NEW HORIZONS IN NEUROLOGY
The First Decade of Molecular Genetics in Neurology: Changmg Clinical Thought and Practice Lewis P. Rowland, MD
Molecular genetics has had a powerful impact on clinical neurology. Definitions of disease are changing from clinical criteria to DNA analysis, resolving questions about the nature of clinically similar but not identical diseases. Genetic counseling is more reliable. Concepts of mendelian inheritance are being tested and new forms of mutation have been discovered to explain anticipation. Nonmendelian forms of inheritance have emerged; concepts of pathogenesis are on a more secure footing; and novel treatments are being explored. Rowland LP. The first decade of molecular genetics in neurology: changing clinical thought and practice. Ann Neurol 1992;32:207-2 14 Molecular genetics entered clinical neurology in 1983 when Gusella and colleagues 111 mapped the gene for Huntington’s disease to chromosome 4. That year saw the first use of “molecular genetics” in the title of an article 121 and the first research paper on the subject 131 in a neurology journal. My task is to summarize the subsequent clinical impact on our field. Clinical neurology has made major contributions to genetics. Reciprocally, molecular genetics has radically altered concepts and practice of clinical neurology.
The Accelerating Pace of Change There is a self-propelling force in molecular genetics. The process of assigning a chromosomal location for a disease gene is called positional mapping, which depends on having DNA probes on each chromosome for linkage analysis. The closer the marker is to the disease gene, the more likely they are to be inherited together. Collins 141 compared the activity to “searching for a needle in a haystack in the dark wearing thick gloves.” Each linkage, however, makes the next one a bit easier. The more probes available, the faster the mapping of any disease. The X-chromosome has been most thoroughly saturated because many X-linked diseases had been identified by pedigree analysis before 1983. Autosomal diseases are now accessible because the number of probes has increased exponentially 151 (Fig). In 1988, 61 diseases of neurological interest had been mapped C6l; now, the number is 129 (Table) 171.
From the Department of Neurology, Neurological Institute, and the H. Houston Merritt Clinical Research Center for Muscular Dystrophy and Related Diseases, columbia-presbYterian New York, NY.
Changing Definitions from Clinical to Molecular Criteria Some diseases are still defined by clinical criteria, but Duchenne muscular dystrophy (DMD) indicates how DNA analysis has changed definitions of related myopathies. Duchenne and Becker dystrophies were considered similar but distinct diseases. Now we know they are allelic, both due to alterations of the same gene product, dystrophin 181. This landmark achievement proved that positional cloning could lead to identification of a previously unknown gene product. Similarly, there had been debates about whether hyperkalemic periodic paralysis and paramyotonia are variations of the same genetic disorder or separate diseases 191. Now, both have been mapped to the same site, proving their allelic identity 110-121. Likewise, infantile spinal muscular atrophy (SMA) (WerdnigHoffmann) and juvenile SMA (Kugelberg-Welander) seem to be allelic 113-151. Proof of allelic identity is not restricted to these clinically similar disorders. Even clinically disparate syndromes may be manifestations of mutations of the same gene product 16, 16, 171. For instance, the clinical manifestations of “Xp2 1 myopathy” or “dystrophinopathy” have extended from the typical limb weakness of young boys to X-linked cramps 118, 191, myoglobinuria 1201, quadriceps myopathy 12 11, or congenital myopathy E22-241. Mild myopathies appear in girls or women who prove to be manifesting gene carriers 125-271 or have a deletion that also af-
Received Mar 27, 1992, and in revised form Apr 7. Accepted for publication Apr 8, 1992. Address correspondence to Dr Rowland, neurologic^ Institute, Columbia-Presbyterian Medical Center, 710 West 168th St. New York, NY 10032-2603.
Copyright 0 1992 by the American Neurological Association 207
Positional Mapping of Neurological Diseasej 1. Charcot-Marie-Tooth, Type IB (118200) (lq2 1.2-q23) Myoglobinuria, phosphofructokinase deficiency (232800) (lcenq32) 'Nemaline myopathy, autosomal dominant (161800) ( 1 41-q23) 2. Hypobetalipoproteinemia (107730) ( 2 ~ 2 4 ~j 2 3 Abetalipoproteinemia, Bassen-Kornzweig syndrome (200100) ( 2 ~ 2 4 ) Myopathy; desmin deficiency (?) (125660) ( 2 ~ 3 5 ) Hyperammonemia (carbamyl phosphate synthetase) (237 300) (2p) 3. GM1 galactosidosis (beta-galactosidase) (230500) ( 3 ~ 1-~14.2) 2 Morquio syndrome B, mucopolysaccharidosis IVB (230500) (3p2 1-p14.2) (beta-galactosidase) Proprionic acidemia B (proprionyl CoA-carboxylase) (232050) (3q13.3-q22) Postanesthetic apnea (succinylcholine sensitivity) (177400) (3q21-q26) *Von Hippel-Lindau disease (193300) (3p25-p24) 4. Huntington disease (143100) ( 4 ~ 1 6 . 3 ) Aspartylglucosaminuria (208400) (492 1-qter) Phenylketonuria, lack of dihydropteridine reductase (261630) (4q16.l-pl5.1) Wolf-Hirschorn syndrome, with mental retardation (194190) ( 4 ~ 1 6 . 3 ) *Facioscapulohumeral muscular dystrophy ( 158900) (4935) 5. Sandhoff disease, hexosaminidase deficiency (268800) (5q13) Tay-Sachs disease, AB variant (lack of GM2 activator protein) (272750) (5q) "Infantile spinal muscular atrophy (WerdnigHoffmann) (253300) (5ql1.2) *Juvenile spinal muscular atrophy (KugelbergWelander) (253400) (5q11.2) 6. Spinocerebellar ataxia; olivopontocerebellar atrophy I. (164400) ( 6 ~1.3-p2 2 1.2) Narcolepsy (161400) ( 6 ~ 2 1 . 3 ) "Juvenile myoclonic epilepsy (254770) (6p2 1) 7. Arginosuccinic aciduria (207900) (7cen-q11.2) Myoglobinuria, phosphoglycerace mutase deficiency (261670) ( 7 ~ 1 3 - p l 2 ) 'Zellweger syndrome (214100) (7ql1.23) 8. Renal tubular acidosis, with periodic paralysis, optic
atrophy, or calcification of basal ganglia (259730) (8q32) (carbonic anhydrase deficiency 11) ) 9. Galactosemia (230400) ( 9 ~ 1 3(galactose-1-phosphate urid yltransferase) Citrullinemia (2 15700) (9q34) 'Friedreich ataxia (229390) (9q13-9q21) 'Tuberous sclerosis (191100) (9q33-9q34) Torsion dystonia, Ashkenazi, dominant (128100) (9~32-9q34) *Amyloidosis V with cranial neuropathy and corneal lattice dystrophy (105 120) (9q32-q34) (gelosin) *Hypomelanosis of Ito (146150) (9q33-qter)
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Positional Mapping of Neurological Diseases (continued) 10. Ornithemia with gyrate atrophy of choroid and retina, mental retardation (258870) (10q26) Metachromatic leucodystrophy; lack of spingolipid activator protein (249900) (10q2 1-q22) "Glioblastoma multiforme (137800) (lop 12q23.2) 11. Myoglobinuria, lactate dehydrogenase deficiency (150000) (1l p 15.4) Myoglobinuria, phosphorylase deficiency (McArdle disease) (232600) ( l l q l 3 ) Acute intermittent porphyria (176000) (1lq23.2-qter) (porphobilinogen deaminase) Pyruvate carboxylase deficiency (Leigh) (266 150) (1Iq) WAGR syndrome (Wilms tumor, aniridia, genitalia abnormality, mental retardation) (194070) (1lp13) 'Tuberous sclerosis (191090) ( l l q 2 3 ) "Ataxia telangiectasia (208900) (1lq22-q23) "Cerebellar ataxia (213200) (llq14-q21) 12. Phenylketonuria (phenylalanine hydroxylase) (261600) (12q-24.1) 13. Wilson disease (277900) ( 1 3 ~ 1 4 ) Propionicacidemia, type A (232000) (13) 14. 'Porphyria variegata (176200) (14q32.1) (protoporphyrogen oxidase) 15. Prader-Willi syndrome (176270) (15ql1) *Angelmann syndrome (234400) ( l 5 q l 1 ) Tay-Sachs disease, GM2 gangliosidosis (272800) (15q22-q25.1) (hexosaminidase A) Dyslexia-1 (127700) (15q 1 1) *Limb-girdle muscular dystrophy (253600) (15q) 16. Tyrosinemia (276600) (16q22.1q22.3) Norum disease (245900) (16q.22.1) Hemoglobin H, with mental retardation (14 1750) (16pter-p 13.5) *Juvenile lipofuscinosis (Batten disease) (1 16820) (16q24-925) 17. Alpha-glucosidase deficiency (Pompe) (232300) (17q23) "Charcot-Marie-Tooth, Type IA (118229) (17~13.1) "Neurofibromatosis 1, peripheral, Von Recklinghausen (162200) (17q11.2) 'Hyperkalemic periodic paralysis (170500) (17q 13.1q13.3) (sodium channel alpha subunit) *Paramyotonia congenita (168300) ( 17ql3.l-q13.5) (247200) 'Lissencephaly (Miller-Dieker) (247200) ( 1 7 ~ 1 3 . 3 ) *&abbe disease (245200) (17) (galactocerebrosidase) 18. Familial amyloidotic polyneuropathy (176300) (18q 11.2q12.1) (transthyretin) Tourette syndrome (137580) (18q22.1) 19. Myotonic muscular dystrophy (160900) (19~13.3) Mannosidosis (248500) (19~13.2-q 12) Susceptibility to poliomyelitis (17 3850) (19q 12-q 13.2) 'Familial Altheimer disease, late onset (104300) ( 19) 'Malignant hyperthermia (ryanodine receptor) (145600) (19 s 12-q13.2)
Positional Mapping of Neurological Diseases (continued)
Positional Mapping of Neurological Diseases (continued)
'Central core disease (117000) (19q13.1) 'Maple syrup disease (248600) (19~13.1-q13.2) 20. Galactosialidosis (256540) (20) "Familial benign neonatal convulsions (12 1200) (20) *Familial prion diseases; Creutzfeldt-Jakob ( 123400); Gerstmann-Scheinker-Strauss(137400) (20pter-p 12) "Cerebral amyloid angiopathy (105 150) (20) 21. Homocystinuria (236200) (21q22.3) Familial Alzheimer disease, early onset (104300) (21qll.2-2 lq21) *Familial amyotrophic lateral sclerosis (105400) (21q.22) 'Progressive myoclonus epilepsy (2lq22) 22. Familial acoustic neuroma, neurofibromatosis-2 (10 1000) (22q 11.2 I-q 13.1) Metachromatic leucodystrophy (250100) (22q 13.31qter) Familial meningioma (156100) (22q12.3-qter) Succinylpurinemic autism (103050) (22) (adenylsuccinase) Hurler-Scheie syndrome (2 52 80) X
*Mental retardation, nonspecific I (309530) (Xp22) "Mental retardation, nonspecific I (309540) (Xqll-ql2) 'Omithine transcarbamylase deficiency (3 11250) (Xp21.1) "Myoglobinuria, phosphoglycerate kinase deficiency (311800) (Xq13) Glycerol kinase deficiency and myopathy (307030) (Xp2 1.1) Duchenne muscular dystrophy ( 3 10200) (Xp2 1.2) (dystrophin) Becker muscular dystrophy (310100) (Xp21.2) (dystrophin) 'McLeod syndrome myopathy (3 14850) (Xp2 1.2p2 1.1) (not dystrophin) Menkes syndrome (305370) (Xp11.4-pll.23) Norrie syndrome, mental retardation (310600) (Xpl I .4) Charcot-Marie-Tooth, X-linked (302800) (Xql3) Pelizaeus-Merzbacher disease (312080) (Xq22) 'Kallmann syndrome (308700) (Xp22.3) Lowe oculocerebral disease (309000) (Xq25) Lesch-Nyhan disease, HPRT deficiency (308000) (Xq26) Hunter disease, mucopolysaccharidosis I1 (3099000) (Xq27.3) Fragile X, mental retardation, Martin-Bell syndrome (309550) (Xq2 7.3) Adrenoleucodystrophy (300100) (Xq28) Emery-Dreifuss muscular dystrophy (3 10300) (Xq28) Kennedy disease, spinobulbar muscular atrophy (30000) (Xq21.3-qI2) (androgen receptor) Sensorimotor peripheral neuropathy, Charcot-MarieTooth X, (302800) (Xq13) Spastic paraplegia (300000) (Xq28) *Myotubular (centronuclear) myopathy (310400) (Xq.28) "Hydrocephalus (30700) (Xq28)
Mitochondrial DNA? "Kearns-Sayre syndrome ( I65 100) (deletion) 'Progressive external ophthalmoplegia ( 165000) (deletion) "Leber hereditary optic atrophy (mutation) (308900) "MERRF (myoclonic epilepsy, ragged red fibers) (mutation) * M E U S (mitochondrial encephalomyopathy, lactic acidosis, stroke) (mutation) 'MNGIE (mitochondrial neuromyopath y, gastrointestinal, encephalopathy) 'NARP (neuropathy, ataxia, retinitis pigmentosa) (mutation) "Cytochrome c oxidase deficiency "Leigh syndrome (266150) "Benign infantile myopathy 'Fatal infantile myopathy *Depletion of mtDNA "Added since 1989 +Except for those marked, the mitochondria1 disorders do not have McKusick numbers in the 10th edition of Mendelian Znheritunce in Man 17). Presentation is by chromosome number, name of disease, McKLusick number (7), gene location, and gene product.
fects genes for glycerol kinase or congenital adrenal insufficiency [6, 28). Mitochondrial diseases provide another example. For years they were debated by lumpers and splitters, mostly in arguments about Kearns-Sayre syndrome (KSS). Is it a unique syndrome? Is it sporadic or heritable? The arguments began to abate in 1988, when (deletions of mitochondrial DNA (mtDNA) were found in most cases of KSS C29-31). Then point mutations (not deletions) were found in other syndromes (mitochondrial encephalomyopathy with lactic acidosis, and stroke, MELAS; and myoclonus epilepsy and ragged red fibers, MERRF), buttressing the splitters, but the clinical manifestations of all three mutations are inconsistent [32, 331. We are not quite ready for diagnosis of mitochondrial diseases by affected gene. Other examples abound. Clinically homogeneous Charcot-Marie-Tooth (CMT) syndromes are genetically heterogeneous; families map to chromosomes 1, 17, X, or elsewhere E343. Clinically indistinguishable families with Alzheimer's disease map to chromosomes 19,21 or somewhere else 135, 36). Despite overlapping clinical features, hyperkalemic and hypokalemic periodic paralysis are not allelic [37). Knowing the gene product affects diagnosis in sometimes unexpected ways. For instance, the distinction between juvenile SMA and limb-girdle rnuscu1a.r dystrophy depended largely on electromyography (EMG). Sometimes, however, EMG findings are ambiguous or erroneous; dystrophin analysis shows that some cases of presumed SMA are really Becker dystrophy 138, 391.
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tions, or by dystrophin histochemistry on a muscle biopsy 126, 27). Presymptomatic diagnosis is available for some autosomal dominant diseases, including familial amyloidotic polyneuropathy [4 1) and Huntington’s disease (HD) [42J Once a family has been identified, DNA analysis can determine whether (but not when) an asymptomatic child or young adult is destined to have the disease. Because there is no effective therapy for either disease, ethical problems have emerged 142,431. Neonatal screening for DMD is being reconsidered. A decade ago, a blot test for CK was recommended for all newborns. However, the lack of specificity of high CK values and the lack of effective therapy weighed against the proposal. Now, DNA analysis can demonstrate a deletion in most affected boys. Neonatal identification might prevent the appearance of a second case in a family. Genetic counseling has already led to a sharp decrease in the incidence of DMD.
0
Z
:I 1978 80
82
84 86
88
1990
Year Cumulative annual number of reports on the diagnosis ofgenetic disease using recombinant DNA. Used with permission from (5)‘
Genetic Counseling Is More Reliable Prenatal diagnosis is no longer restricted to assaying the enzyme affected in an autosomal recessive disease; deletions or mutations can be detected by DNA analysis of amniocytes. Even when that is not possible, family haplotype analysis is often feasible. If the protein is not expressed in amniocytes, it may be possible to induce the gene product; for instance, insertion and expression of a gene called “ M y o D induces normal amniocytes to produce dystrophin, which can be tested for diagnosis r40). Carrier detection has improved for diseases in which the gene product was not known. For instance, DMD carriers were identified by calf hypertrophy or high creatine kinase (CK) values, neither method satisfactory. Now, carriers can be identified by DNA dele210 Annals of Neurology Vol 32
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Genetic Concepts New, non-mendelian forms of human inheritance have been identified: maternal inheritance of mitochondrial diseases C29-31) and the prion diseases, which are both heritable and transmissible {44}. Identification of the gene for prion protein extended the clinical concept from dementia to familial insomnia C45). As for traditional mendelian inheritance, presumptions have been tested. For instance, dominant diseases are expressed in the hemizygote. In theory, clinical manifestations should be worse in a homozygous person and this is true for some diseases, including CMT [46). However, in HD, DNA analysis ascertained the homozygosity of affected individuals who had two affected parents, and the disease was not more severe than usual C42); a double dose of the gene was not more harmful than a single dose. The mutant gene may produce a harmful product (still unidentified) in contrast to the view that hereditary diseases arise from functional lack of a normal gene product. Genetic heterogeneity of clinical syndromes can be determined. Although CMT is heterogeneous, all HD seems to be the same, worldwide; all families studied have mapped to the same location on chromosome 4 1471. SMA may also be homogeneous but about 9% of juvenile-onset families are not linked to 5q, implying either misdiagnosis or locus heterogeneity 1151. Older classifications, based solely on clinical criteria, are being chipped away. Limb-girdle muscular dystrophy is one example. It was a necessary classification in 1954 [48). Then, some cases proved to be neurogenic (Kugelberg-Welander); others were metabolic myopathies due to identifiable enzyme or mitochondrial abnor>malities.Now, DNA analysis indicates that some carry the dystrophin mutation 126).
New Genetic Principles DNA analysis of human diseases has uncovered several new genetic principles. 1. Imprinting is the term for the asymmetrical impact of one parental chromosome of an autosomal pair 149). This effect is seen most dramatically in the Prader-Willi syndrome, a form of mental retardation with delayed motor development and later obesity. This condition, mapped to 15qll-12, appears if the patient inherits a mutated maternal gene. However, if the paternal chromosome bears the mutant gene, an entirely different condition emerges as the Angelmann syndrome of malformations and hyperactivity 149, 501. The gene products may differ; one is probably a GABA-receptor T50J. Another possible manifestation of imprinting is seen in HD. The rigid juvenile form is seen if the maternal gene is transmitted; later-onset chorea results if the paternal gene is transmitted [5 1). Imprinting has also been invoked in congenital myotonic dystrophy, which appears only if the mother is the affected parent, but Koch and associates [52) concluded that the congenital form is more likely to occur if the mother is severely affected, which they consider inconsistent with imprinting. 2. The success of positional chromosome mapping has validated the concept that gene products can be uncovered by linkage analysis. Because the gene product is not known at the start of the process, this has been called “reverse genetics,” in contrast to “forward genetics,” in which prior knowledge of the gene product (for instance, hemoglobin or an enzyme) made it easier to identify the complementary DNA (cDNA). 3. Previously, knowing the gene product before the gene was mapped was a feature of recessive diseases. However, mapping the m y loid precursor protein (APP) made it a candidate gene product for familial Alzheimer’s disease 135, 361. After a period of uncertainty, APP mutations were found in young-onset families 153, 541 and insertion of the mutated gene into transgenic mice was said to induce overexpression and deposition of amyloid in brain 155-581 but these claims are now under a cloud of retractions [59]. Aberrant processing of APP may lead to secretion of amyloidogenic fragments 160J. Candidate gene products were also important in mapping malignant hyperthermia (ryanodine receptor) 161-65 1, hyperkalemic periodic paralysis (sodium channel) 1661, X-linked bulbospinal muscular atrophy (Kennedy syndrome) (androgen receptor) [671, and Pelizaeus-Merzbacher disease (proteolipid protein) [681. 4. The two-step theory ofoncogenesis was bolstered by studies of neurofibromatosis 1691. 5. A new theory of twinning emerged from Duchenne studies 1701. 6. Labile genes. As disease genes have been mapped and cloned, new forms of mutation have been identi-
fied. For instance, in CMT there is duplication of DIVA on chromosome 17p [461; a CAG repeat is amplified in the Kennedy syndrome 1671; and CGG repeats are amplified in the fragile X syndrome [71,721. Myotonic dystrophy shows amplification of a CTG repeat 173751. The repeats seem unstable and liable to spontaneous mutation. These novel mutations are not merely curiosities; they are being used for diagnosis and counseling 1761. The DNA patterns in the fragile X syndrome have affected the way we think about “anticipation,”the tendency for dominant diseases to appear at an earlier age in succeeding generations. In this X-linked mental retardation, 30% of carrier women are mildly affected and 20% of men with the mutant gene are clinically normal. These normal transmitting males (NTM) may pass the mutation to daughters who are also asymptomatic, but their children, grandsons of the NTM, are often affected. Brothers of an NTM have a 9% risk of mental retardation but 50% of grandsons are affected; this is the “Sherman paradox,” which has been resolved by molecular genetics 17 11. Normal individuals have 6-54 CGG repeats within the fragile mental retardation-1 (FMR-1) gene. In asymptomatic female carriers, there are 52 to 200 repeats, thought to be “premutations” because they are unstable and tend to mutate. Small inserts of 100 to 600 bp are found in NTMs; large inserts of 600 to 4,000 bp are found in affected males. During oogenesis, there is “amplification of the premutation”, the number of inserts increases in carrier women. ‘The risk of expansion during oogenesis to the full mutation associated with mental retardation increases with the number of repeats, and this variation in risk accounts for the Sherman paradox” [71]. The actual mutation in fragile X affects an exon of the gene, which is methylated in affected individuals, resulting in loss of expression of the mRNA for FMR-1. In myotonic muscular dystrophy 173-761, amplification of the repeat also increases in succeeding generations and, in siblings, the number of repeats parallels clinical severity, explaining how anticipation is related to “potentiation”; symptoms begin earlier and are more severe in succeeding generations. Pathogenesis Identifying the gene product has ended debate about theories of pathogenesis; for instance, that DMD is neurogenic. The discovery of dystrophin, a protein never known before DNA was analyzed, and its localization in the muscle surface membranes reinforced the theory that the primary disorder affects the sarcolemma [77]. Similarly, malignant hyperthermia may be explained by altered function of the ryanodine receptor, a key protein in the sarcoplasmic reticulum and involved in
New Horizons: Rowland: Molecular Genetics in Neurology 21 1
regulation of cytoplasmic levels of calcium ion [611. How altered function of the androgen receptor accounts for a form of SMA is more mysterious. In myotonic dystrophy, the amplified CTG repeat is found at the 3’ end of a protein kinase [78), an enzyme implicated 20 years ago 179, SO?. In attempts to understand how the mutations induce the clinical disorders, trangenic mice have been used to study Alzheimer’s disease 155-58, 811, prion diseases [82), familial amyloidotic polyneuropathy [83, 841, and Lesch-Nyhan syndrome.
Therapy Although therapy is a central goal of medical research, molecular genetics has yet to deliver an effective treatment for any inherited disease. Perhaps there will be none; even though there has been complete knowledge of the affected gene product in sickle cell disease, treatment is still symptomatic, not curative. Nevertheless there is a mood of hope and expectation. The most dramatic therapeutic attempts involve DMD, including trials of gene replacement by myoblast implantation {SS] or by intramuscular injection of the gene in a vector [SC). These concepts are in their infancy and not yet practical. However, once we understand the gene product, it becomes possible to consider rational therapy. Problems Remain Therapy is not the only challenge. We still do not know the central functions of dystrophin, beta-amyloid, or several other gene products. In some conditions it is difficult to understand how lack of the gene product causes symptoms; a striking example is the relation of the androgen receptor to dysfunction and loss of motor neurons. Another manifestation of ignorance is the divergent clinical expression of mutations at the same site, such as malignant hyperthermia and central core disease at 15q23, or the several clinical syndromes that result from mutations of mtDNA. Other clinical problems bear upon function of the gene product. Here are some examples: how to relate the D N A mutation to a specific enzyme or structural protein and thence to clinical manifestations in any disease, especially mitochondrial disease; how mild and severe cases arise in the same family; or why there is such variability of expression in autosomal dominant and mitochondrial diseases; how heteroplasmy of mitochondrial DNA, the mixture of normal and mutated mitochondrial genomes in the same cell, leads to normal or abnormal function. We do not understand why there is genetic heterogeneity, how mutations at different loci cause virtually identical clinical syndromes, as in CMT. Or how clinical syndromes as similar as the myopathies of McLeod syndrome and DMD arise from 212 Annals of Neurology Vol 32
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two different mutations at nearby but not identical loci or gene products. Until we do understand the pathogenesis of clinical symptoms in molecular terms, rational therapy may be elusive. The final incongruity may be the inability of any society to pay for the research or the applications; the tools are at hand.
Looking to the Future As Yogi Berra said, “It is difficult to predict, especially about the future.” We can be certain that more genes will be mapped and more gene products identified as the Human Genome Project progresses. The many different genetic forms of mental retardation will be unravelled. Diseases of interest will not be restricted to rare monogenic disorders. Alzheimer’s disease, Parkinson’s disease, and other public health problems are already under scrutiny. Autoimmune diseases, including multiple sclerosis and myasthenia gravis, are being evaluated for disease-susceptibility genes. Alcoholism, schizophrenia, and bipolar disease are also open to DNA research. The techniques of recombinant D NA are being applied in other areas. In viral infections, DNA analysis is already vital for diagnosis, as well as preparation of vaccines and antiviral drugs. Analysis of neuronal receptors and channel proteins leads to better drugs for neurological disorders. Growth factors, identified and produced by recombinant DNA methods, are being tested in human diseases, some by brain implantation @7]. Neurologically important proteins can be produced in pure form and in quantity sufficient to make antibodies for analysis of heritable diseases (such as dystrophin or prion proteins) and perhaps for therapy. For autoimmune disorders such as myasthenia and multiple sclerosis, immunogenic peptides are identified and produced for similar purposes 1881. Two challenges are foremost: How does the molecular abnormality lead to symptoms of a disease? Can we devise effective therapy? With accelerating research progress 1891, the next decennial review may answer these questions. Supported by Center grants from the National Institute of Neurological Diseases and Stroke and the Muscular Dystrophy Association. The author’s views have been tempered by active associates: S. Bressman, S. DiMauro, T. Conrad Gilliam, W. G. Johnson, T. Nygaard, E. Schon, K. Wilhelmsen. The errors are my own.
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heterogeneity between Huntington disease and G8. Genomics 1989;5:304-308 48. Walton JN. Nattrass FJ. On the classification, natural history and treatment of the myopathies. Brain 1954;77:169-231 49. Hall JG. Genomic imprinting: review and relevance to human diseases. Am J Hum Genet 1990;46:857-873 50. Saitoh S, KubotaT, OhtaT, et al. Familial Angelmann syndrome caused by an imprinted submicroscopic deletion encompassing the GABA-A receptor beta-3 subunit gene. Lancet 1992 (in press) S 1. Conneally PM. Aging genes and diseases of the aging nervous system. In Rowland LP, ed. Amyotrophic lateral sclerosis and other motor neuron diseases. Raven Press, New York, 1991. (Adv Neural 1991;56:233-238.) 52. Koch MC, Grimm T, Harley HG, Harper PS. Genetic risks for children of women with myotonic dystrophy. Am J Hum Genet 1991;48:1084- 1091 53. Chartier-Hardin MC, Crawford F, Houlden H , et al. Early-onset Alzheimer’s disease caused by mutations at codon 717 of the beta-amyloid precursor protein gene. Nature 1991;353: 844-846 54. Murrell J, Farlow M, Ghetti B, Benson MD. A mutation in the amyloid precursor protein associated with hereditary Alzheimer’s disease. Science 1991;254:97-99 55. Quon D , Wang Y, Catalan0 R, et al. Formation of beta-amyloid protein deposits in brains of transgenic mice. Nature 1991; 352~239-241 56. Wirak DO, Bayney R, Ramabhadran TV, et al. Deposits of amyloid beta protein in the central nervous system of transgenic mice. Science 1991;253:323-325 57. Kawabata S, Higins GA, Gordon JW. Amyloid plaques, neurofibrillary tangles and neuronal loss in brains of transgenic mice over-expressing a C-terminal fragment of human amyloid precursor protein. Nature 1991;354:4 76-4 78 58. Sandhu FA, Salim M, Zain SB. Expression of the human betaamyloid protein of Alzheimer’s disease specifically in the brains of transgenic mice. J Biol Chem 1991;266:21331-21334 59. Marx J. Major setback for Alzheimer’s models. Science 1992; 255:1200-1202 60. Estus S, Golde TE, Kunishita T, et al. Potentially amyloidogenic, carboxyl-terminal derivatives of the amyloid precursor protein. Science 1992;255:726-728 61. MacLennan DH, Duff C, Corzato F, et al. The ryanodine receptor gene: a candidate gene for the predisposition to malignant hyperthermia. Nature 1990;343:559-56 1 62. Healy JMS, Heffron JJA, Lhane M, et al. Diagnosis of susceptibility to malignant hyperthermia with flanking DNA markers. BMJ 1991;303:1225-1228 63. Kausch K, Grimm T, Janka M, et al. Evidence for linkage of the central core disease locus to chromosome 19q. J Neural Sci 1990;98:549 64. Levitt RC, Nouri N , Jedlicka AE, et al. Evidence for genetic heterogeneity in malignant hyperthermia susceptibility. Genomics 1991;11:543-547 65. MacKenzie AE, Allen G, Lahey D, et al. A comparison of caffeine halothane muscle contracture test with the molecular genetic diagnosis of malignant hyperthermia. Anesthesiology 199l ; 74:4-8 66. Rojas CV, Wang J, Schwartz LS, et al. A met-to-val mutation in the skeletal muscle N a + channel alpha subunit in hyperkalemic periodic paralysis. Nature 1991;354:387-389 67. La Spada AR, Wilson EM, Lubahn DB, et al. Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature 1991;352:77-79 68. Raskind WH, Williams CA, Hudson LD, Bird TD. Complete
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deletion of the proteolipid protein gene in a family with Xlinked Pelizaeus-Merzbacher disease. Am J Hum Genet 1991; 49: 1355- 1360 69. Wallace MR, Marchuk DA, Andersen LB, et al. Type 1 neurofibromatosis gene; identification of a large transcript disrupted in three NF1 patients. Science 1991;249:181-186 70. Lupsl JR, Garcia CA, Zoghbi HY, et al. Discordance of muscular dystrophy in monozygotic female twins: evidence supporting asymmetric splitting of the inner cell mass in a manifesting carrier of Duchenne dystrophy. Am J Hum Genet 1991;40: 354-364 71. Fu YH, Kuhl DPA, Pizzuli A, et al. Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell 1991;67:1057-1058 72. Kremer E, Pritchard M, Lynch M, et al. Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG)n. Science 1991;252: I7 11- 17 14 73. Harley HG, Brook JD, Rundle SA, et al. Expansion of an unstable DNA region and phenotypic variation in myotonic dysrrophy. Nature 1992;355:545-546 74. Buxton J, Shelbourne P, Davies J. et al. Detection ofan unstable fragment of DNA specific to individuals with myotonic dystrophy. Nature 1992;355:547-548 75. Asianidis C, Jansen G , Amemiya C, et al. Cloning of the essential myotonic dystrophy region and mapping of the putarive defect. Nature 1992;355:548-55 1 76. Rousseau F, Heitz D, Biancalana V, et al. Direct diagnosis by DNA analysis of the fragile X syndrome of mental retardation. N Engl J Med 1991;325:1673-1681 77. Rowland LP. Biochemistry of muscle membranes in Duchenne muscular dystrophy. Muscle Nerve 1980;3:3-20 78. Brook JD, McCurrach ME, Harley HG, et al. Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3’end of a transcript encoding a protein kinase family member. Cell 1992;69:799-808 79. Roses AD, Appel SH. Protein kinase activity in erythrocyte ghosts of patients with myotonic muscular dystrophy. Proc Nat Acad Sci USA 1973;70:1855-1859 80. Roses AD, Appel SH. Muscle membrane protein kinase in myotonic muscular dystrophy. Nature 1974;250:245-247 81. Sofroniew MV, Staley K. Transgenic modelling of neurodegenerative events gathers momentum. TINS 1991;14:513-514 82. Hsiao KK,Scott M, Foster D, et al. Spontaneous neurodegeneration in transgenic mice with mutant prion protein of Gerstmann-Straussler syndrome. Science 1990;250:1587-1 590 83. Shimada K, Maeda S, Murakami T, et al. Transgenic mouse model of familial amyloidotic polyneuropathy. Mol Biol Med 1989;6:333-343 84. Yi S, Takahashi K, Naito M, et al. Systemic amyloidosis in transgenic mice carrying the human mutant transthyretin (Met30) gene. Pathologic similarity to human familial amyloidotic polyneuropathy, type 1. Am J Pathol 1991;138: 403-412 85. Partridge TA. Myoblast transfer: a possible therapy for inherited myopathies? Muscle Nerve 1991;14:197-212 86. Ascadi G, Dickson G, Love DR, et al. Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs. Nature 1991;352:815 87. Gage FH, Fisher LJ. Intracerebral grafting: a tool for the neurobiologist. Cell 1991;6:1-12 88. Steinman L, Oksenberg JR, Bernard CCA. Association of susceptibility to multiple sclerosis with TCR genes. Immunol Today 1992;13-15 89. Harper PS. The Human Genome Project and medical genetics. J Med Genet 1992;29:1-2
The First Decade of Molecular Genetics in Neurology: Changmg Clinical Thought and Practice Lewis P. Rowland, MD
Molecular genetics has had a powerful impact on clinical neurology. Definitions of disease are changing from clinical criteria to DNA analysis, resolving questions about the nature of clinically similar but not identical diseases. Genetic counseling is more reliable. Concepts of mendelian inheritance are being tested and new forms of mutation have been discovered to explain anticipation. Nonmendelian forms of inheritance have emerged; concepts of pathogenesis are on a more secure footing; and novel treatments are being explored. Rowland LP. The first decade of molecular genetics in neurology: changing clinical thought and practice. Ann Neurol 1992;32:207-2 14 Molecular genetics entered clinical neurology in 1983 when Gusella and colleagues 111 mapped the gene for Huntington’s disease to chromosome 4. That year saw the first use of “molecular genetics” in the title of an article 121 and the first research paper on the subject 131 in a neurology journal. My task is to summarize the subsequent clinical impact on our field. Clinical neurology has made major contributions to genetics. Reciprocally, molecular genetics has radically altered concepts and practice of clinical neurology.
The Accelerating Pace of Change There is a self-propelling force in molecular genetics. The process of assigning a chromosomal location for a disease gene is called positional mapping, which depends on having DNA probes on each chromosome for linkage analysis. The closer the marker is to the disease gene, the more likely they are to be inherited together. Collins 141 compared the activity to “searching for a needle in a haystack in the dark wearing thick gloves.” Each linkage, however, makes the next one a bit easier. The more probes available, the faster the mapping of any disease. The X-chromosome has been most thoroughly saturated because many X-linked diseases had been identified by pedigree analysis before 1983. Autosomal diseases are now accessible because the number of probes has increased exponentially 151 (Fig). In 1988, 61 diseases of neurological interest had been mapped C6l; now, the number is 129 (Table) 171.
From the Department of Neurology, Neurological Institute, and the H. Houston Merritt Clinical Research Center for Muscular Dystrophy and Related Diseases, columbia-presbYterian New York, NY.
Changing Definitions from Clinical to Molecular Criteria Some diseases are still defined by clinical criteria, but Duchenne muscular dystrophy (DMD) indicates how DNA analysis has changed definitions of related myopathies. Duchenne and Becker dystrophies were considered similar but distinct diseases. Now we know they are allelic, both due to alterations of the same gene product, dystrophin 181. This landmark achievement proved that positional cloning could lead to identification of a previously unknown gene product. Similarly, there had been debates about whether hyperkalemic periodic paralysis and paramyotonia are variations of the same genetic disorder or separate diseases 191. Now, both have been mapped to the same site, proving their allelic identity 110-121. Likewise, infantile spinal muscular atrophy (SMA) (WerdnigHoffmann) and juvenile SMA (Kugelberg-Welander) seem to be allelic 113-151. Proof of allelic identity is not restricted to these clinically similar disorders. Even clinically disparate syndromes may be manifestations of mutations of the same gene product 16, 16, 171. For instance, the clinical manifestations of “Xp2 1 myopathy” or “dystrophinopathy” have extended from the typical limb weakness of young boys to X-linked cramps 118, 191, myoglobinuria 1201, quadriceps myopathy 12 11, or congenital myopathy E22-241. Mild myopathies appear in girls or women who prove to be manifesting gene carriers 125-271 or have a deletion that also af-
Received Mar 27, 1992, and in revised form Apr 7. Accepted for publication Apr 8, 1992. Address correspondence to Dr Rowland, neurologic^ Institute, Columbia-Presbyterian Medical Center, 710 West 168th St. New York, NY 10032-2603.
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Positional Mapping of Neurological Diseasej 1. Charcot-Marie-Tooth, Type IB (118200) (lq2 1.2-q23) Myoglobinuria, phosphofructokinase deficiency (232800) (lcenq32) 'Nemaline myopathy, autosomal dominant (161800) ( 1 41-q23) 2. Hypobetalipoproteinemia (107730) ( 2 ~ 2 4 ~j 2 3 Abetalipoproteinemia, Bassen-Kornzweig syndrome (200100) ( 2 ~ 2 4 ) Myopathy; desmin deficiency (?) (125660) ( 2 ~ 3 5 ) Hyperammonemia (carbamyl phosphate synthetase) (237 300) (2p) 3. GM1 galactosidosis (beta-galactosidase) (230500) ( 3 ~ 1-~14.2) 2 Morquio syndrome B, mucopolysaccharidosis IVB (230500) (3p2 1-p14.2) (beta-galactosidase) Proprionic acidemia B (proprionyl CoA-carboxylase) (232050) (3q13.3-q22) Postanesthetic apnea (succinylcholine sensitivity) (177400) (3q21-q26) *Von Hippel-Lindau disease (193300) (3p25-p24) 4. Huntington disease (143100) ( 4 ~ 1 6 . 3 ) Aspartylglucosaminuria (208400) (492 1-qter) Phenylketonuria, lack of dihydropteridine reductase (261630) (4q16.l-pl5.1) Wolf-Hirschorn syndrome, with mental retardation (194190) ( 4 ~ 1 6 . 3 ) *Facioscapulohumeral muscular dystrophy ( 158900) (4935) 5. Sandhoff disease, hexosaminidase deficiency (268800) (5q13) Tay-Sachs disease, AB variant (lack of GM2 activator protein) (272750) (5q) "Infantile spinal muscular atrophy (WerdnigHoffmann) (253300) (5ql1.2) *Juvenile spinal muscular atrophy (KugelbergWelander) (253400) (5q11.2) 6. Spinocerebellar ataxia; olivopontocerebellar atrophy I. (164400) ( 6 ~1.3-p2 2 1.2) Narcolepsy (161400) ( 6 ~ 2 1 . 3 ) "Juvenile myoclonic epilepsy (254770) (6p2 1) 7. Arginosuccinic aciduria (207900) (7cen-q11.2) Myoglobinuria, phosphoglycerace mutase deficiency (261670) ( 7 ~ 1 3 - p l 2 ) 'Zellweger syndrome (214100) (7ql1.23) 8. Renal tubular acidosis, with periodic paralysis, optic
atrophy, or calcification of basal ganglia (259730) (8q32) (carbonic anhydrase deficiency 11) ) 9. Galactosemia (230400) ( 9 ~ 1 3(galactose-1-phosphate urid yltransferase) Citrullinemia (2 15700) (9q34) 'Friedreich ataxia (229390) (9q13-9q21) 'Tuberous sclerosis (191100) (9q33-9q34) Torsion dystonia, Ashkenazi, dominant (128100) (9~32-9q34) *Amyloidosis V with cranial neuropathy and corneal lattice dystrophy (105 120) (9q32-q34) (gelosin) *Hypomelanosis of Ito (146150) (9q33-qter)
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Positional Mapping of Neurological Diseases (continued) 10. Ornithemia with gyrate atrophy of choroid and retina, mental retardation (258870) (10q26) Metachromatic leucodystrophy; lack of spingolipid activator protein (249900) (10q2 1-q22) "Glioblastoma multiforme (137800) (lop 12q23.2) 11. Myoglobinuria, lactate dehydrogenase deficiency (150000) (1l p 15.4) Myoglobinuria, phosphorylase deficiency (McArdle disease) (232600) ( l l q l 3 ) Acute intermittent porphyria (176000) (1lq23.2-qter) (porphobilinogen deaminase) Pyruvate carboxylase deficiency (Leigh) (266 150) (1Iq) WAGR syndrome (Wilms tumor, aniridia, genitalia abnormality, mental retardation) (194070) (1lp13) 'Tuberous sclerosis (191090) ( l l q 2 3 ) "Ataxia telangiectasia (208900) (1lq22-q23) "Cerebellar ataxia (213200) (llq14-q21) 12. Phenylketonuria (phenylalanine hydroxylase) (261600) (12q-24.1) 13. Wilson disease (277900) ( 1 3 ~ 1 4 ) Propionicacidemia, type A (232000) (13) 14. 'Porphyria variegata (176200) (14q32.1) (protoporphyrogen oxidase) 15. Prader-Willi syndrome (176270) (15ql1) *Angelmann syndrome (234400) ( l 5 q l 1 ) Tay-Sachs disease, GM2 gangliosidosis (272800) (15q22-q25.1) (hexosaminidase A) Dyslexia-1 (127700) (15q 1 1) *Limb-girdle muscular dystrophy (253600) (15q) 16. Tyrosinemia (276600) (16q22.1q22.3) Norum disease (245900) (16q.22.1) Hemoglobin H, with mental retardation (14 1750) (16pter-p 13.5) *Juvenile lipofuscinosis (Batten disease) (1 16820) (16q24-925) 17. Alpha-glucosidase deficiency (Pompe) (232300) (17q23) "Charcot-Marie-Tooth, Type IA (118229) (17~13.1) "Neurofibromatosis 1, peripheral, Von Recklinghausen (162200) (17q11.2) 'Hyperkalemic periodic paralysis (170500) (17q 13.1q13.3) (sodium channel alpha subunit) *Paramyotonia congenita (168300) ( 17ql3.l-q13.5) (247200) 'Lissencephaly (Miller-Dieker) (247200) ( 1 7 ~ 1 3 . 3 ) *&abbe disease (245200) (17) (galactocerebrosidase) 18. Familial amyloidotic polyneuropathy (176300) (18q 11.2q12.1) (transthyretin) Tourette syndrome (137580) (18q22.1) 19. Myotonic muscular dystrophy (160900) (19~13.3) Mannosidosis (248500) (19~13.2-q 12) Susceptibility to poliomyelitis (17 3850) (19q 12-q 13.2) 'Familial Altheimer disease, late onset (104300) ( 19) 'Malignant hyperthermia (ryanodine receptor) (145600) (19 s 12-q13.2)
Positional Mapping of Neurological Diseases (continued)
Positional Mapping of Neurological Diseases (continued)
'Central core disease (117000) (19q13.1) 'Maple syrup disease (248600) (19~13.1-q13.2) 20. Galactosialidosis (256540) (20) "Familial benign neonatal convulsions (12 1200) (20) *Familial prion diseases; Creutzfeldt-Jakob ( 123400); Gerstmann-Scheinker-Strauss(137400) (20pter-p 12) "Cerebral amyloid angiopathy (105 150) (20) 21. Homocystinuria (236200) (21q22.3) Familial Alzheimer disease, early onset (104300) (21qll.2-2 lq21) *Familial amyotrophic lateral sclerosis (105400) (21q.22) 'Progressive myoclonus epilepsy (2lq22) 22. Familial acoustic neuroma, neurofibromatosis-2 (10 1000) (22q 11.2 I-q 13.1) Metachromatic leucodystrophy (250100) (22q 13.31qter) Familial meningioma (156100) (22q12.3-qter) Succinylpurinemic autism (103050) (22) (adenylsuccinase) Hurler-Scheie syndrome (2 52 80) X
*Mental retardation, nonspecific I (309530) (Xp22) "Mental retardation, nonspecific I (309540) (Xqll-ql2) 'Omithine transcarbamylase deficiency (3 11250) (Xp21.1) "Myoglobinuria, phosphoglycerate kinase deficiency (311800) (Xq13) Glycerol kinase deficiency and myopathy (307030) (Xp2 1.1) Duchenne muscular dystrophy ( 3 10200) (Xp2 1.2) (dystrophin) Becker muscular dystrophy (310100) (Xp21.2) (dystrophin) 'McLeod syndrome myopathy (3 14850) (Xp2 1.2p2 1.1) (not dystrophin) Menkes syndrome (305370) (Xp11.4-pll.23) Norrie syndrome, mental retardation (310600) (Xpl I .4) Charcot-Marie-Tooth, X-linked (302800) (Xql3) Pelizaeus-Merzbacher disease (312080) (Xq22) 'Kallmann syndrome (308700) (Xp22.3) Lowe oculocerebral disease (309000) (Xq25) Lesch-Nyhan disease, HPRT deficiency (308000) (Xq26) Hunter disease, mucopolysaccharidosis I1 (3099000) (Xq27.3) Fragile X, mental retardation, Martin-Bell syndrome (309550) (Xq2 7.3) Adrenoleucodystrophy (300100) (Xq28) Emery-Dreifuss muscular dystrophy (3 10300) (Xq28) Kennedy disease, spinobulbar muscular atrophy (30000) (Xq21.3-qI2) (androgen receptor) Sensorimotor peripheral neuropathy, Charcot-MarieTooth X, (302800) (Xq13) Spastic paraplegia (300000) (Xq28) *Myotubular (centronuclear) myopathy (310400) (Xq.28) "Hydrocephalus (30700) (Xq28)
Mitochondrial DNA? "Kearns-Sayre syndrome ( I65 100) (deletion) 'Progressive external ophthalmoplegia ( 165000) (deletion) "Leber hereditary optic atrophy (mutation) (308900) "MERRF (myoclonic epilepsy, ragged red fibers) (mutation) * M E U S (mitochondrial encephalomyopathy, lactic acidosis, stroke) (mutation) 'MNGIE (mitochondrial neuromyopath y, gastrointestinal, encephalopathy) 'NARP (neuropathy, ataxia, retinitis pigmentosa) (mutation) "Cytochrome c oxidase deficiency "Leigh syndrome (266150) "Benign infantile myopathy 'Fatal infantile myopathy *Depletion of mtDNA "Added since 1989 +Except for those marked, the mitochondria1 disorders do not have McKusick numbers in the 10th edition of Mendelian Znheritunce in Man 17). Presentation is by chromosome number, name of disease, McKLusick number (7), gene location, and gene product.
fects genes for glycerol kinase or congenital adrenal insufficiency [6, 28). Mitochondrial diseases provide another example. For years they were debated by lumpers and splitters, mostly in arguments about Kearns-Sayre syndrome (KSS). Is it a unique syndrome? Is it sporadic or heritable? The arguments began to abate in 1988, when (deletions of mitochondrial DNA (mtDNA) were found in most cases of KSS C29-31). Then point mutations (not deletions) were found in other syndromes (mitochondrial encephalomyopathy with lactic acidosis, and stroke, MELAS; and myoclonus epilepsy and ragged red fibers, MERRF), buttressing the splitters, but the clinical manifestations of all three mutations are inconsistent [32, 331. We are not quite ready for diagnosis of mitochondrial diseases by affected gene. Other examples abound. Clinically homogeneous Charcot-Marie-Tooth (CMT) syndromes are genetically heterogeneous; families map to chromosomes 1, 17, X, or elsewhere E343. Clinically indistinguishable families with Alzheimer's disease map to chromosomes 19,21 or somewhere else 135, 36). Despite overlapping clinical features, hyperkalemic and hypokalemic periodic paralysis are not allelic [37). Knowing the gene product affects diagnosis in sometimes unexpected ways. For instance, the distinction between juvenile SMA and limb-girdle rnuscu1a.r dystrophy depended largely on electromyography (EMG). Sometimes, however, EMG findings are ambiguous or erroneous; dystrophin analysis shows that some cases of presumed SMA are really Becker dystrophy 138, 391.
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tions, or by dystrophin histochemistry on a muscle biopsy 126, 27). Presymptomatic diagnosis is available for some autosomal dominant diseases, including familial amyloidotic polyneuropathy [4 1) and Huntington’s disease (HD) [42J Once a family has been identified, DNA analysis can determine whether (but not when) an asymptomatic child or young adult is destined to have the disease. Because there is no effective therapy for either disease, ethical problems have emerged 142,431. Neonatal screening for DMD is being reconsidered. A decade ago, a blot test for CK was recommended for all newborns. However, the lack of specificity of high CK values and the lack of effective therapy weighed against the proposal. Now, DNA analysis can demonstrate a deletion in most affected boys. Neonatal identification might prevent the appearance of a second case in a family. Genetic counseling has already led to a sharp decrease in the incidence of DMD.
0
Z
:I 1978 80
82
84 86
88
1990
Year Cumulative annual number of reports on the diagnosis ofgenetic disease using recombinant DNA. Used with permission from (5)‘
Genetic Counseling Is More Reliable Prenatal diagnosis is no longer restricted to assaying the enzyme affected in an autosomal recessive disease; deletions or mutations can be detected by DNA analysis of amniocytes. Even when that is not possible, family haplotype analysis is often feasible. If the protein is not expressed in amniocytes, it may be possible to induce the gene product; for instance, insertion and expression of a gene called “ M y o D induces normal amniocytes to produce dystrophin, which can be tested for diagnosis r40). Carrier detection has improved for diseases in which the gene product was not known. For instance, DMD carriers were identified by calf hypertrophy or high creatine kinase (CK) values, neither method satisfactory. Now, carriers can be identified by DNA dele210 Annals of Neurology Vol 32
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Genetic Concepts New, non-mendelian forms of human inheritance have been identified: maternal inheritance of mitochondrial diseases C29-31) and the prion diseases, which are both heritable and transmissible {44}. Identification of the gene for prion protein extended the clinical concept from dementia to familial insomnia C45). As for traditional mendelian inheritance, presumptions have been tested. For instance, dominant diseases are expressed in the hemizygote. In theory, clinical manifestations should be worse in a homozygous person and this is true for some diseases, including CMT [46). However, in HD, DNA analysis ascertained the homozygosity of affected individuals who had two affected parents, and the disease was not more severe than usual C42); a double dose of the gene was not more harmful than a single dose. The mutant gene may produce a harmful product (still unidentified) in contrast to the view that hereditary diseases arise from functional lack of a normal gene product. Genetic heterogeneity of clinical syndromes can be determined. Although CMT is heterogeneous, all HD seems to be the same, worldwide; all families studied have mapped to the same location on chromosome 4 1471. SMA may also be homogeneous but about 9% of juvenile-onset families are not linked to 5q, implying either misdiagnosis or locus heterogeneity 1151. Older classifications, based solely on clinical criteria, are being chipped away. Limb-girdle muscular dystrophy is one example. It was a necessary classification in 1954 [48). Then, some cases proved to be neurogenic (Kugelberg-Welander); others were metabolic myopathies due to identifiable enzyme or mitochondrial abnor>malities.Now, DNA analysis indicates that some carry the dystrophin mutation 126).
New Genetic Principles DNA analysis of human diseases has uncovered several new genetic principles. 1. Imprinting is the term for the asymmetrical impact of one parental chromosome of an autosomal pair 149). This effect is seen most dramatically in the Prader-Willi syndrome, a form of mental retardation with delayed motor development and later obesity. This condition, mapped to 15qll-12, appears if the patient inherits a mutated maternal gene. However, if the paternal chromosome bears the mutant gene, an entirely different condition emerges as the Angelmann syndrome of malformations and hyperactivity 149, 501. The gene products may differ; one is probably a GABA-receptor T50J. Another possible manifestation of imprinting is seen in HD. The rigid juvenile form is seen if the maternal gene is transmitted; later-onset chorea results if the paternal gene is transmitted [5 1). Imprinting has also been invoked in congenital myotonic dystrophy, which appears only if the mother is the affected parent, but Koch and associates [52) concluded that the congenital form is more likely to occur if the mother is severely affected, which they consider inconsistent with imprinting. 2. The success of positional chromosome mapping has validated the concept that gene products can be uncovered by linkage analysis. Because the gene product is not known at the start of the process, this has been called “reverse genetics,” in contrast to “forward genetics,” in which prior knowledge of the gene product (for instance, hemoglobin or an enzyme) made it easier to identify the complementary DNA (cDNA). 3. Previously, knowing the gene product before the gene was mapped was a feature of recessive diseases. However, mapping the m y loid precursor protein (APP) made it a candidate gene product for familial Alzheimer’s disease 135, 361. After a period of uncertainty, APP mutations were found in young-onset families 153, 541 and insertion of the mutated gene into transgenic mice was said to induce overexpression and deposition of amyloid in brain 155-581 but these claims are now under a cloud of retractions [59]. Aberrant processing of APP may lead to secretion of amyloidogenic fragments 160J. Candidate gene products were also important in mapping malignant hyperthermia (ryanodine receptor) 161-65 1, hyperkalemic periodic paralysis (sodium channel) 1661, X-linked bulbospinal muscular atrophy (Kennedy syndrome) (androgen receptor) [671, and Pelizaeus-Merzbacher disease (proteolipid protein) [681. 4. The two-step theory ofoncogenesis was bolstered by studies of neurofibromatosis 1691. 5. A new theory of twinning emerged from Duchenne studies 1701. 6. Labile genes. As disease genes have been mapped and cloned, new forms of mutation have been identi-
fied. For instance, in CMT there is duplication of DIVA on chromosome 17p [461; a CAG repeat is amplified in the Kennedy syndrome 1671; and CGG repeats are amplified in the fragile X syndrome [71,721. Myotonic dystrophy shows amplification of a CTG repeat 173751. The repeats seem unstable and liable to spontaneous mutation. These novel mutations are not merely curiosities; they are being used for diagnosis and counseling 1761. The DNA patterns in the fragile X syndrome have affected the way we think about “anticipation,”the tendency for dominant diseases to appear at an earlier age in succeeding generations. In this X-linked mental retardation, 30% of carrier women are mildly affected and 20% of men with the mutant gene are clinically normal. These normal transmitting males (NTM) may pass the mutation to daughters who are also asymptomatic, but their children, grandsons of the NTM, are often affected. Brothers of an NTM have a 9% risk of mental retardation but 50% of grandsons are affected; this is the “Sherman paradox,” which has been resolved by molecular genetics 17 11. Normal individuals have 6-54 CGG repeats within the fragile mental retardation-1 (FMR-1) gene. In asymptomatic female carriers, there are 52 to 200 repeats, thought to be “premutations” because they are unstable and tend to mutate. Small inserts of 100 to 600 bp are found in NTMs; large inserts of 600 to 4,000 bp are found in affected males. During oogenesis, there is “amplification of the premutation”, the number of inserts increases in carrier women. ‘The risk of expansion during oogenesis to the full mutation associated with mental retardation increases with the number of repeats, and this variation in risk accounts for the Sherman paradox” [71]. The actual mutation in fragile X affects an exon of the gene, which is methylated in affected individuals, resulting in loss of expression of the mRNA for FMR-1. In myotonic muscular dystrophy 173-761, amplification of the repeat also increases in succeeding generations and, in siblings, the number of repeats parallels clinical severity, explaining how anticipation is related to “potentiation”; symptoms begin earlier and are more severe in succeeding generations. Pathogenesis Identifying the gene product has ended debate about theories of pathogenesis; for instance, that DMD is neurogenic. The discovery of dystrophin, a protein never known before DNA was analyzed, and its localization in the muscle surface membranes reinforced the theory that the primary disorder affects the sarcolemma [77]. Similarly, malignant hyperthermia may be explained by altered function of the ryanodine receptor, a key protein in the sarcoplasmic reticulum and involved in
New Horizons: Rowland: Molecular Genetics in Neurology 21 1
regulation of cytoplasmic levels of calcium ion [611. How altered function of the androgen receptor accounts for a form of SMA is more mysterious. In myotonic dystrophy, the amplified CTG repeat is found at the 3’ end of a protein kinase [78), an enzyme implicated 20 years ago 179, SO?. In attempts to understand how the mutations induce the clinical disorders, trangenic mice have been used to study Alzheimer’s disease 155-58, 811, prion diseases [82), familial amyloidotic polyneuropathy [83, 841, and Lesch-Nyhan syndrome.
Therapy Although therapy is a central goal of medical research, molecular genetics has yet to deliver an effective treatment for any inherited disease. Perhaps there will be none; even though there has been complete knowledge of the affected gene product in sickle cell disease, treatment is still symptomatic, not curative. Nevertheless there is a mood of hope and expectation. The most dramatic therapeutic attempts involve DMD, including trials of gene replacement by myoblast implantation {SS] or by intramuscular injection of the gene in a vector [SC). These concepts are in their infancy and not yet practical. However, once we understand the gene product, it becomes possible to consider rational therapy. Problems Remain Therapy is not the only challenge. We still do not know the central functions of dystrophin, beta-amyloid, or several other gene products. In some conditions it is difficult to understand how lack of the gene product causes symptoms; a striking example is the relation of the androgen receptor to dysfunction and loss of motor neurons. Another manifestation of ignorance is the divergent clinical expression of mutations at the same site, such as malignant hyperthermia and central core disease at 15q23, or the several clinical syndromes that result from mutations of mtDNA. Other clinical problems bear upon function of the gene product. Here are some examples: how to relate the D N A mutation to a specific enzyme or structural protein and thence to clinical manifestations in any disease, especially mitochondrial disease; how mild and severe cases arise in the same family; or why there is such variability of expression in autosomal dominant and mitochondrial diseases; how heteroplasmy of mitochondrial DNA, the mixture of normal and mutated mitochondrial genomes in the same cell, leads to normal or abnormal function. We do not understand why there is genetic heterogeneity, how mutations at different loci cause virtually identical clinical syndromes, as in CMT. Or how clinical syndromes as similar as the myopathies of McLeod syndrome and DMD arise from 212 Annals of Neurology Vol 32
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two different mutations at nearby but not identical loci or gene products. Until we do understand the pathogenesis of clinical symptoms in molecular terms, rational therapy may be elusive. The final incongruity may be the inability of any society to pay for the research or the applications; the tools are at hand.
Looking to the Future As Yogi Berra said, “It is difficult to predict, especially about the future.” We can be certain that more genes will be mapped and more gene products identified as the Human Genome Project progresses. The many different genetic forms of mental retardation will be unravelled. Diseases of interest will not be restricted to rare monogenic disorders. Alzheimer’s disease, Parkinson’s disease, and other public health problems are already under scrutiny. Autoimmune diseases, including multiple sclerosis and myasthenia gravis, are being evaluated for disease-susceptibility genes. Alcoholism, schizophrenia, and bipolar disease are also open to DNA research. The techniques of recombinant D NA are being applied in other areas. In viral infections, DNA analysis is already vital for diagnosis, as well as preparation of vaccines and antiviral drugs. Analysis of neuronal receptors and channel proteins leads to better drugs for neurological disorders. Growth factors, identified and produced by recombinant DNA methods, are being tested in human diseases, some by brain implantation @7]. Neurologically important proteins can be produced in pure form and in quantity sufficient to make antibodies for analysis of heritable diseases (such as dystrophin or prion proteins) and perhaps for therapy. For autoimmune disorders such as myasthenia and multiple sclerosis, immunogenic peptides are identified and produced for similar purposes 1881. Two challenges are foremost: How does the molecular abnormality lead to symptoms of a disease? Can we devise effective therapy? With accelerating research progress 1891, the next decennial review may answer these questions. Supported by Center grants from the National Institute of Neurological Diseases and Stroke and the Muscular Dystrophy Association. The author’s views have been tempered by active associates: S. Bressman, S. DiMauro, T. Conrad Gilliam, W. G. Johnson, T. Nygaard, E. Schon, K. Wilhelmsen. The errors are my own.
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