Chapter 12: The Molecular, Biochemical, and Cellular Basis of Genetic Disease PDF

C H A P T E R 12 The Molecular, Biochemical, and Cellular Basis of Genetic Disease In this chapter, we extend our examination of the...

C H A P T E R 12 The Molecular, Biochemical, and Cellular Basis of Genetic Disease In this chapter, we extend our examination of the molec- Housekeeping Proteins and Specialty Proteins ular and biochemical basis of genetic disease beyond the hemoglobinopathies to include other diseases and the in Genetic Disease abnormalities in gene and protein function that cause Proteins can be separated into two general classes on them. In Chapter 11, we presented an outline of the the basis of their pattern of expression: housekeeping general mechanisms by which mutations cause disease proteins, which are present in virtually every cell and (see Fig. 11-1) and reviewed the steps at which muta- have fundamental roles in the maintenance of cell struc- tions can disrupt the synthesis or function of a protein ture and function; and tissue-specific specialty proteins, (see Table 11-2). Those outlines provide a framework which are produced in only one or a limited number of for understanding the pathogenesis of all genetic disease. cell types and have unique functions that contribute to However, mutations in other classes of proteins often the individuality of the cells in which they are expressed. disrupt cell and organ function by processes that differ Most cell types in humans express 10,000 to 15,000 from those illustrated by the hemoglobinopathies, and protein-coding genes. Knowledge of the tissues in which we explore them in this chapter. a protein is expressed, particularly at high levels, is often To illustrate these other types of disease mechanisms, useful in understanding the pathogenesis of a disease. we examine here well-known disorders such as phenyl- Two broad generalizations can be made about the ketonuria, cystic fibrosis, familial hypercholesterolemia, relationship between the site of a protein’s expression Duchenne muscular dystrophy, and Alzheimer disease. and the site of disease. In some instances, less common disorders are included First (and somewhat intuitively), mutation in a tissue- because they best demonstrate a specific principle. The specific protein most often produces a disease importance of selecting representative disorders becomes restricted to that tissue. However, there may be sec- apparent when one considers that to date, mutations in ondary effects on other tissues, and in some cases almost 3000 genes have been associated with a clinical mutations in tissue-specific proteins may cause abnor- phenotype. In the coming decade, one anticipates that malities primarily in organs that do not express many more of the approximately 20,000 to 25,000 the protein at all; ironically, the tissue expressing the coding genes in the human genome will be shown to be mutant protein may be left entirely unaffected by the associated with both monogenic and genetically complex pathological process. This situation is exemplified by diseases. 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 DISEASES DUE TO MUTATIONS IN enzyme), and not the liver, that is damaged by the DIFFERENT CLASSES OF PROTEINS high blood levels of phenylalanine resulting from the Proteins carry out an astounding number of different lack of hepatic PAH. Consequently, one cannot nec- functions, some of which are presented in Figure 12-1. essarily infer that disease in an organ results from Mutations in virtually every functional class of protein mutation in a gene expressed principally or only in can lead to genetic disorders. In this chapter, we describe that organ, or in that organ at all. important genetic diseases that affect representative pro- Second, although housekeeping proteins are expressed teins selected from the groups shown in Figure 12-1; in most or all tissues, the clinical effects of mutations many other of the proteins listed, as well as the diseases in housekeeping proteins are frequently limited to associated with them, are described in the Cases section. one or just a few tissues, for at least two reasons. In 215 216 THOMPSON & THOMPSON GENETICS IN MEDICINE ORGANELLES NUCLEUS Mitochondria Developmental transcription factors Oxidative phosphorylation Pax6 ND1 protein of electron transport chain -aniridia - Leber hereditary optic neuropathy Genome integrity Translation of mitochondrial proteins BRCA1, BRCA2 tRNAleu - breast cancer - MELAS DNA mismatch repair proteins 12S RNA - hereditary nonpolyposis colon cancer - sensorineural deafness RNA translation regulation Peroxisomes FMRP (RNA binding to suppress Peroxisome biogenesis translation) 12 proteins - fragile X syndrome - Zellweger syndrome Chromatin-associated proteins Lysosomes MeCP2 (transcriptional repression) Lysosomal enzymes - Rett syndrome Hexosaminidase A Tumor suppressors - Tay-Sachs disease Rb protein α-L-iduronidase deficiency - retinoblastoma - Hurler syndrome Oncogenes BCR-Abl oncogene EXTRACELLULAR PROTEINS - chronic myelogenous leukemia Transport β-globin - sickle cell disease - b-thalassemia Morphogens Sonic hedgehog CELL SURFACE - holoprosencephaly Hormone receptors Protease inhibition Androgen receptor α1-Antitrypsin - androgen insensitivity - emphysema, liver disease Growth factor receptors Hemostasis FGFR3 receptor Factor VIII - achondroplasia - hemophilia A CYTOPLASM Metabolic receptors Hormones Metabolic enzymes LDL receptor Insulin Phenylalanine hydroxylase - hypercholesterolemia - rare forms of type 2 diabetes mellitus - PKU Ion transport Extracellular matrix Adenosine deaminase CFTR Collagen type 1 - severe combined immunodeficiency - cystic fibrosis - osteogenesis imperfecta Cytoskeleton Antigen presentation Inflammation, infection response Dystrophin HLA locus DQβ1 Complement factor H - Duchenne muscular dystrophy - type 1 diabetes mellitus - age-related macular degeneration 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 func- tion. CFTR, Cystic fibrosis transmembrane regulator; FMRP, fragile X mental retardation protein; HLA, human leukocyte antigen; LDL, low-density lipoprotein; MELAS, mitochondrial encepha- lomyopathy with lactic acidosis and strokelike episodes; PKU, phenylketonuria. most such instances, a single or a few tissue(s) may genes that one might consider as essential to every be affected because the housekeeping protein in ques- cell, such as actin, can result in viable offspring. tion is normally expressed abundantly there and serves a specialty function in that tissue. This situa- tion is illustrated by Tay-Sachs disease, as discussed DISEASES INVOLVING ENZYMES later; the mutant enzyme in this disorder is hexos- Enzymes are the catalysts that mediate the efficient aminidase A, which is expressed in virtually all cells, conversion of a substrate to a product. The diversity but its absence leads to a fatal neurodegeneration, of substrates on which enzymes act is huge. Accord- leaving non-neuronal cell types unscathed. In other ingly, the human genome contains more than 5000 instances, another protein with overlapping biologi- genes that encode enzymes, and there are hundreds cal activity may also be expressed in the unaffected of human diseases—the so-called enzymopathies—that tissue, thereby lessening the impact of the loss of involve enzyme defects. We first discuss one of the function of the mutant gene, a situation known as best-known groups of inborn errors of metabolism, the genetic redundancy. Unexpectedly, even mutations in hyperphenylalaninemias.  CHAPTER 12 — THE MOLECULAR, BIOCHEMICAL, AND CELLULAR BASIS OF GENETIC DISEASE 217 GTP GTP-cyclohydrolase DHNP Protein (diet, endogenous) 6-PT synthase Sepiapterin 6-PT reductase BH4 Phenylalanine Tyrosine phe tyr CO2 + H2O phe Phenylalanine hydroxylase hydroxylase BH4 BH4 4αOHBH4 tyr L-dopa dopamine NE E tyr DHPR qBH2 PCD hydroxylase BH4 trp 5-OH trp serotonin trp hydroxylase Figure 12-2 The biochemical pathways affected in the hyperphenylalaninemias. BH4, tetrahydro- biopterin; 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, epineph- rine; 5-OH trp, 5-hydroxytryptophan. TABLE 12-1 Locus Heterogeneity in the Hyperphenylalaninemias Biochemical Defect Incidence/106 Births Enzyme Affected Treatment Mutations in the Gene Encoding Phenylalanine Hydroxylase Classic PKU 5-350 PAH Low-phenylalanine diet* (depending on the population) Variant PKU Less than classic PKU PAH Low-phenylalanine diet (less restrictive than that required to treat PKU* Non-PKU 15-75 PAH None, or a much less restrictive low-phenylalanine hyperphenylalaninemia diet* Mutations in Genes Encoding Enzymes of Tetrahydrobiopterin Metabolism Impaired BH4 recycling a 18% represent the majority of PAH mutations in Asian popu- 9% E6nt–96a>g lations (Fig. 12-4). The remaining disease-causing muta- 14% tions are individually rare. To record and make this Figure 12-4 The nature and identity of PAH mutations in popu- information publicly available, a PAH database has lations of European and Asian descent (the latter from China, been developed by an international consortium. Korea, and Japan). The one-letter amino acid code is used (see The allelic heterogeneity at the PAH locus has major Table 3-1). See Sources & Acknowledgments. 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 heterogene- ity accounts for much of the enzymatic and phenotypic normal, and the hyperphenylalaninemia results from a heterogeneity observed in this patient population. Thus, defect in one of the steps in the biosynthesis or regenera- mutations that eliminate or dramatically reduce PAH tion of BH4, the cofactor for PAH (see Table 12-1 and activity generally cause classic PKU, whereas greater Fig. 12-2). The association of a single biochemical phe- residual enzyme activity is associated with milder phe- notype, such as hyperphenylalaninemia, with mutations notypes. However, homozygous patients with certain in different genes, is an example of locus heterogeneity PAH mutations have been found to have phenotypes (see Table 11-1). The proteins encoded by genes that ranging all the way from classic PKU to non-PKU hyper- manifest locus heterogeneity generally act at different phenylalaninemia. Accordingly, it is now clear that steps in a single biochemical pathway, another principle other unidentified biological variables—undoubtedly of genetic disease illustrated by the genes associated including modifier genes—generate variation in the phe- with hyperphenylalaninemia (see Fig. 12-2). BH4- notype seen with any specific genotype. This lack of a deficient patients were first recognized because they strict genotype-phenotype correlation, initially some- developed profound neurological problems in early what surprising, is now recognized to be a common life, despite the successful administration of a low- feature of many single-gene diseases and highlights the phenylalanine diet. This poor outcome is due in part to fact that even monogenic traits like PKU are not geneti- the requirement for the BH4 cofactor of two other cally “simple” disorders. enzymes, tyrosine hydroxylase and tryptophan hydrox- ylase. These hydroxylases are critical for the synthesis Defects in Tetrahydrobiopterin Metabolism. In 1% to of the monoamine neurotransmitters dopamine, norepi- 3% of hyperphenylalaninemic patients, the PAH gene is nephrine, epinephrine, and serotonin (see Fig. 12-2). Homozygous Patient: {Mutation A/Mutation A} , where Mutation A eliminates PAH activity Compound Heterozygous Patient: {Mutation A/Mutation B} , where Mutation A eliminates PAH activity but Mutation B allows partial activity. Sandhoff is more severe: both hex’s and HexB 220 THOMPSON & THOMPSON GENETICS IN MEDICINE The locus heterogeneity of hyperphenylalaninemia is treatment of many inborn errors of enzyme metabolism, of great significance because the treatment of patients as discussed further in Chapter 13. In the general case, with a defect in BH4 metabolism differs markedly from a cofactor comes into contact with the protein compo- subjects with mutations in PAH, in two ways. First, nent of an enzyme (termed an apoenzyme) to form the because the PAH enzyme of individuals with BH4 defects active holoenzyme, which consists of both the cofactor is itself normal, its activity can be restored by large and the otherwise inactive apoenzyme. Illustrating this doses of oral BH4, leading to a reduction in their plasma strategy, BH4 supplementation has been shown to exert phenylalanine levels. This practice highlights the prin- its beneficial effect through one or more mechanisms, ciple of product replacement in the treatment of some all of which result from the increased amount of the genetic disorders (see Chapter 13). Consequently, phe- cofactor that is brought into contact with the mutant nylalanine restriction can be significantly relaxed in PAH apoenzyme. These mechanisms include stabiliza- the diet of patients with defects in BH4 metabolism, tion of the mutant enzyme, protection of the enzyme and some patients actually tolerate a normal (i.e., a from degradation by the cell, and increase in the cofac- phenylalanine-unrestricted) diet. Second, one must also tor supply for a mutant enzyme that has a low affinity try to normalize the neurotransmitters in the brains of for BH4. these patients by administering the products of tyrosine hydroxylase and tryptophan hydroxylase, L-dopa and Newborn Screening. PKU is the prototype of genetic 5-hydroxytryptophan, respectively (see Fig. 12-2 and diseases for which mass newborn screening is justified Table 12-1). (see Chapter 18) because it is relatively common in some Remarkably, mutations in sepiapterin reductase, populations (up to approximately 1 in 2900 live births), an enzyme in the BH4 synthesis pathway, do not mass screening is feasible, failure to treat has severe cause hyperphenylalaninemia. In this case, only dopa- consequences (profound developmental delay), and responsive dystonia is seen, due to impaired synthesis treatment is effective if begun early in life. To allow time of dopamine and serotonin (see Fig. 12-2). It is thought for the postnatal increase in blood phenylalanine levels that alternative pathways exist for the final step in BH4 to occur, the test is performed after 24 hours of age. synthesis, bypassing the sepiapterin reductase deficiency Blood from a heel prick is assayed in a central labora- in peripheral tissues, an example of genetic redundancy. tory for blood phenylalanine levels and measurement of For these reasons, all hyperphenylalaninemic infants the phenylalanine-to-tyrosine ratio. Positive test results must be screened to determine whether their hyperphe- must be confirmed quickly because delays in treatment nylalaninemia is the result of an abnormality in PAH or beyond 4 weeks postnatally have profound effects on in BH4 metabolism. The hyperphenylalaninemias thus intellectual outcome. illustrate the critical importance of obtaining a specific molecular diagnosis in all patients with a genetic disease Maternal Phenylketonuria. Originally, the low-phenyl- phenotype—the underlying genetic defect may not be alanine diet was discontinued in mid-childhood for what one first suspects, and the treatment can vary most patients with PKU. Subsequently, however, it was accordingly. discovered that almost all offspring of women with PKU not receiving treatment are clinically abnormal; Tetrahydrobiopterin Responsiveness in PAH Muta- most are severely delayed developmentally, and many tions. Many hyperphenylalaninemia patients with have microcephaly, growth impairment, and malforma- mutations in the PAH gene (rather than in BH4 metabo- tions, particularly of the heart. As predicted by princi- lism) will also respond to large oral doses of BH4 cofac- ples of mendelian inheritance, all of these children are tor, with a substantial decrease in plasma phenylalanine. heterozygotes. Thus their neurodevelopmental delay is BH4 supplementation is therefore an important adjunct not due to their own genetic constitution but to the therapy for PKU patients of this type, allowing them a highly teratogenic effect of elevated levels of phenylala- less restricted dietary intake of phenylalanine. The nine in the maternal circulation. Accordingly, it is patients most likely to respond are those with significant imperative that women with PKU who are planning residual PAH activity (i.e., patients with variant PKU pregnancies commence a low-phenylalanine diet before and non-PKU hyperphenylalaninemia), but even a conceiving. minority of patients with classic PKU are also respon- sive. The presence of residual PAH activity does not, Lysosomal Storage Diseases: A Unique Class however, necessarily guarantee an effect of BH4 admin- istration on plasma phenylalanine levels. Rather, the of Enzymopathies degree of BH4 responsiveness will depend on the specific Lysosomes are membrane-bound organelles containing properties of each mutant PAH protein, reflecting the an array of hydrolytic enzymes involved in the degrada- allelic heterogeneity underlying PAH mutations. tion of a variety of biological macromolecules. Muta- The provision of increased amounts of a cofactor is tions in these hydrolases are unique because they lead a general strategy that has been employed for the to the accumulation of their substrates inside the The presence of residual PAH activity in patients with PAH mutations does not guarantee a response to BH₄ administration. While BH₄ supplementation can help to increase PAH enzyme activity and lower plasma phenylalanine levels, the degree of responsiveness to BH₄ is influenced by the properties of the mutant PAH (type of mutation)  CHAPTER 12 — THE MOLECULAR, BIOCHEMICAL, AND CELLULAR BASIS OF GENETIC DISEASE 221 lysosome, where the substrates remain trapped because A (hex A). Although the enzyme is ubiquitous, the their large size prevents their egress from the organelle. disease has its clinical impact almost solely on the brain, Their accumulation and sometimes toxicity interferes the predominant site of GM2 ganglioside synthesis. Cat- with normal cell function, eventually causing cell death. alytically active hex A is the product of a three-gene Moreover, the substrate accumulation underlies one system (see Fig. 12-5). These genes encode the α and β uniform clinical feature of these diseases—their unre- subunits of the enzyme (the HEXA and HEXB genes, lenting progression. In most of these conditions, sub- respectively) and an activator protein that must associ- strate storage increases the mass of the affected tissues ate with the substrate and the enzyme before the enzyme and organs. When the brain is affected, the picture is can cleave the terminal N-acetyl-β-galactosamine residue one of neurodegeneration. The clinical phenotypes are from the ganglioside. very distinct and often make the diagnosis of a storage The clinical manifestations of defects in the three disease straightforward. More than 50 lysosomal hydro- genes are indistinguishable, but they can be differenti- lase or lysosomal membrane transport deficiencies, ated by enzymatic analysis. Mutations in the HEXA almost all inherited as autosomal recessive conditions, gene affect the α subunit and disrupt hex A activity to have been described. Historically, these diseases were cause Tay-Sachs disease (or less severe variants of hex untreatable. However, bone marrow transplantation A deficiency). Defects in the HEXB gene or in the gene and enzyme replacement therapy have dramatically encoding the activator protein impair the activity of improved the prognosis of these conditions (see Chapter both hex A and hex B (see Fig. 12-5) to produce Sand- 13). hoff disease or activator protein deficiency (which is very rare), respectively. Tay-Sachs Disease The clinical course of Tay-Sachs disease is tragic. Tay-Sachs disease (Case 43) is one of a group of het- Affected infants appear normal until approximately 3 erogeneous lysosomal storage diseases, the GM2 gan- to 6 months of age but then gradually undergo progres- gliosidoses, that result from the inability to degrade a sive neurological deterioration until death at 2 to 4 sphingolipid, GM2 ganglioside (Fig. 12-5). The biochem- years. The effects of neuronal death can be seen directly ical lesion is a marked deficiency of hexosaminidase in the form of the so-called cherry-red spot in the The GM2 gangliosidoses Disease Tay-Sachs disease and Sandhoff disease and Activator later-onset variants later-onset variants deficiency Affected gene α (chr 15) β (chr 5) activator (chr 5) Polypeptide α subunit β subunit activator Isozyme: subunits Hex A: αβ Hex B: ββ activator Active αβ enzyme complex GM2 ganglioside N-acetylgalactosamine - galactose - glucose - ceramide Cleavage site NANA 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 gan- glioside substrate and present it to the enzyme. Hex A, Hexosaminidase A; hex B, hexosaminidase B; NANA, N-acetyl neuraminic acid. See Sources & Acknowledgments. Although all three conditions share similar symptoms, enzymatic analysis can differentiate them 222 THOMPSON & THOMPSON GENETICS IN MEDICINE... – Arg – Ile – Ser – Try – Gly – Pro – Asp –... Normal HEXA allele... CGT ATA TCC TAT GCC CCT GAC...... CGT ATA TCT ATC CTA TGC CCC TGA C... Tay-Sachs allele... – Arg – Ile – Ser – Ile – Leu – Cys – Pro – Stop Altered reading frame 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. retina (Case 43). In contrast, HEXA alleles associated in other populations. A founder effect or heterozygote with some residual activity lead to later-onset forms of advantage is the most likely explanation for this high neurological disease, with manifestations including frequency (see Chapter 9). Because most Ashkenazi lower motor neuron dysfunction and ataxia due to spi- Jewish carriers will have one of the three common nocerebellar degeneration. In contrast to the infantile alleles, a practical benefit of the molecular characteriza- disease, vision and intelligence usually remain normal, tion of the disease in this population is the degree to A fetus with one pseudodeficiency allele and although psychosis develops in one third of these which carrier screening has been simplified. one Tay-Sachs mutation might be mistakenly patients. Finally, pseudodeficiency alleles (discussed diagnosed as affected because laboratory next) do not cause disease at all. assays show low Hex A activity. Altered Protein Function due to Abnormal Hex A Pseudodeficiency Alleles and Their Clinical Sig- nificance. An unexpected consequence of screening for Post-translational Modification Tay-Sachs carriers in the Ashkenazi Jewish population A Loss of Glycosylation: I-Cell Disease was the discovery of a unique class of hex A alleles, the Some proteins have information contained in their so-called pseudodeficiency alleles. Although the two primary amino acid sequence that directs them to their pseudodeficiency alleles are clinically benign, individu- subcellular residence, whereas others are localized on als identified as pseudodeficient in screening tests are the basis of post-translational modifications. This latter genetic compounds with a pseudodeficiency allele on mechanism is true of the acid hydrolases found in lyso- one chromosome and a common Tay-Sachs mutation somes, but this form of cellular trafficking was unrec- on the other chromosome. These individuals have a ognized until the discovery of I-cell disease, a severe low level of hex A activity (approximately 20% of autosomal recessive lysosomal storage disease. The dis- controls) that is adequate to prevent GM2 ganglioside order has a range of phenotypic effects involving facial accumulation in the brain. The importance of hex A features, skeletal changes, growth retardation, and intel- pseudodeficiency alleles is twofold. First, they compli- lectual disability and survival of less than 10 years (Fig. cate prenatal diagnosis because a pseudodeficient fetus 12-7). The cytoplasm of cultured skin fibroblasts from could be incorrectly diagnosed as affected. More gener- I-cell patients contains numerous abnormal lysosomes, ally, the recognition of the hex A pseudodeficiency or inclusions, (hence the term inclusion cells or I cells). alleles indicates that screening programs for other In I-cell disease, the cellular levels of many lysosomal genetic diseases must recognize that comparable alleles acid hydrolases are greatly diminished, and instead they may exist at other loci and may confound the correct are found in excess in body fluids. This unusual situa- characterization of individuals in screening or diagnos- tion arises because the hydrolases in these patients tic tests. have not been properly modified post-translationally. A typical hydrolase is a glycoprotein, the sugar moiety Population Genetics. In many single-gene diseases, containing mannose residues, some of which are phos- some alleles are found at higher frequency in some phorylated. The mannose-6-phosphate residues are populations than in others (see Chapter 9). This situa- essential for recognition of the hydrolases by receptors tion is illustrated by Tay-Sachs disease, in which three on the cell and lysosomal membrane surface. In I-cell alleles account for 99% of the mutations found in Ash- disease, there is a defect in the enzyme that transfers kenazi Jewish patients, the most common of which (Fig. a phosphate group to the mannose residues. The 12-6) accounts for 80% of cases. Approximately 1 in fact that many enzymes are affected is consistent with 27 Ashkenazi Jews is a carrier of a Tay-Sachs allele, and the diversity of clinical abnormalities seen in these the incidence of affected infants is 100 times higher than patients.  CHAPTER 12 — THE MOLECULAR, BIOCHEMICAL, AND CELLULAR BASIS OF GENETIC DISEASE 223 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 fre- quently capable of restoring some residual activity to the mutant enzyme, for example by increasing the stabil- ity 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 Figure 12-7 I-cell disease facies and habitus in an 18-month-old girl. See Sources & Acknowledgments. Chapter 13). Impaired Cofactor Binding: Homocystinuria due to Cystathionine Synthase Deficiency Gains of Glycosylation: Mutations That Create Homocystinuria due to cystathionine synthase defi- New (Abnormal) Glycosylation Sites ciency (Fig. 12-8) was one of the first aminoacidopathies In contrast to the failure of protein glycosylation exem- to be recognized. The clinical phenotype of this autoso- plified by I-cell disease, it has been shown that an unex- mal recessive condition is often dramatic. The most pectedly high proportion (approximately 1.5%) of the common features include dislocation of the lens, intel- missense mutations that cause human disease may be lectual disability, osteoporosis, long bones, and throm- associated with abnormal gains of N-glycosylation due boembolism of both veins and arteries, a phenotype that to mutations creating new consensus N-glycosylation can be confused with Marfan syndrome, a disorder of sites in the mutant proteins. That such novel sites can connective tissue (Case 30). The accumulation of homo- actually lead to inappropriate glycosylation of the cysteine is believed to be central to most, if not all, of mutant protein, with pathogenic consequences, is high- the pathology. lighted by the rare autosomal recessive disorder, men- Homocystinuria was one of the first genetic diseases delian susceptibility to mycobacterial disease (MSMD). shown to be vitamin responsive; pyridoxal phosphate is MSMD patients have defects in any one of a the cofactor of the enzyme, and the administration of number of genes that regulate the defense against some large amounts of pyridoxine, the vitamin precursor of infections. Consequently, they are susceptible to dis- the cofactor, often ameliorates the biochemical abnor- seminated infections upon exposure to moderately viru- mality and the clinical disease (see Chapter 13). In many lent mycobacterial species, such as the bacillus patients, the affinity of the mutant enzyme for pyridoxal Calmette-Guérin (BCG) used throughout the world as phosphate is reduced, indicating that altered conforma- a vaccine against tuberculosis, or to nontuberculous tion of the protein impairs cofactor binding. environmental bacteria that do not normally cause Not all cases of homocystinuria result from muta- illness. Some MSMD patients carry missense mutations tions in cystathionine synthase. Mutations in five dif- in the gene for interferon-γ receptor 2 (IFNGR2) that ferent enzymes of cobalamin (vitamin B12) or folate generate novel N-glycosylation sites in the mutant metabolism can also lead to increased levels of homo- IFNGR2 protein. These novel sites lead to the synthesis cysteine in body fluids. These mutations impair the pro- of an abnormally large, overly glycosylated receptor. vision of the vitamin B12 cofactor, methylcobalamin The mutant receptors reach the cell surface but fail to (methyl-B12), or of methyl-H4-folate (see Fig. 12-8) and respond to interferon-γ. Mutations leading to gains of thus represent another example (like the defects in BH4 glycosylation have also been found to lead to a loss of synthesis that lead to hyperphenylalaninemia) of genetic protein function in several other monogenic disorders. diseases due to defects in the biogenesis of enzyme The discovery that removal of the abnormal polysac- cofactors. The clinical manifestation of these disorders charides restores function to the mutant IFNGR2 pro- is variable but includes megaloblastic anemia, develop- teins in MSMD offers hope that disorders of this type mental delay, and failure to thrive. These conditions, all 224 THOMPSON & THOMPSON GENETICS IN MEDICINE Cystathionine synthase Methionine Homocysteine Cystathionine Cysteine Pyridoxal Methionine phosphate synthase Methyl-B12 Vitamin B6 H4-folate Methyl-H4-folate 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 cystathio- nine 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). of which are autosomal recessive, are often partially or Z allele (Glu342Lys) is relatively common. The reason completely treatable with high doses of vitamin B12. for the relatively high frequency of the Z allele in white populations is unknown, but analysis of DNA haplo- types suggests a single origin with subsequent spread Mutations of an Enzyme Inhibitor: throughout northern Europe. Given the increased risk α1-Antitrypsin Deficiency for emphysema, α1AT deficiency is an important public α1-Antitrypsin (α1AT) deficiency is an important auto- health problem, affecting an estimated 60,000 persons somal recessive condition associated with a substantial in the United States alone. risk for chronic obstructive lung disease (emphysema) The α1AT gene is expressed principally in the liver, (Fig. 12-9) and cirrhosis of the liver. The α1AT protein which normally secretes α1AT into plasma. Approxi- belongs to a major family of protease inhibitors, the mately 17% of Z/Z homozygotes present with neonatal serine protease inhibitors or serpins; SERPINA1 is the jaundice, and approximately 20% of this group subse- formal gene name. Notwithstanding the specificity sug- quently develop cirrhosis. The liver disease associated gested by its name, α1AT actually inhibits a wide spec- with the Z allele is thought to result from a novel prop- trum of proteases, particularly elastase released from erty of the mutant protein—its tendency to aggregate, neutrophils in the lower respiratory tract. trapping it within the rough endoplasmic reticulum In white populations, α1AT deficiency affects approx- (ER) of hepatocytes. The molecular basis of the Z imately 1 in 6700 persons, and approximately 4% are protein aggregation is a consequence of structural carriers. A dozen or so α1AT alleles are associated with changes in the protein that predispose to the formation an increased risk for lung or liver disease, but only the of long beadlike necklaces of mutant α1AT polymers. 1.0 All females Cumulative probability of survival 0.8 (mostly M/M) 0.6 Z/Z nonsmokers All males (mostly M/M) 0.4 Z/Z smokers 0.2 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 speci- 0 20 30 40 50 60 70 80 90 100 fied ages of smokers, with or without α1-antitrypsin deficiency. See Sources & Acknowledgments. Age (years)  CHAPTER 12 — THE MOLECULAR, BIOCHEMICAL, AND CELLULAR BASIS OF GENETIC DISEASE 225 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. 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 envi- ronmental factor, cigarette smoke, dramatically influ- ences 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, sur- vival 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 inflamma- tory cells, thus reducing its affinity for elastase by 2000-fold. The field of ecogenetics, illustrated by α1AT defi- Figure 12-10 A posteroanterior chest radiograph of an individual carrying two Z alleles of the α1AT gene, showing the hyperinfla- ciency, is concerned with the interaction between envi- tion and basal hyperlucency characteristic of emphysema. See ronmental factors and different human genotypes. This Sources & Acknowledgments. 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 envi- Thus, like the sickle cell disease mutation in β-globin ronmental agents (e.g., drugs, foods, industrial chemi- (see Chapter 11), the Z allele of α1AT is a clear example cals, and viruses). At present, the most highly developed of a mutation that confers a novel property on the area of ecogenetics is that of pharmacogenetics, pre- protein (in both of these examples, a tendency to aggre- sented in Chapter 16. gate) (see Fig. 11-1). Both sickle cell disease and the α1AT deficiency asso- Dysregulation of a Biosynthetic Pathway: Acute ciated with homozygosity for the Z allele are examples of inherited conformational diseases. These disorders Intermittent Porphyria occur when a mutation causes the shape or size of a Acute intermittent porphyria (AIP) is an autosomal protein to change in a way that predisposes it to self- dominant disease associated with intermittent neuro- association and tissue deposition. Notably, some frac- logical dysfunction. The primary defect is a deficiency tion of the mutant protein is invariably correctly folded of porphobilinogen (PBG) deaminase, an enzyme in the in these disorders, including α1AT deficiency. Note biosynthetic pathway of heme, required for the synthe- that not all conformational diseases are single-gene sis of both hemoglobin and hepatic cytochrome p450 disorders, as illustrated, for example, by nonfamilial drug-metabolizing enzymes (Fig. 12-11). All individuals Alzheimer disease (discussed later) and prion diseases. with AIP have an approximately 50% reduction in PBG The lung disease associated with the Z allele of α1AT deaminase enzymatic activity, whether their disease is deficiency is due to the alteration of the normal balance clinically latent (90% of patients throughout their life- between elastase and α1AT, which allows progressive time) or clinically expressed (approximately 10%). This degradation of the elastin of alveolar walls (Fig. 12-10). reduction is consistent with the autosomal dominant Two mechanisms contribute to the elastase α1AT imbal- inheritance pattern (see Chapter 7). Homozygous defi- ance. First, the block in the hepatic secretion of the Z ciency of PBG deaminase, a critical enzyme in heme protein, although not complete, is severe, and Z/Z biosynthesis, would presumably be incompatible with patients have only approximately 15% of the normal life. AIP illustrates one molecular mechanism by which plasma concentration of α1AT. Second, the Z protein an autosomal dominant disease may manifest only has only approximately 20% of the ability of the normal episodically. α1AT protein to inhibit neutrophil elastase. The infu- The pathogenesis of the nervous system disease is sion of normal α1AT is used in some patients to augment uncertain but may be mediated directly by the increased 226 THOMPSON & THOMPSON GENETICS IN MEDICINE Clinically latent AIP: No symptoms ALA 50% reduction Glycine + succinyl CoA ALA PBG Hydroxymethylbilane Heme synthetase PBG deaminase Clinically expressed AIP: Postpubertal neurological symptoms Drugs, chemicals, steroids, fasting, etc. ALA 50% reduction Glycine + succinyl CoA ALA PBG Hydroxymethylbilane Heme synthetase PBG deaminase 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 porpho- bilinogen (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. See Sources & Acknowledgments. levels of δ-aminolevulinic acid (ALA) and PBG that (LDL) receptor as the polypeptide affected in the most accumulate due to the 50% reduction in PBG deaminase common form of familial hypercholesterolemia. This (see Fig. 12-11). The peripheral, autonomic, and central disorder, which leads to a greatly increased risk for nervous systems are all affected, and the clinical mani- myocardial infarction, is characterized by elevation of festations are diverse. Indeed, this disorder is one of the plasma cholesterol carried by LDL, the principal cho- great mimics in clinical medicine, with manifestations lesterol transport protein in plasma. Goldstein and ranging from acute abdominal pain to psychosis. Brown’s discovery has cast much light on normal cho- Clinical crises in AIP are elicited by a variety of pre- lesterol metabolism and on the biology of cell surface cipitating factors: drugs (most prominently the barbitu- receptors in general. LDL receptor deficiency is repre- rates, and to this extent, AIP is a pharmacogenetic sentative of a number of disorders now recognized to disease; see Chapter 18); some steroid hormones (clini- result from receptor defects. cal disease is rare before puberty or after menopause); and catabolic states, including reducing diets, intercur- Familial Hypercholesterolemia: rent illnesses, and surgery. The drugs provoke the clini- cal manifestations by interacting with drug-sensing A Genetic Hyperlipidemia nuclear receptors in hepatocytes, which then bind to Familial hypercholesterolemia is one of a group of meta- transcriptional regulatory elements of the ALA synthe- bolic disorders called the hyperlipoproteinemias. These tase gene, increasing the production of both ALA and diseases are characterized by elevated levels of plasma PBG. In normal individuals the drug-related increase in lipids (cholesterol, triglycerides, or both) carried by apo- ALA synthetase is beneficial because it increases heme lipoprotein B (apoB)-containing lipoproteins. Other synthesis, allowing greater formation of hepatic cyto- monogenic hyperlipoproteinemias, each with distinct chrome P450 enzymes that metabolize many drugs. In biochemical and clinical phenotypes, have also been patients with AIP, however, the increase in ALA synthe- recognized. tase causes the accumulation of ALA and PBG because In addition to mutations in the LDL receptor gene of the 50% reduction in PBG deaminase activity (see (Table 12-2), abnormalities in three other genes can Fig. 12-11). The fact that half of the normal activity of also lead to familial hypercholesterolemia (Fig. 12-12). PBG deaminase is inadequate to cope with the increased Remarkably, all four of the genes associated with famil- requirement for heme synthesis in some situations ial hypercholesterolemia disrupt the function or abun- accounts for both the dominant inheritance of the con- dance either of the LDL receptor at the cell surface dition and the episodic nature of the clinical illness. or of apoB, the major protein component of LDL and a ligand for the LDL receptor. Because of its impor- tance, we first review familial hypercholesterolemia DEFECTS IN RECEPTOR PROTEINS due to mutations in the LDL receptor. We also discuss The recognition of a class of diseases due to defects in mutations in the PCSK9 protease gene; although gain- receptor molecules began with the identification by of-function mutations in this gene cause hypercholester- Goldstein and Brown of the low-density lipoprotein olemia, the greater importance of PCSK9 lies in the fact  CHAPTER 12 — THE MOLECULAR, BIOCHEMICAL, AND CELLULAR BASIS OF GENETIC DISEASE 227 TABLE 12-2 Four Genes Associated with Familial Hypercholesterolemia Typical LDL Cholesterol Level Mutant Gene Product Pattern of Inheritance Effect of Disease-Causing Mutations (Normal Adults: ≈120 mg/dL) LDL receptor Autosomal dominant Loss of function Heterozygotes: 350 mg/dL Homozygotes: 700 mg/dL Apoprotein B-100 Autosomal dominant* Loss of function Heterozygotes: 270 mg/dL Homozygotes: 320 mg/dL ARH adaptor protein Autosomal recessive† Loss of function Homozygotes: 470 mg/dL PCSK9 protease Autosomal dominant Gain of function Heterozygotes: 225 mg/dL *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. 2. Apoprotein B-100 1. Mature LDL surrounding a receptor cholesterol ester core 3. ARH adaptor protein, Vesicle required for clustering Golgi the LDL receptor in the complex clathrin-coated pit 4. PCSK9: a protease Endoplasmic that targets the LDL reticulum receptor for lysosomal degradation 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). that several common loss-of-function sequence variants arcus corneae (deposits of cholesterol around the periph- lower plasma LDL cholesterol levels, conferring sub- ery of the cornea). Few diseases have been as thoroughly stantial protection from coronary heart disease. characterized; the sequence of pathological events from the affected locus to its effect on individuals and popula- Familial Hypercholesterolemia due to Mutations tions has been meticulously documented. in the LDL Receptor Mutations in the LDL receptor gene (LDLR) are the Genetics. Familial hypercholesterolemia due to muta- most common cause of familial hypercholesterol- tions in the LDLR gene is inherited as an autosomal emia (Case 16). The receptor is a cell surface protein semidominant trait. Both homozygous and heterozy- responsible for binding LDL and delivering it to the cell gous phenotypes are known, and a clear gene dosage interior. Elevated plasma concentrations of LDL choles- effect is evident; the disease manifests earlier and much terol lead to premature atherosclerosis (accumulation of more severely in homozygotes than in heterozygotes, cholesterol by macrophages in the subendothelial space reflecting the greater reduction in the number of LDL of major arteries) and increased risk for heart attack and receptors and the greater elevation in plasma LDL cho- stroke in both untreated heterozygote and homozygote lesterol (Fig. 12-13). Homozygotes may have clinically carriers of mutant alleles. Physical stigmata of familial significant coronary heart disease in childhood and, if hypercholesterolemia include xanthomas (cholesterol untreated, few live beyond the third decade. The hetero- deposits in skin and tendons) (Case 16) and premature zygous form of the disease, with a population frequency 228 THOMPSON & THOMPSON GENETICS IN MEDICINE (Fig. 12-14). Receptor-bound LDL is brought into the cell by endocytosis of the coated pits, which ultimately s s te te go ro e o evolve into lysosomes in which LDL is hydrolyzed to te at yg zy h e b lig oz al m release free cholesterol. The increase in free intracellular om O or H N 1000 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 800 metabolism or membrane synthesis may be re-esterified for storage as cholesteryl esters, a process stimulated by the activation of acyl coenzyme A : cholesterol acyl- transferase (ACAT). The increase in intracellular choles- Plasma cholesterol (mg/dL) terol also reduces synthesis of the LDL receptor (see 600 Fig. 12-14). Classes of Mutations in the LDL Receptor More than 1100 different mutations have been identi- 400 fied 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 200 substitutions, small insertions, or deletions; structural rearrangements account for only 2% to 10% of the Mean LDLR alleles in most populations. The mature LDL _ +2 SD receptor has five distinct structural domains that for the 0 most part have distinguishable functions that mediate Figure 12-13 Gene dosage in low-density lipoprotein (LDL) defi- the steps in the life cycle of an LDL receptor, shown in ciency. Shown is the distribution of total plasma cholesterol levels Figure 12-14. Analysis of the effect on the receptor of in 49 patients homozygous for deficiency of the LDL receptor, mutations in each domain has played an important role their parents (obligate heterozygotes), and normal controls. See Sources & Acknowledgments. in defining the function of each domain. These studies exemplify the important contribution that genetic anal- ysis can make in determining the structure-function rela- of approximately 2 per 1000, is one of the most common tionships of a protein. single-gene disorders. Heterozygotes have levels of Fibroblasts cultured from affected patients have been plasma cholesterol that are approximately twice those used to characterize the mutant receptors and the result- of controls (see Fig. 12-13). Because of the inherited ing disturbances in cellular cholesterol metabolism. nature of familial hypercholesterolemia, it is important LDLR mutations can be grouped into six classes, to make the diagnosis in the approximately 5% of sur- depending on which step of the normal cellular itinerary vivors of premature (G in the Largely Maternal optic neuropathy adult life due to optic nerve ND4 subunit of complex I homoplasmic (LHON) atrophy; some recovery of of the electron transport vision, depending on the chain; this mutation, with mutation. Strong sex bias: ≈50% two others, accounts for of male carriers have visual loss more than 90% of cases. vs. ≈10% of females. Leigh syndrome Early-onset progressive Point mutations in the Heteroplasmic Maternal neurodegeneration with ATPase subunit 6 gene hypotonia, developmental delay, optic atrophy, and respiratory abnormalities MELAS Myopathy, mitochondrial Point mutations in Heteroplasmic Maternal encephalomyopathy, lactic tRNAleu(UUR), a mutation acidosis, and stroke like hot spot, most commonly episodes; may present only as 3243A>G diabetes mellitus and deafness MERRF (Case 33) Myoclonic epilepsy with ragged- Point mutations in tRNAlys, Heteroplasmic Maternal red muscle fibers, myopathy, most commonly 8344A>G ataxia, sensorineural deafness, dementia Deafness Progressive sensorineural deafness, 1555A>G mutation in the Homoplasmic Maternal often induced by aminoglycoside 12S rRNA gene antibiotics; nonsyndromic 7445A>G mutation in the Homoplasmic Maternal sensorineural deafness 12S rRNA gene Kearns-Sayre Progressive myopathy, progressive The ≈5-kb large deletion (see Heteroplasmic Generally sporadic, syndrome (KSS) external ophthalmoplegia of Fig. 12-26) likely due to early onset, cardiomyopathy, maternal gonadal heart block, ptosis, retinal mosaicism pigmentation, ataxia, diabetes mtDNA, Mitochondrial DNA; rRNA, ribosomal RNA; tRNA, transfer RNA. Mutations in tRNA and rRNA Genes of the Mitochon- that depend on intact oxidative phosphorylation to drial Genome. Mutations in the noncoding tRNA and satisfy high demands for metabolic energy. This pheno- rRNA genes of mtDNA are of general significance typic focus reflects the central role of the oxidative because they illustrate that not all disease-causing muta- phosphorylation complex in the production of cellular tions in humans occur in genes that encode pro- energy. Consequently, decreased production of ATP teins (Case 33). More than 90 pathogenic mutations characterizes many diseases of mtDNA and is likely to have been identified in 20 of the 22 tRNA genes of underlie the cell dysfunction and cell death that occur the mtDNA, and they are the most common cause in mtDNA diseases. The evidence that mechanisms of oxidative phosphorylation abnormalities in humans other than decreased energy production contribute to (see Fig. 12-26 and Table 12-7). The resulting phe- the pathogenesis of mtDNA diseases is either indirect or notypes are those generally associated with mtDNA weak, but the generation of reactive oxygen species as defects. The tRNA mutations include 18 substitutions a byproduct of faulty oxidative phosphorylation may in the tRNAleu(UUR) gene, some of which, like the common also contribute to the pathology of mtDNA disorders. 3243A>G mutation, cause a phenotype referred to as A substantial body of evidence indicates that there is a MELAS, an acronym for mitochondrial encephalomy- phenotypic threshold effect associated with mtDNA het- opathy with lactic acidosis and strokelike episodes (see eroplasmy (see Fig. 7-25); a critical threshold in the Fig. 12-26 and Table 12-7); others are associated pre- proportion of mtDNA molecules carrying the detrimen- dominantly with myopathy. An example of a 12S rRNA tal mutation must be exceeded in cells from the affected mutation is a homoplasmic substitution (see Table 12-7) tissue before clinical disease becomes apparent. The that causes sensorineural prelingual deafness after expo- threshold appears to be approximately 60% for disor- sure to aminoglycoside antibiotics (see Fig. 12-26). ders due to deletions in mtDNA and approximately 90% for diseases due to other types of mutations. The Phenotypes of Mitochondrial Disorders The neuromuscular system is the one most commonly Oxidative Phosphorylation and mtDNA Diseases. affected by mutations in mtDNA; the consequences Mitochondrial mutations generally affect those tissues can include encephalopathy, myopathy, ataxia, retinal 250 THOMPSON & THOMPSON GENETICS IN MEDICINE degeneration, and loss of function of the external ocular It is likely that much of the phenotypic variation muscles. Mitochondrial myopathy is characterized by observed among patients with mutations in mitochon- so-called ragged-red (muscle) fibers, a histological phe- drial genes will be explained by the fact that the proteins notype due to the proliferation of structurally and bio- within mitochondria are remarkably heterogeneous chemically abnormal mitochondria in muscle fibers. The between tissues, differing on average by approximately spectrum of mitochondrial disease is broad and, as illus- 25% between any two organs. This molecular hetero- trated in Figure 12-27, may include liver dysfunction, geneity is reflected in biochemical heterogeneity. For bone marrow failure, pancreatic islet cell deficiency and example, whereas much of the energy generated by diabetes, deafness, and other disorders. brain mitochondria derives from the oxidation of ketones, skeletal muscle mitochondria preferentially use fatty acids as their fuel. HETEROPLASMY AND MITOCHONDRIAL DISEASE Heteroplasmy accounts for three general characteristics Interactions between the Mitochondrial and of genetic disorders of mtDNA that are of importance to Nuclear Genomes their pathogenesis. First, female carriers of heteroplasmic mtDNA point Because both the nuclear and mitochondrial genomes mutations or of mtDNA duplications usually transmit contribute polypeptides to oxidative phosphorylation, it some mutant mtDNAs to their offspring. is not surprising that the phenotypes associated with Second, the fraction of mutant mtDNA molecules mutations in the nuclear genes are often indistinguish- inherited by each child of a carrier mother is very vari- able. This is because the number of mtDNA molecules able from those due to mtDNA mutations. Moreover, within each oocyte is reduced before being subse- mtDNA depends on many nuclear genome–encoded quently amplified to the huge total seen in mature proteins for its replication and the maintenance of its oocytes. This restriction and subsequent amplification integrity. Genetic evidence has highlighted the direct of mtDNA during oogenesis is termed the mitochon- nature of the relationship between the nuclear and drial genetic bottleneck. Consequently, the variability in the percentage of mutant mtDNA molecules seen in mtDNA genomes. The first indication of this interaction the offspring of a mother carrying a mtDNA mutation was provided by the identification of the syndrome of arises, at least in part, from the sampling of only a autosomally transmitted deletions in mtDNA. Muta- subset of the mtDNAs during oogenesis. tions in at least two genes have been associated with Third, despite the variability in the degree of hetero- this phenotype. The protein encoded by one of these plasmy arising from the bottleneck, mothers with a high proportion of mutant mtDNA molecules are genes, amusingly called Twinkle, appears to be a DNA more likely to have clinically affected offspring than primase or helicase. The product of the second gene is are mothers with a lower proportion, as one would a mitochondrial-specific DNA polymerase γ, whose loss predict from the random sampling of mtDNA mole- of function is associated with both dominant and reces- cules through the bottleneck. Nevertheless, even women sive multiple deletion syndromes. carrying low proportions of pathogenic mtDNA mol- ecules have some risk for having an affected child A second autosomal disorder, the mtDNA depletion because the bottleneck can lead to the sampling and syndrome, is the result of mutations in any of six nuclear subsequent expansion, by chance, of even a rare genes that lead to a reduction in the number of copies mutant mtDNA species. 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 Unexplained and Unexpected Phenotypic Variation in metabolize nucleotides appropriately in the mitochon- mtDNA Diseases. As seen in Table 12-7, heteroplasmy drion. For example, both myopathic and hepatocerebral is the rule for many mtDNA diseases. Heteroplasmy phenotypes result from mutations in the nuclear genes leads to an unpredictable and variable fraction of for mitochondrial thymidine kinase and deoxyguano- mutant mtDNA being present in any particular tissue, sine kinase. Because mutations in the six genes identified undoubtedly accounting for much of the pleiotropy and to date account for only a minority of affected individu- variable expressivity of mtDNA mutations (see Box). An als, additional genes must also be involved in this example is provided by what appears to be the most disorder. common mtDNA mutation, the 3243A>G substitution Apart from the insights that these rare disorders in the tRNAleu(UUR) gene just mentioned in the context of provide into the biology of the mitochondrion, the iden- the MELAS phenotype. This mutation leads predomi- tification of the affected genes facilitates genetic counsel- nantly to diabetes and deafness in some families, whereas ing and prenatal diagnosis in some families and suggests, in others it causes a disease called chronic progressive in some instances, potential treatments. For example, external ophthalmoplegia. Moreover, a very small frac- the blood thymidine level is markedly increased in thy- tion (G substitution. an excess of substrate rather than a deficiency of the  CHAPTER 12 — THE MOLECULAR, BIOCHEMICAL, AND CELLULAR BASIS OF GENETIC DISEASE 251 product plays a major role in the pathogenesis of the syndrome (Case 17), GAA in Friedreich ataxia, and disease. CUG in myotonic dystrophy 1 (Fig. 12-28). Although the initial nucleotide repeat diseases to be Nuclear Genes Can Modify the Phenotype of mtDNA described are all due to the expansion of three nucleo- Diseases. Although heteroplasmy is a major source of tide repeats, other disorders have now been found to phenotypic variability in mtDNA diseases (see Box), result from the expansion of longer repeats; these include additional factors, including alleles at nuclear loci, must a tetranucleotide (CCTG) in myotonic dystrophy 2 (a also play a role. Strong evidence for the existence close genocopy of myotonic dystrophy 1) and a penta- of such factors is provided by families carrying muta- nucleotide (ATTCT) in spinocerebellar atrophy 10. tions associated with Leber hereditary optic neuropathy Because the affected gene is passed from generation to (LHON; see Table 12-7), which is generally homo- generation, the number of repeats may expand to a plasmic (thus ruling out heteroplasmy as the explana- degree that is pathogenic, ultimately interfering with tion for the observed phenotypic variation). LHON is normal gene expression and function. The intergenera- expressed phenotypically as rapid, painless bilateral loss tional expansion of the repeats accounts for the phe- of central vision due to optic nerve atrophy in young nomenon of anticipation, the appearance of the disease adults (see Table 12-7 and Fig. 12-26). Depending on at an earlier age as it is transmitted through a family. the mutation, there is often some recovery of vision, but The biochemical mechanism most commonly proposed the pathogenic mechanisms of the optic nerve damage to underlie the expansion of unstable repeat sequences are unclear. is slipped mispairing (Fig. 12-29). Remarkably, the There is a striking and unexplained increase in the repeat expansions appear to occur both in proliferating penetrance of the disease in males; approximately 50% cells such as spermatogonia (during meiosis) and in of male carriers but only approximately 10% of female nonproliferating somatic cells such as neurons. Conse- carriers of a LHON mutation develop symptoms. The quently, expansion can occur, depending on the disease, variation in penetrance and the male bias of the LHON during both DNA replication (as shown in Fig. 12-29) phenotype are determined by a haplotype on the short and genome maintenance (i.e., DNA repair). arm of the X chromosome. The gene at this nuclear- The clinical phenotypes of Huntington disease and encoded modifier locus has not yet been identified, but fragile X syndrome are presented in Chapter 7 and in it is contained, notably, in a haplotype that is common their respective Cases. For reasons that are gradually in the general population. When the protective haplo- becoming apparent, particularly in the case of fragile X type is transmitted from a typically unaffected mother syndrome, diseases due to the expansion of unstable to individuals who have inherited the LHON mtDNA repeats are primarily neurological; the clinical presenta- mutation from that mother, the phenotype is substan- tions include ataxia, cognitive defects, dementia, nystag- tially ameliorated. Thus males who carry the high-risk mus, parkinsonism, and spasticity. Nevertheless, other X-linked haplotype as well as a LHON mtDNA muta- systems are sometimes involved, as illustrated by some tion (other than the one associated with the most severe of the diseases discussed here. LHON phenotype [see Table 12-7]) are thirty-fivefold more likely to develop visual failure than those who The Pathogenesis of Diseases due to Unstable carry the low-risk X-linked haplotype. These observa- Repeat Expansions tions are of general significance because they demon- Diseases of unstable repeat expansion are diverse in strate the powerful effect that modifier loci can have on their pathogenic mechanisms and can be divided into the phenotype of a monogenic disease. 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 Diseases due to the Expansion of Unstable Class 2: disorders resulting from expansions of non- Repeat Sequences coding repeats that confer novel properties on the The inheritance pattern of diseases due to unstable RNA repeat expansions was presented in Chapter 7, with Class 3: diseases due to repeat expansion of a codon emphasis on the unusual genetics of this unique group such as CAG (for glutamine) that confers novel prop- of almost 20 disorders. These features include the erties on the affected protein unstable and dynamic nature of the mutations, which are due to the expansion, within the transcribed Class 1: Diseases due to the Expansion of region of the affected gene, of repeated sequences such Noncoding Repeats That Cause a Loss of as the codon for glutamine (CAG) in Huntington Protein Expression disease (Case 24) and most of a group of neurode- Fragile X Syndrome. In the X-linked fragile X syn- generative disorders called the spinocerebellar ataxias, drome, the expansion of the CGG repeat in the 5′ or due to the expansion of trinucleotides in noncoding untranslated region (UTR) of the FMR1 gene to more regions of RNAs, including CGG in fragile X than 200 copies leads to excessive methylation of 252 THOMPSON & THOMPSON GENETICS IN MEDICINE AUG stop pre-mRNA 59 59 UTR intron exon intron 39 UTR 39 (CGG)n (GAA)n (CCUG)n (CAG)n (CUG)n (CGG)n>200 (CGG)n60 to 200 (GAA)n≥200 (CCTG)n≥75 (CAG)n≥40 (CTG)n≥50 Fragile X Fragile X Friedreich Myotonic Huntington Myotonic syndrome tremor/ataxia ataxia dystrophy 2 disease dystrophy 1 syndrome Transcriptional 2 to 5-fold increase Impaired Expanded polyglutamine Expanded CUG silencing in FMR1 mRNA transcriptional tracts in the huntingtin repeats in the = loss-of- = ? gain-of- elongation protein confer novel RNA confer novel function RNA function = loss of properties on the protein properties mutation frataxin function on the RNA Loss of Neuronal Increased Fe Increased and/or Expanded CUG RNA binding intranuclear in mitochondria, promiscuous protein:protein repeats bind increased = impaired inclusions reduced heme interactions with transcription amounts of RNA-binding translational synthesis, factors → loss of their function proteins → impaired RNA repression of reduced activity splicing of key proteins target RNAs of Fe-S complex containing proteins Figure 12-28 The locations of the trinucleotide repeat expansions and the sequence of each tri- nucleotide 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. See Sources & Acknowledgments. cytosines in the promoter, an epigenetic modification synaptic plasticity, the capacity to alter the strength of of the DNA that silences transcription of the gene (see a synaptic connection, a process critical to learning and Figs. 7-22 and 12-28). Remarkably, the epigenetic memory. silencing appears to be mediated by the mutant FMR1 mRNA itself. The initial step in the silencing of FMR1 Fragile X Tremor/Ataxia Syndrome. Remarkably, the results from the FMR1 mRNA, containing the tran- pathogenesis of disease in individuals with less pro- scribed CGG repeat, hybridizing with the complemen- nounced CGG repeat expansion (60 to 200 repeats) in tary CGG-repeat sequence of the FMR1 gene, to form the FMR1 gene, causing the clinically distinct fragile X an RNA : DNA duplex. The mechanisms that subse- tremor/ataxia syndrome (FXTAS), is entirely different quently maintain the silencing of the FMR1 gene are from that of the fragile X syndrome itself. Although unknown. The loss of the fragile X mental retardation decreased translational efficiency impairs the expres- protein (FMRP) is the cause of the intellectual disability sion of the FMRP protein in FXTAS, this reduction and learning deficits