrate accumulates (e.g., phenylpyruvate in phenylketonuria).

• Diffusible versus 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, which remains trapped within its organelle or cell). The pathological 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 unmetabolized substrate or its derivatives can move freely throughout the body, damaging cells that may normally have no relationship to the affected enzyme, as in PKU.

• Loss of multiple enzyme activities

A patient with a single-gene defect may have a 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); and 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 pathological and clinical features resulting from an enzyme defect are often shared by diseases due to deficiencies of 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 etiological relationship between the two diseases may not be immediately obvious. For example, partial deficiency of the purine enzyme hypoxanthine-guanine phosphoribosyltransferase causes only hyperuricemia, whereas a complete deficiency causes hyperuricemia as well as a profound neurological disease, Lesch-Nyhan syndrome, which resembles cerebral palsy.

Variant PKU includes patients who require only some dietary phenylalanine restriction but to a lesser degree than that required in classic PKU, because their increases in blood phenylalanine levels are more moderate and less damaging to the brain. In contrast to classic PKU, in which the plasma phenylalanine levels are greater than 1000 μmol/L when the patient is receiving a normal diet, non-PKU hyperphenylalaninemia is defined by plasma phenylalanine concentrations above the upper limit of normal (120 μmol/L), but less than the levels seen in classic PKU. If the increase in non-PKU hyperphenylalaninemia is small (<400 μmol/L), no treatment is required; these individuals come to clinical attention only because they are identified by newborn screening (see Chapter 17). Their normal phenotype has been the best indication of the “safe” level of plasma phenylalanine that must not be exceeded in treating classic PKU. The association of these three clinical phenotypes with mutations in the PAHgene is a clear example of allelic heterogeneity leading to clinical heterogeneity (see Table 12-1).

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 700 different mutations worldwide—has been identified in patients with hyperphenylalaninemia associated with classic PKU, variant PKU, and non-PKU hyperphenylalaninemia (see Table 12-1). Seven mutations account for a majority of known mutant alleles in populations of European descent, whereas six others represent the majority of PAHmutations in Asian populations (Fig. 12-4). The remaining disease-causing mutations are individually rare. To record and make this information publicly available, a PAH database has been developed by an international consortium.


FIGURE 12-4 The nature and identity of PAH mutations in populations of European and Asian descent (the latter from China, Korea, and Japan). The one-letter amino acid code is used (see Table 3-1). SeeSources & Acknowledgments.

The allelic heterogeneity at the PAH locus has major clinical consequences. Most important is the fact that most hyperphenylalaninemic subjects 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 observed in this patient population. Thus, mutations 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 mutations have been found to have phenotypes ranging all the way from classic PKU to non-PKU hyperphenylalaninemia. Accordingly, it is now clear that other unidentified biological variables—undoubtedly including modifier genes—generate variation in the phenotype seen with any specific genotype. This lack of a strict genotype-phenotype correlation, initially somewhat surprising, is now recognized to be a common feature of many single-gene diseases and highlights the fact that even monogenic traits like PKU are not genetically “simple” disorders.

Defects in Tetrahydrobiopterin Metabolism.

In 1% to 3% of hyperphenylalaninemic patients, 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 12-1 and Fig. 12-2). The association of a single biochemical phenotype, such as hyperphenylalaninemia, with mutations in different genes, is an example of locus heterogeneity (see Table 11-1). 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 the genes associated with hyperphenylalaninemia (see Fig. 12-2). BH4-deficient patients were first recognized because they developed profound neurological 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 of 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. 12-2).

The locus heterogeneity of hyperphenylalaninemia is of great significance because the treatment of patients with a defect in BH4 metabolism differs markedly from subjects with mutations in PAH, in two ways. First, because the PAH enzyme of individuals with BH4 defects is itself normal, its activity can be restored by large doses of oral BH4, leading to a reduction in their plasma phenylalanine levels. This practice highlights the principle of product replacement in the treatment of some genetic disorders (see Chapter 13). Consequently, phenylalanine restriction can be significantly relaxed in the diet of patients with defects in BH4 metabolism, and some patients actually tolerate a normal (i.e., a phenylalanine-unrestricted) diet. Second, one must also 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. 12-2 and Table 12-1).

Remarkably, mutations in sepiapterin reductase, an enzyme in the BH4 synthesis pathway, do not cause hyperphenylalaninemia. In this case, only dopa-responsive dystonia is seen, due to impaired synthesis of dopamine and serotonin (see Fig. 12-2). It is thought that alternative pathways exist for the final step in BH4 synthesis, bypassing the sepiapterin reductase deficiency in peripheral tissues, an example of genetic redundancy.

For these reasons, all hyperphenylalaninemic infants must be screened to determine whether their hyperphenylalaninemia is the result of an abnormality in PAH or in BH4 metabolism. 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 in PAH Mutations.

Many hyperphenylalaninemia patients with mutations 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 them a less restricted dietary intake of phenylalanine. The patients most likely to respond are those with significant residual PAH activity (i.e., patients with variant PKU and non-PKU hyperphenylalaninemia), but even a minority of patients with classic PKU are also responsive. The presence of residual PAH activity does not, however, necessarily guarantee an effect of BH4 administration on plasma phenylalanine levels. Rather, the degree of BH4 responsiveness will depend on the specific properties of each mutant PAH protein, reflecting the allelic heterogeneity underlying PAH mutations.

The provision of increased amounts of a cofactor is a general strategy that has been employed for the treatment of many inborn errors of enzyme metabolism, as discussed further in Chapter 13. In the general case, 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 has been shown to exert its beneficial effect through one or more mechanisms, all of which result from the increased amount of the cofactor that is brought into contact with the mutant PAH apoenzyme. These mechanisms include stabilization of the mutant enzyme, protection of the enzyme from degradation by the cell, and increase in the cofactor supply for a mutant enzyme that has a low affinity for BH4.

Newborn Screening.

PKU is the prototype of genetic diseases for which mass newborn screening is justified (see Chapter 18) because it is relatively common in some populations (up to approximately 1 in 2900 live births), mass screening is feasible, failure to treat has severe consequences (profound developmental delay), and treatment is effective if begun early in life. To allow time for the postnatal increase in blood phenylalanine levels to occur, the test is performed after 24 hours of age. Blood from a heel prick is assayed in a central laboratory for blood phenylalanine levels and measurement of the 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.

Maternal Phenylketonuria.

Originally, the low-phenylalanine diet was discontinued in mid-childhood for most patients with PKU. Subsequently, however, it was discovered that almost all offspring of women with PKU not receiving 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, all of these children are heterozygotes. Thus their neurodevelopmental delay is not due to their own genetic constitution but to the highly teratogenic effect of elevated levels of phenylalanine in the maternal circulation. Accordingly, it is imperative that women with PKU who are planning pregnancies commence a low-phenylalanine diet before conceiving.

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 biological macromolecules. Mutations 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 13).

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. 12-5). 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. 12-5). These genes encode the α and β subunits of the enzyme (the HEXA and HEXB genes, 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.


FIGURE 12-5 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. SeeSources & Acknowledgments.

The clinical manifestations of defects in the three genes are indistinguishable, but they can be differentiated by enzymatic analysis. Mutations 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. 12-5) 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 approximately 3 to 6 months of age but then gradually undergo progressive neurological deterioration until death at 2 to 4 years. The effects of neuronal death can be seen directly in the form of the so-called cherry-red spot in the retina (Case 43). In contrast, HEXA alleles associated with some residual activity lead to later-onset forms of neurological 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) do not cause disease at all.

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 so-called 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 mutation on the other chromosome. These individuals have a low level of hex A activity (approximately 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 single-gene diseases, some alleles are found at higher frequency in some populations than in others (see Chapter 9). This situation is illustrated by Tay-Sachs disease, in which three alleles account for 99% of the mutations found in Ashkenazi Jewish patients, the most common of which (Fig. 12-6) accounts for 80% of cases. Approximately 1 in 27 Ashkenazi Jews is a carrier of a Tay-Sachs allele, and the incidence of affected infants is 100 times higher than in other populations. A founder effect or heterozygote advantage is the most likely explanation for this high frequency (see Chapter 9). 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.


FIGURE 12-6 Four-base insertion (TATC) in the hexosaminidase A (hex A) gene in Tay-Sachs disease, leading to a frameshift mutation. This mutation 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.

Altered Protein Function due to Abnormal Post-translational Modification

A Loss of Glycosylation: 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 post-translational 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 (Fig. 12-7). The cytoplasm of cultured skin fibroblasts from I-cell patients contains numerous abnormal lysosomes, or inclusions, (hence the term inclusion cells or I cells).


FIGURE 12-7 I-cell disease facies and habitus in an 18-month-old girl. SeeSources & Acknowledgments.

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. This unusual situation arises because the hydrolases in these patients have not been properly modified post-translationally. 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.

Gains of Glycosylation: Mutations 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 (approximately 1.5%) of the missense mutations that cause human disease may be associated with abnormal gains of N-glycosylation due to mutations 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 Calmette-Guérin (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 mutations 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. Mutations 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 biological activity only after they associate with cofactors, such as BH4 in the case of PAH, as discussed earlier. Mutations 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 13).

Impaired Cofactor Binding: Homocystinuria due to Cystathionine Synthase Deficiency

Homocystinuria due to cystathionine synthase deficiency (Fig. 12-8) 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.


FIGURE 12-8 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 13).

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 13). 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 mutations in cystathionine synthase. Mutations in five different enzymes of cobalamin (vitamin B12) or folate metabolism can also lead to increased levels of homocysteine in body fluids. These mutations impair the provision of the vitamin B12 cofactor, methylcobalamin (methyl-B12), or of methyl-H4-folate (see Fig. 12-8) and thus represent another example (like the defects in BH4 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.

Mutations 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. 12-9) 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.


FIGURE 12-9 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. SeeSources & Acknowledgments.

In white populations, α1AT deficiency affects approximately 1 in 6700 persons, and approximately 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 (Glu342Lys) is relatively common. The reason for the relatively high frequency of the Z allele in white 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 approximately 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 mutation in β-globin (see Chapter 11), the Z allele of α1AT is a clear example of a mutation that confers a novel property on the protein (in both of these examples, a tendency to aggregate) (see Fig. 11-1).

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 mutation 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 Alzheimer disease (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. 12-10). 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 approximately 15% of the normal plasma concentration of α1AT. Second, the Z protein has only approximately 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 rectify the elastase:α1AT imbalance. At present, it is still uncertain whether progression of the lung disease is slowed by α1AT augmentation.


FIGURE 12-10 A posteroanterior chest radiograph of an individual carrying two Z alleles of the α1AT gene, showing the hyperinflation and basal hyperlucency characteristic of emphysema. SeeSources & Acknowledgments.

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 monogenetic disease. Thus, for persons with the Z/Z genotype, survival after 60 years of age is approximately 60% in nonsmokers but only approximately 10% in smokers (see Fig. 12-9). 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 likely to be 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 16.

Dysregulation of a Biosynthetic Pathway: Acute Intermittent Porphyria

Acute intermittent porphyria (AIP) is an autosomal dominant disease associated with intermittent neurological 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 p450 drug-metabolizing enzymes (Fig. 12-11). All individuals with AIP have an approximately 50% reduction in PBG deaminase enzymatic activity, whether their disease is clinically latent (90% of patients throughout their lifetime) or clinically expressed (approximately 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 incompatible with life. AIP illustrates one molecular mechanism by which an autosomal dominant disease may manifest only episodically.


FIGURE 12-11 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. The residual PBG deaminase activity (approximately 50% of controls) is overloaded, and the accumulation of ALA and PBG causes clinical disease. CoA, Coenzyme A. SeeSources & Acknowledgments.

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. 12-11). 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 18); 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. 12-11). 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 (apoB)-containing lipoproteins. Other monogenic hyperlipoproteinemias, each with distinct biochemical and clinical phenotypes, have also been recognized.

In addition to mutations in the LDL receptor gene (Table 12-2), abnormalities in three other genes can also lead to familial hypercholesterolemia (Fig. 12-12). 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 apoB, the major protein component of LDL and a ligand for the LDL receptor. Because of its importance, we first review familial hypercholesterolemia due to mutations in the LDL receptor. We also discuss mutations in the PCSK9 protease gene; although gain-of-function mutations 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.

TABLE 12-2

Four Genes Associated with Familial Hypercholesterolemia


*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.


FIGURE 12-12 The four proteins associated with familial hypercholesterolemia. The low-density lipoprotein (LDL) receptor binds apoprotein B-100. Mutations 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 clathrin-coated pits requires the ARH adaptor protein, which links the receptor to the endocytic machinery of the coated pit. Homozygous mutations 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).

Familial Hypercholesterolemia due to Mutations in the LDL Receptor

Mutations 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 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 pathological events from the affected locus to its effect on individuals and populations has been meticulously documented.


Familial hypercholesterolemia due to mutations 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. 12-13). Homozygotes may have clinically significant coronary heart disease in childhood and, if untreated, few live beyond the third decade. The heterozygous form of the disease, with a population frequency of approximately 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. 12-13). Because of the inherited nature of familial hypercholesterolemia, it is important to make the diagnosis in the approximately 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 approximately 1 in 20 has familial hypercholesterolemia; most such individuals have an uncharacterized hypercholesterolemia due to multiple common genetic variants, as presented in Chapter 8.


FIGURE 12-13 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. SeeSources & Acknowledgments.

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. 12-14). 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 CoA) reductase. Cholesterol not required for cellular metabolism or membrane synthesis may be re-esterified 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. 12-14).


FIGURE 12-14 The cell biology and biochemical role of the low-density lipoprotein (LDL) receptor and the six classes of mutations 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 CoA reductase, 3-hydroxy-3-methylglutaryl coenzyme A reductase. SeeSources & Acknowledgments.

Classes of Mutations in the LDL Receptor

More than 1100 different mutations have been identified in the LDLR gene, and these are distributed throughout the gene and protein sequence. Not all of the reported mutations 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 Figure 12-14. Analysis of the effect on the receptor of mutations in each domain has played an important role in defining the function 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 mutations can be grouped into six classes, depending on which step of the normal cellular itinerary of the receptor is disrupted by the mutation (see Fig. 12-14).

• Class 1 mutations are null alleles that prevent the synthesis of any detectable receptor; they are the most common type of disease-causing mutations at this locus. In the remaining five classes, the receptor is synthesized normally, but its function is impaired.

• Mutations in class 2 (like those in classes 4 and 6) define features of the polypeptide critical to its subcellular localization. The relatively common class 2 mutations 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 mutant receptors reach the cell surface but are incapable of binding LDL.

• Class 4 mutations impair localization of the receptor to the coated pit, and consequently the bound LDL is not internalized. These mutations 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 mutations are recycling-defective alleles. Receptor recycling requires the dissociation of the receptor and the bound LDL in the endosome. Mutations 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 mutations 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. Mutations 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 Potential Drug Target for Lowering LDL Cholesterol

Rare cases of autosomal dominant familial hypercholesterolemia have been found to result from gain-of-function missense mutations 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. 12-12). Consequently, the increase in PSCK9 activity associated with gain-of-function mutations 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 mutations 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 12-3). For example, in the African American population one of two PCSK9nonsense variants is found in 2.6% of all subjects; each variant is associated with a mean reduction in LDL cholesterol of approximately 40%. This reduction in LDL cholesterol has a powerful protective effect against coronary artery disease, reducing the risk by approximately 90%; only approximately 1% of African American 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 (Arg46Leu) is more common in white populations (3.2% of subjects) but appears to confer only approximately 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 cholesterol levels of 20 to 40 mg/dL would significantly decrease the incidence of coronary heart disease in the population. The strong protective effect of PCSK9 loss-of-function alleles, together with the apparent absence of any clinical sequelae in subjects with a total absence of PCSK9 activity, has made PCSK9 a strong candidate target for drugs that inactivate or diminish the activity of the enzyme.

TABLE 12-3

PCSK9 Variants Associated with Low LDL Cholesterol Levels


LDL, Low-density lipoprotein.

Derived from Cohen JC, Boerwinkle E, Mosley TH, Hobbs H: Sequence variants in PCSK9, low LDL, and protection against coronary heart disease, N Engl J Med 354:1264–1272, 2006.

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 13). Newer therapies include monoclonal antibodies that directly target PCSK9, which lower LDL cholesterol by an additional 60% in clinical trials.

Transport Defects

Cystic Fibrosis

Since the 1960s, cystic fibrosis (CF) has been one of the most publicly visible of all human monogenic diseases (Case 12). It is the most common fatal autosomal recessive genetic disorder of children in white populations, with an incidence of approximately 1 in 2500 white births (and thus a carrier frequency of approximately 1 in 25), whereas it is much less prevalent in other ethnic groups, such as African Americans (1 in 15,000 births) and Asian Americans (1 in 31,000 births). The isolation of the CF gene (called CFTR, for CF transmembrane regulator) (see Chapter 10) more than 25 years ago was one of the first illustrations of the power of molecular genetic and genomic approaches to identify disease genes. Physiological 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 Phenotypes 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 infants). 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 mutations in the CFTR gene.

The pancreatic defect in CF is a maldigestion syndrome due to the deficient secretion of pancreatic enzymes (lipase, trypsin, chymotrypsin). Approximately 5% to 10% 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 phenotypes are observed in CF patients. For example, neonatal lower intestinal tract obstruction (meconium ileus) occurs in 10% to 20% of CF newborns. The genital tract is also affected; females with CF have some reduction in fertility, but more than 95% of CF males 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 mutant alleles in the CFTR gene. Similarly, some individuals with idiopathic chronic pancreatitis are carriers of mutations in CFTR, yet lack other clinical signs of CF.

The CFTR Gene and Protein.

The CFTR gene has 27 exons and spans approximately 190 kb of DNA. The CFTR protein encodes a large integral membrane protein of approximately 170 kD (Fig. 12-15). The protein belongs to the so-called ABC (ATP [adenosine triphosphate]–binding cassette) family of transport proteins. At least 22 ABC transporters have been implicated in mendelian disorders and complex trait phenotypes.


FIGURE 12-15 The structure of the CFTR gene and a schematic of the CFTR protein. Selected mutations are shown. The exons, introns, and domains of the protein are not drawn to scale. ΔF508 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. SeeSources & Acknowledgments.

The CFTR chloride channel has five domains, shown in Figure 12-15: 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 mutations in each of them (see Fig. 12-15). 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.

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 physiological 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

Mutations in the Cystic Fibrosis Transmembrane Regulator Polypeptide.

The most common CF mutation is a deletion of a phenylalanine residue at position 508 (ΔF508) in the first ATP-binding fold (NBD1; see Fig. 12-15), accounting for approximately 70% of all CF alleles in white populations. In these populations, only seven other mutations are more frequent than 0.5%, and the remainder are each quite rare. Mutations of all types have been identified, but the largest single group (nearly half) are missense substitutions. The remainder are point mutations 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 mutations that are disease-causing is uncertain because few have been subjected to functional analysis. However, a new project called the Clinical and Functional Translation of CFTR (CFTR2 project; has succeeded in assigning pathogenicity to more than 125 CFTR mutations, which together account for at least 96% of all CFTR alleles worldwide.

Although the specific biochemical abnormalities associated with most CF mutations 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 Figure 12-15.

• Class 1 mutations are null alleles—no CFTR polypeptide is produced. This class includes alleles with premature stop codons or that generate highly 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 mutations impair the folding of the CFTR protein, thereby arresting its maturation. The ΔF508 mutant typifies this class; this misfolded protein cannot exit from the endoplasmic reticulum. However, the biochemical phenotype of the ΔF508 protein is complex, because it also exhibits defects in stability and activation in addition to impaired folding.

• Class 3 mutations allow normal delivery of the CFTR protein to the cell surface, but disrupt its function (see Fig. 12-15). The prime example is the Gly551Asp mutation that impedes the opening and closing of the CFTR ion channel at the cell surface. This mutation is particularly notable because, although it constitutes only approximately 2% of CFTR alleles, the drug ivacaftor has been shown to be remarkably effective in correcting the function of the mutant Gly551Asp protein at the cell surface, resulting in both physiological and clinical improvements (see Chapter 13).

• Class 4 mutations are located in the membrane-spanning domains and, consistent with this localization, have defective chloride ion conduction.

• Class 5 mutations reduce the number of CFTR transcripts.

• Class 6 mutant proteins are synthesized normally but are unstable at the cell surface.

A Cystic Fibrosis Genocopy: Mutations 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 mutations in the epithelial sodium channel gene SCNN1, a so-called 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 mutations are not identified in a particular case, abnormalities in SCNNI must be considered.

Genotype-Phenotype Correlations in Cystic Fibrosis.

Because all patients with the classic form of CF appear to have mutations 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 ΔF508 mutation 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 Arg117His (see Fig. 12-15), 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 ΔF508 mutation, 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 8. Overall, the evidence indicates that TGFB1alleles 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 CFTR mutant allele frequency of 1 in 50 that is observed in white populations (see Chapter 9). The disease is much less frequent in nonwhites, although it has been reported in Native Americans, African Americans, and Asians (e.g., approximately 1 in 90,000 Hawaiians of Asian descent). The ΔF508 allele is the only one found to date that is common in virtually all white populations, but its frequency among all mutant alleles varies significantly in different European populations, from 88% in Denmark to 45% in southern Italy.

In populations in which the ΔF508 allele frequency is approximately 70% of all mutant alleles, approximately 50% of patients are homozygous for the ΔF508 allele; an additional 40% are genetic compounds for ΔF508 and another mutant allele. In addition, approximately 70% of CF carriers have the ΔF508 mutation. As noted earlier, except for ΔF508, other mutations at the CFTR locus are rare, although in specific populations, some alleles are relatively common.

Population Screening.

The complex issues raised by considering population screening for diseases such as CF are discussed in Chapter 18. At present, CF meets most of the criteria for a newborn screening program, except it is not yet clear that early identification of affected infants significantly improves long-term prognosis. Nevertheless, the advantages of early diagnosis (such as improved nutrition from the provision of pancreatic enzymes) have led some jurisdictions to implement newborn screening programs. It is generally agreed that universal screening for carriers should not be considered until at least 90% of the mutations in a population can be detected. Although population screening for couples has been underway in the United States for several years, the sensitivity of carrier screening for CF has only recently surpassed 90%.

Genetic Analysis of Families of Patients and Prenatal Diagnosis.

The high frequency of the ΔF508 allele is useful when CF patients without a family history present for DNA diagnosis. The identification of the ΔF508 allele, in combination with a panel of 127 common mutations suggested by the American College of Medical Genetics, 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 presentation), carrier detection, and prenatal diagnosis. Given the vast knowledge of CF mutations in many populations, direct mutation detection is the method of choice for genetic analysis. Nevertheless, if linkage is used in the absence of knowing the specific mutation, accurate diagnosis is possible in virtually all families. 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 17).

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 pharmacological interventions, including the drug ivacaftor, that modulate CFTR function in some patients (see Chapter 13). Alternatively, gene transfer therapy may be possible in 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 year or two of life but develop muscle weakness by 3 to 5 years of age (Fig. 12-16), 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 DMD boys) 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 to 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 approximately 20 points.


FIGURE 12-16 Pseudohypertrophy of the calves due to the replacement of normal muscle tissue with connective tissue and fat in an 8-year-old boy with Duchenne muscular dystrophy. SeeSources & Acknowledgments.

The Clinical Phenotype of Becker Muscular Dystrophy.

Becker muscular dystrophy (BMD) is also due to mutations in the dystrophin gene, but the BMD alleles produce a much milder phenotype. Patients are said to have BMD if they are still walking at the age of 16 years. There is significant variability in the progression of the disease, and some patients remain ambulatory for many years. In general, patients with BMD carry mutated 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. 12-17). In contrast, patients with DMD have little or no detectable dystrophin.


FIGURE 12-17 Microscopic visualization of the effect of mutations 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. SeeSources & Acknowledgments.

The Genetics of Duchenne Muscular Dystrophy and Becker Muscular Dystrophy


DMD has an incidence of approximately 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 approximately 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 16). The great majority of carrier females have no clinical manifestations, although approximately 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 mutation on that chromosome.

BMD accounts for approximately 15% of the mutations 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 approximately 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 approximately 10%) represent new mutations.

The DMD Gene and Its Product.

The most remarkable feature of the DMD gene is its size, estimated to be 2300 kb, 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) dystrophin transcript encodes a huge 427-kD protein (Fig. 12-18). 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.


FIGURE 12-18 A representation of the full-length dystrophin protein, the corresponding cDNA, and the distribution of representative deletions in patients with Becker muscular dystrophy (BMD) and Duchenne muscular dystrophy (DMD). Partial duplications of the gene (not shown) account for approximately 6% of DMD or BMD alleles. The actin-binding domain links the protein to the filamentous actin cytoskeleton. The rod domain presumably acts as a spacer between the N-terminal and C-terminal domains. The cysteine-rich domain mediates protein-protein interactions. The C-terminal domain, which associates with a large transmembrane glycoprotein complex (see Fig. 12-19), is also found in three dystrophin-related proteins (DRPs): utrophin (DRP-1), DRP-2, and dystrobrevin. The protein domains are not drawn to scale.

The Molecular and Physiological Defects in Becker Muscular Dystrophy and Duchenne Muscular Dystrophy.

The most common molecular defects in patients with DMD are deletions (60% of alleles) (see Figs. 12-18 and 12-19), 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 (see Fig. 12-18). 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 mutations account for approximately one third of the alleles and are randomly distributed throughout the gene.


FIGURE 12-19 Diagnosis of Duchenne muscular dystrophy (DMD) involves screening for deletions and duplications by a procedure called multiplex ligation-dependent probe amplification (MLPA). MLPA allows the simultaneous analysis of all 79 exons of the DMD gene in a single DNA sample and can detect exon deletions and duplications in males or females. Each amplification peak represents a single DMD gene exon, after separation of the amplification products by capillary electrophoresis. Top panel, The amplification profiles of 16 exons of a normal male sample. Control (C) DNAs are included at each end of the scan. The MLPA DNA fragments elute according to size, which is why the exons are not numbered sequentially. Bottom panel, The corresponding amplification profile from a DMD patient with a deletion of exons 46 and 47. SeeSources & Acknowledgments.

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 (DGC).

Dystrophin is a structural protein that anchors the DGC at the cell membrane. The DGC is a veritable constellation of polypeptides associated with a dozen genetically distinct muscular dystrophies (Fig. 12-20). 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. Mutations in several of these proteins cause autosomal recessive limb girdle muscular dystrophies and other congenital muscular dystrophies (Fig. 12-20).


FIGURE 12-20 In muscle, dystrophin links the extracellular matrix (laminin) to the actin cytoskeleton. Dystrophin interacts with a multimeric complex composed of the dystroglycans (DG), the sarcoglycans, the syntrophins, and dystrobrevin. The α,β-dystroglycan complex is a receptor for laminin and agrin in the extracellular matrix. The function of the sarcoglycan complex is uncertain, but it is integral to muscle function; mutations in the sarcoglycans have been identified in limb girdle muscular dystrophies (LGMDs) types 2C, 2D, 2E, and 2F. Mutations in laminin type 2 (merosin) cause a congenital muscular dystrophy (CMD). The branched structures represent glycans. The WW domain of dystrophin is a tryptophan-rich, protein-binding motif.

That each component of the DGC is affected by mutations that cause other types of muscular dystrophies highlights the principle that no protein functions in isolation but rather is a component of a biological pathway or a multiprotein complex. Mutations in the genes encoding other components of a pathway or a complex often lead to genocopies, much as we saw previously in the case of CF.

Post-translational Modification of the Dystrophin Glycoprotein Complex.

Five of the muscular dystrophies associated with the DGC result from mutations in glycosyltransferases, leading to hypoglycosylation of α-dystroglycan (see Fig. 12-20). That five proteins are required for the post-translational 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 post-translational modifications for the normal function of most proteins.

Clinical Applications of Gene Testing in Muscular Dystrophy

Prenatal Diagnosis and Carrier Detection.

With gene-based 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 mutation 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. 12-19). In most other families, point mutations 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, approximately 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 mutation in her lymphocytes, the usual explanation is that he has a new mutation at the DMD locus. However, approximately 5% to 15% of such cases appear to be due to maternal germline mosaicism, in which case the recurrence risk is significant (see Chapter 7).


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 13.

Mutations 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. 12-21). The combined incidence of all forms of the disease is approximately 1 per 10,000. Approximately 95% of affected individuals have heterozygous mutations 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 mutation at the locus. The major phenotypes and genotypes associated with mutations in the type I collagen genes are outlined in Table 12-4.


FIGURE 12-21 Radiograph of a premature (26 weeks' 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. SeeSources & Acknowledgments.

TABLE 12-4

Summary of the Genetic, Biochemical, and Molecular Features of the Types of Osteogenesis Imperfecta due to Mutations in Type 1 Collagen Genes


*A few patients with type I disease have substitutions of glycine in one of the type I collagen chains.

Rare cases are autosomal recessive.

mRNA, Messenger RNA.

Modified from Byers PH: Disorders of collagen biosynthesis and structure. In Scriver CR, Beaudet AL, Sly WS, Valle D, editors: The metabolic basis of inherited disease, ed 6, New York, 1989, McGraw-Hill, pp 2805–2842; and Byers PH: Brittle bones—fragile molecules: disorders of collagen structure and expression. Trends Genet 6:293–300, 1990.

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. 12-22).


FIGURE 12-22 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. SeeSources & Acknowledgments.

Proteins composed of subunits, like collagen, are often subject to mutations 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, mutations 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 carboxyl terminus, and triple helix formation progresses toward the amino terminus. Consequently, mutations that alter residues in the carboxyl-terminal part of the triple helical domain are more disruptive because they interfere earlier with the propagation of the triple helix (Fig. 12-23). Second, the post-translational 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 mutation, 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 of 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. 12-21).


FIGURE 12-23 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 mutations. The small vertical bars on each procollagen chain indicate post-translational modifications (see text). Column 3, The effect of mutations on the biochemical processing of collagen. OI, Osteogenesis imperfecta; Proα1M, a proα1 chain with a missense mutation; Proα2M, a proα2 chain with a missense mutation; Proα10, a proα1 chain null allele. OI, Osteogenesis imperfecta.

Molecular Abnormalities of Collagen in Osteogenesis Imperfecta

More than 2000 different mutations 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 12-4). For the type I collagen genes, the mutations 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 mutations that result in production by cells of approximately half the normal amount of type I procollagen. Most of these mutations result in 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. 12-23). Missense mutations 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 mutations that produce structurally abnormal proα1(I) or proα2(I) chains (see Fig. 12-23 and Table 12-4). Most of these patients have substitutions in the triple helix that replace a glycine with a more bulky 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. 12-23). 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 Mutations

Three additional forms of clinically defined OI (types V, VI, and VII) do not result from mutations in type I collagen genes but involve defects in other genes. These 5% of OI subjects with normal collagen genes have either dominant mutations in the IFITM5 gene (encoding interferon-induced transmembrane protein 5) or biallelic mutations in any of almost 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 mutations in type I collagen genes that cause OI act in a dominant manner. This group of disorders illustrates the genetic complexities that result when mutations 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. 12-23). 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. 12-23). 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. Mutations such as the proα1(I) missense allele (proα1M) shown in Figure 12-23 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 mutation 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 Case 8 and Case 30 for other examples of dominant negative alleles).

Although mutations 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 mutations, to cause the severe perinatal lethal phenotype (see Table 12-4). 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 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.

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 8 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 approximately 1.4% of persons in developed countries and is responsible for at least 100,000 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 the increased risk in relatives of affected individuals is not due to mendelian inheritance; rather, as described in Chapter 8, 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, do 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 12-5). Mutations in three of these genes—encoding the β-amyloid precursor protein (βAPP), presenilin 1, and presenilin 2—lead to autosomal dominant AD. The fourth gene, APOE, encodes apolipoprotein E (apoE), the protein component of several plasma lipoproteins. Mutations in APOE are not associated with monogenic AD. Rather, as we saw in Chapter 8, 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).

TABLE 12-5

Genes and Proteins Associated with Inherited Susceptibility to Alzheimer Disease


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, Valle D, editors: The molecular and metabolic bases of inherited disease, ed 8, New York, 2000, McGraw-Hill; and Martin JB: Molecular basis of the neurodegenerative disorders. N Engl J Med 340:1970–1980, 1999.

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 βAPP, called the Aβ peptide, appears to be at the center of AD pathogenesis, and the currently available experimental evidence suggests that the βAPP, 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 pathological 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 βAPP protein (see Table 12-5), 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 apoE (see Table 12-5). 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 of the causes of the neuronal degeneration in AD, mutations 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 βAPP and its corresponding gene are summarized in Table 12-5. βAPP is a single-pass intracellular transmembrane protein found in 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 approximately 90% of βAPP is cleavage by the α-secretase (Fig. 12-24), an event that precludes the formation of the Aβ peptide, because α-secretase cleaves within the Aβ peptide domain. The other approximately 10% of βAPP 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.


FIGURE 12-24 The normal processing of β-amyloid precursor protein (βAPP)and the effect on processing of missense mutations in the βAPP gene associated with familial Alzheimer disease. The ovals show the locations of the missense mutations. SeeSources & Acknowledgments.

In monogenic AD due to missense substitutions in the gene encoding βAPP (APP), however, several mutations 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 neuropathological changes of AD by 40 years of age. Moreover, mutations in the AD genes presenilin 1 and presenilin 2 (see Table 12-5) also lead to increased production of Aβ42. Notably, the amount of the neurotoxic Aβ42 peptide is increased in the serum of individuals with mutations in the βAPP, presenilin 1, and presenilin 2 genes; furthermore, in cultured cell systems, the expression of mutant βAPP, presenilin 1, and presenilin 2 increases the relative production of Aβ42 peptide by twofold to tenfold.

The central role of the Aβ42 peptide in AD is highlighted by the discovery of a coding mutation (Ala673Thr) in the APP gene (Fig. 12-25) 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 Thr673 to the β-secretase cleavage site (see Fig. 12-25).


FIGURE 12-25 The topology of the amyloid precursor protein (βAPP), 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 mutations are shown in highlighted circles, whereas the amino acid residues representing various missense mutations are shown in boxes. The mutated amino acid residues are near the sites of β-, α-, and γ-secretase cleavage (black arrowheads). The mutations lead to the accumulation of toxic peptide Aβ42 rather than the wild-type Aβ40 peptide. The location of the protective allele Ala673Thr is indicated by the dashed arrowSeeSources & Acknowledgments.

The Presenilin 1 and 2 Genes

The genes encoding presenilin 1 and presenilin 2 (see Table 12-5) were identified in families with autosomal dominant AD. Presenilin 1 is required for γ-secretase cleavage of βAPP derivatives. Indeed, some evidence suggests that presenilin 1 is a critical cofactor protein of γ-secretase. The mutations 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 mutations is that the age at onset with the latter is much more variable (presenilin 1, 35 to 60 years; presenilin 2, 40 to 85 years); indeed, in one family, an asymptomatic octogenarian carrying a presenilin 2 mutation transmitted the disease to his offspring. The basis of this variation is partly dependent on the number of APOE ε4 alleles (see Table 12-5 and later discussion) carried by individuals with a presenilin 2 mutation; 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 8, 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 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 apoE binds to the Aβ peptide. The APOE protein has three common forms encoded by corresponding APOE alleles (Table 12-6). 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 approximately 10 to 15 years earlier than in the general population; see Chapter 8). 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) (see Fig. 8-11 and Table 8-14). In contrast, the ε2 allele has a protective effect and correspondingly is more common in elderly subjects who are unaffected by AD (see Table 12-6).

TABLE 12-6

Amino Acid Substitutions Underlying the Three Common Apolipoprotein E Polymorphisms


These figures are estimates, with differences in allele frequencies that vary with ethnicity in control populations, and with age, gender, and ethnicity 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). Available at:

The mechanisms underlying these effects are not known, but apoE polymorphisms may influence the processing of βAPP 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 neurological 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 18).

Other Genes Associated with AD

One significant modifier of AD risk, the TREM2 gene (which encodes the so-called 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 mutations the second most common contributor to classic late-onset 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 10) 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 βAPP.

Genome-wide association studies analyses (see Chapter 10), on the other hand, have greatly expanded the number of genes believed to be associated with AD, identifying at least nine 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)

The mtDNA Genome and the Genetics of mtDNA Diseases

The general characteristics of the mtDNA genome and the features of the inheritance of disorders caused by mutations 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. 12-26). Most cells have at least 1000 mtDNA molecules, distributed among hundreds of individual mitochondria, with multiple copies of mtDNA per mitochondrion. In addition to encoding two types of ribosomal RNA (rRNA) and 22 transfer RNAs (tRNAs), mtDNA encodes 13 proteins that are subunits of oxidative phosphorylation.


FIGURE 12-26 Representative disease-causing mutations and deletions in the human mtDNA genome, shown in relation to the location of the genes encoding the 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), and 13 proteins of the oxidative phosphorylation complex. Specific alleles are indicated when they are the predominant or only alleles associated with the phenotype or particular features of it. OH and OL are the origins of replication of the two DNA strands, respectively; 12S, 12S ribosomal RNA; 16S, 16S ribosomal RNA. The locations of each of the tRNAs are indicated by the single-letter code for their corresponding amino acids. The 13 oxidative phosphorylation polypeptides encoded by mitochondrial DNA (mtDNA) include components of complex I: NADH dehydrogenase (ND1, ND2, ND3, ND4, ND4L, ND5, and ND6); complex III: cytochrome b (cyt b); complex IV: cytochrome c oxidase I or cytochrome c (COI, COII, COIII); and complex V: ATPase 6 and 8 (A6, A8). The disease abbreviations used in this figure (e.g., MELAS, MERRF, LHON) are explained in Table 12-7. CPEO, Chronic progressive external ophthalmoplegia; NARP, neuropathy, ataxia, and retinitis pigmentosa. SeeSources & Acknowledgments.

Mutations in mtDNA can be inherited maternally (see Chapter 7) or acquired as somatic mutations. The diseases that result from mutations in mtDNA show distinctive patterns of inheritance due to three features of mitochondrial chromosomes:

• Replicative segregation

• Homoplasmy and heteroplasmy

• Maternal inheritance

Replicative segregation refers to the fact that the multiple copies of mtDNA in each mitochondrion replicate and sort randomly among newly synthesized mitochondria, which in turn are distributed randomly between the daughter cells (see Fig. 7-25). Homoplasmy is the situation in which a cell contains a pure population of normal mtDNA or of mutant mtDNA, whereas heteroplasmy describes the presence of a mixture of mutant and normal mtDNA molecules within a cell. Thus the phenotype associated with a mtDNA mutation will depend on the relative proportion of normal and mutant mtDNA in the cells of a particular tissue (see Fig. 7-25). As a result, mitochondrial disorders are generally characterized by reduced penetrance, variable expression, and pleiotropy. The maternal inheritance of mtDNA (discussed in greater detail in Chapter 7; see Fig. 7-24) reflects the fact that sperm mitochondria are generally eliminated from the embryo, so that mtDNA is almost always inherited entirely from the mother; paternal inheritance of mtDNA disease is highly unusual and has been well documented in only one instance.

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 100 nuclear genes are associated with disorders of the respiratory chain. Thus diseases of oxidative phosphorylation arise not only from mutations in the mitochondrial genome but also from mutations in nuclear genes that encode oxidative phosphorylation 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. Mutations 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 nuclear genome mutations (see Chapter 7).

Mutations in mtDNA and Disease

The sequence of the mtDNA genome and the presence of pathogenic mutations in mtDNA have been known for over three decades. Unexpected and still unexplained, however, is the fact that the mtDNA genome mutates at a rate approximately tenfold greater than does nuclear DNA. The range of clinical disease resulting from mtDNA mutations is diverse (Fig. 12-27), although neuromuscular disease predominates. More than 100 different rearrangements and approximately 100 different point mutations that are disease-related have been identified in mtDNA. The prevalence of mtDNA mutations has been shown, in at least one population, to be approximately 1 per 8000. Representative mutations and the diseases associated with them are presented in Figure 12-26 and Table 12-7. In general, as illustrated in the sections to follow, three types of mutations have been identified in mtDNA: rearrangements that generate deletions or duplications of the mtDNA molecule; point mutations in tRNA or rRNA genes that impair mitochondrial protein synthesis; and missense mutations in the coding regions of genes that alter the activity of an oxidative phosphorylation protein.


FIGURE 12-27 The range of affected tissues and clinical phenotypes associated with mutations in mitochondrial DNA (mtDNA). SeeSources & Acknowledgments.

TABLE 12-7

Representative Examples of Disorders due to Mutations in Mitochondrial DNA and Their Inheritance


mtDNA, Mitochondrial DNA; rRNA, ribosomal RNA; tRNA, transfer RNA.

Deletions of mtDNA and Disease.

In most cases, mtDNA deletions that cause disease, such as Kearns-Sayre syndrome (see Table 12-7), 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 approximately 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 mtDNAs in their germ cells have such a severe phenotype that they rarely reproduce.

The importance of deletions in mtDNA as a cause of disease has recently been highlighted by the discovery that somatic mtDNA deletions are common in dopaminergic neurons of the substantia nigra, both in normal aging individuals and perhaps to a greater extent in individuals with Parkinson disease. The deletions that have occurred in individual neurons from Parkinson disease patients have been shown to be unique, indicating that clonal expansion of the different mtDNA deletions occurred in each cell. These findings indicate that somatic deletions of the mtDNA may contribute to the loss of dopaminergic neurons in the aging substantia nigra and raise the possibility that the common sporadic form of Parkinson disease results from a greater than normal accumulation of deleted mtDNA molecules in the substantia nigra, with a consequently more severe impairment of oxidative phosphorylation. At present, the mechanisms leading to the deletions and their clonal expansions are entirely unclear.

Mutations in tRNA and rRNA Genes of the Mitochondrial Genome.

Mutations in the noncoding tRNA and rRNA genes of mtDNA are of general significance because they illustrate that not all disease-causing mutations in humans occur in genes that encode proteins (Case 33). More than 90 pathogenic mutations have been identified in 20 of the 22 tRNA genes of the mtDNA, and they are the most common cause of oxidative phosphorylation abnormalities in humans (see Fig. 12-26and Table 12-7). The resulting phenotypes are those generally associated with mtDNA defects. The tRNA mutations include 18 substitutions in the tRNAleu(UUR) gene, some of which, like the common 3243A>G mutation, cause a phenotype referred to as MELAS, an acronym for mitochondrial encephalomyopathy with lactic acidosis and strokelike episodes (see Fig. 12-26 and Table 12-7); others are associated predominantly with myopathy. An example of a 12S rRNA mutation is a homoplasmic substitution (see Table 12-7) that causes sensorineural prelingual deafness after exposure to aminoglycoside antibiotics (see Fig. 12-26).

The Phenotypes of Mitochondrial Disorders

Oxidative Phosphorylation and mtDNA Diseases.

Mitochondrial mutations generally affect those 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 cellular energy. Consequently, decreased production of ATP characterizes many diseases of mtDNA and is likely to underlie the cell dysfunction and cell death that occur in mtDNA diseases. 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. 7-25); a critical threshold in the proportion of mtDNA molecules carrying the detrimental mutation must be exceeded in cells from the affected tissue before clinical disease becomes apparent. The threshold appears to be approximately 60% for disorders due to deletions in mtDNA and approximately 90% for diseases due to other types of mutations.

The neuromuscular system is the one most commonly affected by mutations in mtDNA; the consequences can include encephalopathy, myopathy, ataxia, retinal degeneration, and loss of function of the external ocular muscles. Mitochondrial myopathy is characterized by so-called ragged-red (muscle) fibers, a histological 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 Figure 12-27, may include liver dysfunction, bone marrow failure, pancreatic islet cell deficiency and diabetes, deafness, and other disorders.

Heteroplasmy and Mitochondrial Disease

Heteroplasmy accounts for three general characteristics of genetic disorders of mtDNA that are of importance to their pathogenesis.

• First, female carriers of heteroplasmic mtDNA point mutations or of mtDNA duplications usually transmit some mutant mtDNAs to their offspring.

• Second, the fraction of mutant mtDNA molecules inherited by each child of a carrier mother is very variable. This is because the number of mtDNA molecules within each 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 mutant 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 mutant mtDNA molecules are more likely to have clinically affected offspring than are mothers with a lower proportion, as one would predict from the random sampling of mtDNA molecules 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 mutant mtDNA species.

Unexplained and Unexpected Phenotypic Variation in mtDNA Diseases.

As seen in Table 12-7, heteroplasmy is the rule for many mtDNA diseases. Heteroplasmy leads to an unpredictable and variable fraction of mutant mtDNA being present in any particular tissue, undoubtedly accounting for much of the pleiotropy and variable expressivity of mtDNA mutations (see Box). An example is provided by what appears to be the most common mtDNA mutation, the 3243A>G substitution in the tRNAleu(UUR) gene just mentioned in the context of the MELAS phenotype. This mutation leads predominantly to 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, particularly in Japanese, has been attributed to the 3243A>G substitution.

It is likely that much of the phenotypic variation observed among patients with mutations in mitochondrial genes will be explained by the fact that the proteins within mitochondria are remarkably heterogeneous between tissues, differing on average by approximately 25% between any two organs. This molecular heterogeneity is reflected in biochemical heterogeneity. For example, whereas much of the energy generated by brain mitochondria derives from the oxidation of ketones, skeletal muscle mitochondria preferentially use fatty acids as their fuel.

Interactions between the Mitochondrial and Nuclear Genomes

Because both the nuclear and mitochondrial genomes contribute polypeptides to oxidative phosphorylation, it is not surprising that the phenotypes associated with mutations in the nuclear genes are often indistinguishable from those due to mtDNA mutations. Moreover, mtDNA depends on many nuclear genome–encoded proteins for its replication and the maintenance of its integrity. Genetic evidence has highlighted the direct nature of the relationship between the nuclear and mtDNA genomes. The first indication of this interaction was provided by the identification of the syndrome of autosomally transmitted deletions in mtDNA. Mutations in at least two genes have been associated with this phenotype. The protein encoded by one of these genes, amusingly called Twinkle, appears to be a DNA primase or helicase. The product of the second gene is a mitochondrial-specific DNA polymerase γ, whose loss of function is associated with both dominant and recessive multiple deletion syndromes.

A second autosomal disorder, the mtDNA depletion syndrome, is the result of mutations in any of six nuclear genes that lead to a reduction in the number of copies of mtDNA (both per mitochondrion and per cell) in various tissues. Several of the affected genes encode proteins required to maintain nucleotide pools or to metabolize nucleotides appropriately in the mitochondrion. For example, both myopathic and hepatocerebral phenotypes result from mutations in the nuclear genes for mitochondrial thymidine kinase and deoxyguanosine kinase. Because mutations in the six genes identified to date account for only a minority of affected individuals, additional genes must also be involved in this disorder.

Apart from the insights that these rare disorders provide into the biology of the mitochondrion, the identification of the affected genes facilitates genetic counseling and prenatal diagnosis in some families and suggests, in some instances, potential treatments. For example, the blood thymidine level is markedly increased in thymidine phosphorylase deficiency, suggesting that lowering thymidine levels might have therapeutic benefits if an excess of substrate rather than a deficiency of the product plays a major role in the pathogenesis of the disease.

Nuclear Genes Can Modify the Phenotype of mtDNA Diseases.

Although heteroplasmy is a major source of phenotypic variability in mtDNA diseases (see Box), additional factors, including alleles at nuclear loci, must also play a role. Strong evidence for the existence of such factors is provided by families carrying mutations associated with Leber hereditary optic neuropathy (LHON; see Table 12-7), 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 12-7 and Fig. 12-26). Depending on the mutation, there is often some recovery of vision, but the pathogenic mechanisms of the optic nerve damage are unclear.

There is a striking and unexplained increase in the penetrance of the disease in males; approximately 50% of male carriers but only approximately 10% of female carriers of a LHON mutation develop symptoms. The variation in penetrance and the male bias of the LHON phenotype are determined by a haplotype on the short arm of the X chromosome. The gene at this nuclear-encoded modifier locus has not yet been identified, but it is contained, notably, in a haplotype that is common in the general population. When the protective haplotype is transmitted from a typically unaffected mother to individuals who have inherited the LHON mtDNA mutation from that mother, the phenotype is substantially ameliorated. Thus males who carry the high-risk X-linked haplotype as well as a LHON mtDNA mutation (other than the one associated with the most severe LHON phenotype [see Table 12-7]) are thirty-fivefold more likely to develop visual failure than those who carry the low-risk X-linked haplotype. These observations are of general significance because they demonstrate the powerful effect that modifier loci can have on the phenotype of a monogenic disease.

Diseases due to the Expansion of Unstable Repeat Sequences

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 20 disorders. These features include the unstable and dynamic nature of the mutations, which are due to the expansion, within the transcribed region of the affected gene, of repeated sequences such as the codon for glutamine (CAG) in Huntington disease (Case 24) and most of a group of neurodegenerative disorders called the spinocerebellar ataxias, or due to the expansion of trinucleotides in noncoding regions of RNAs, including CGG in fragile X syndrome (Case 17), GAA in Friedreich ataxia, and CUG in myotonic dystrophy 1 (Fig. 12-28).


FIGURE 12-28 The locations of the trinucleotide repeat expansions and the sequence of each trinucleotide in five representative trinucleotide 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. SeeSources & Acknowledgments.

Although the initial nucleotide repeat diseases to be described are all due to the expansion of three nucleotide repeats, other disorders have now 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) and a pentanucleotide (ATTCT) in spinocerebellar atrophy 10. 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 as it is transmitted through a family. The biochemical mechanism most commonly proposed to underlie the expansion of unstable repeat sequences is slipped mispairing (Fig. 12-29). 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. 12-29) and genome maintenance (i.e., DNA repair).


FIGURE 12-29 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).

The clinical phenotypes of Huntington disease and fragile X syndrome are presented in Chapter 7 and in their respective Cases. For reasons that are gradually becoming apparent, particularly in the case of fragile X syndrome, diseases due to the expansion of unstable repeats are primarily neurological; the clinical presentations include ataxia, cognitive 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 and can be divided into three 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, the 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, an epigenetic modification of the DNA that silences transcription of the gene (see Figs. 7-22 and 12-28). Remarkably, the epigenetic silencing appears to be mediated by the mutant 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 fragile X mental retardation protein (FMRP) is the cause of the intellectual disability and learning deficits and the non-neurological features of the clinical phenotype, including 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, and the disruption of these processes is likely to underlie the intellectual disability and learning abnormalities seen in fragile X patients. 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, the pathogenesis of disease in individuals with less pronounced CGG repeat expansion (60 to 200 repeats) in the FMR1 gene, causing the clinically distinct fragile X tremor/ataxia syndrome (FXTAS),is entirely different from that of the fragile X syndrome itself. Although decreased translational efficiency impairs the expression of the FMRP protein in FXTAS, this reduction cannot be responsible for the disease because males with full mutations and virtually complete loss of function of the FMR1 gene never develop FXTAS. Rather, the evidence suggests that FXTAS results from the twofold to fivefold increased levels of the FMR1 mRNA present in these patients, representing a gain-of-function mutation. 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. 12-28). Myotonic dystrophy 2 (DM2) is 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. 12-28). 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 region 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, and indeed more than a dozen distinct pre-mRNAs have been shown to 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. Even though our knowledge of the abnormal processes underlying DM1 and DM2 is still incomplete, these molecular insights offer the 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 pathological process is caused by the expansion—to more than 40 repeats—of the codon CAG in the HD gene, resulting in long polyglutamine tracts in the mutant protein, huntingtin (see Figs. 7-20and 7-21). The bulk of evidence suggests that the mutant proteins with expanded polyglutamine sequences are novel property mutants (see Chapter 11), the expanded tract conferring 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 mutant 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 actually be protective rather than pathogenic.

A unifying model of the neuronal death mediated by polyglutamine expansion in huntingtin is not at hand. Many cellular processes have been shown to be disrupted by mutant huntingtin in its soluble or its 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 that lead to correction of the phenotype. For example, it has been found that mutant huntingtin abnormally associates with a mitochondrial fission protein, GTPase dynamin-related protein 1 (DRP1) in Huntington disease patients, leading to multiple mitochondrial abnormalities. Remarkably, in mice, these defects are rescued by reducing DRP1 GTPase activity, suggesting both that DRP1 as a therapeutic target for the disorder and that mitochondrial abnormalities play important roles in Huntington disease.

Concluding Comments

Despite the substantial progress in our understanding of the molecular events that underlie the pathology of the unstable repeat expansion diseases, we are only beginning to dissect the pathogenic complexity of these important conditions. It is clear that the study of animal models of these disorders is providing critical insights into these disorders, insights that will undoubtedly lead to therapies to prevent or to reverse the pathogenesis of these slowly developing disorders in the near future. We begin to explore the concepts relevant to the treatment of disease in the next chapter.

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Useful Websites

Mutation Databases

Clinical and functional translation of CFTR (CFTR2 project).

Collagen mutation database.

Cystic fibrosis and CFTR gene mutation database.

Human mitochondrial genome database.

Phenylalanine hydroxylase mutation database.

The Human Gene Mutation Database.


1. One mutant allele at the LDL receptor locus (leading to familial hypercholesterolemia) encodes an elongated protein that is approximately 50,000 Da larger than the normal 120,000-Da 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. Are autosomal dominant PSCK9 gain-of-function mutations that cause familial hypercholesterolemia deficiency phenocopies, or genocopies, of familial hypercholesterolemia due to autosomal dominant mutations in the LDL receptor gene? Explain your answer.

3. In discussing the nucleotide changes found to date in the coding region of the CF gene, we stated that some of the changes (the missense changes) found so far are only “putative” disease-causing mutations. What criteria would one need to fulfill before knowing that a nucleotide change is pathogenic and not a benign polymorphism?

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 if 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 ΔF508 mutation? (Assume that 85% of CF mutations could be detected at the time you are consulted and that his parents are from northern Europe, where the ΔF508 allele has a frequency of 0.70.)

c. If he does not have the ΔF508 mutation, 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 ethnic 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 image-year-old girl, T.N., has been noted to have 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. Females with DMD are rare. Identify three mechanisms of mutation that could account for the occurrence of DMD in a female.

8. In patients with osteogenesis imperfecta, explain why the missense mutations at glycine positions in the triple helix of type I collagen are confined to a limited number of other amino acid residues (Ala, Ser, Cys, Arg, Val, Asp).

9. Glucose-6-phosphate dehydrogenase (G6PD) is encoded by an X-linked gene. G6PD loss-of-function mutations can lead to hemolysis on exposure to some drugs, fava beans, and other compounds (see Chapter 18). Electrophoresis of red blood cell hemolysates shows that some females have two G6PD bands, but males have a single band. Explain this observation and the possible pathological and genetic significance of the finding of two bands in an African American female.

10. 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 enzymes. Explain how a single autosomal recessive mutation 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?

11. The effect of a dominant negative allele illustrates one general mechanism by which mutations in a protein cause dominantly inherited disease. What other mechanism is commonly associated with dominance in genes encoding the subunits of multimeric proteins?

12. The clinical effects of mutations 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.

13. The relationship between the site at which a protein is expressed and the site of pathological change in a genetic disease may be unpredictable. In addition, the tissue that lacks the mutant protein may even be left unaffected by disease. Give examples of this latter phenomenon and discuss them.

14. The two pseudodeficiency alleles of hex A are Arg247Trp and Arg249Trp. What is the probable reason that the missense substitutions of these alleles are so close together in the protein?

15. Why are gain-of-function mutations in proteins, as seen with the autosomal dominant PCSK9 mutations that cause hypercholesterolemia, almost always missense mutations?

16. What are the possible explanations for the presence of three predominant alleles for Tay-Sachs disease 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?

17. All of the known loci associated with Alzheimer disease do not account for the implied genetic risk. Identify at least three other sources of genetic variation that may account for the genetic contribution to AD.

18. Propose a molecular therapy that might counteract the effect of the CUG expansions in the RNAs of myotonic dystrophy 1 and 2 and that would reduce the binding of RNA-binding proteins to the CUG repeats. Anticipate some possible undesirable effects of your proposed therapy.