Am. J. Hum. Genet. 50:887-891, 1992

Invited Editorial: Phenotypic Heterogeneity and the Single Gene Graeme K. Suthers and Kay E. Davies Institute of Molecular Medicine, John Radcliffe Hospital, Oxford

Diagnostic precision is fundamental to medical research. In genetic parlance, the mutant phenotype must be defined accurately if the mutant genotype is to be elucidated. However, diagnostic precision provides no guarantee that the relationship between phenotype and underlying genotype will be straightforward. For example, conditions such as tuberous sclerosis (TSC; MIM 191100) and hereditary motor and sensory neuropathy type 1 (CMT: MIM 118220) are caused by mutations at two or more loci (Haines et al. 1991; Vance 1991). Conversely, similarities in the clinical presentation of two apparently distinct disorders may suggest that the different phenotypes represent allelic heterogeneity at a single locus. Genetic linkage studies in families with Duchenne and Becker muscular dystrophies (DMD and BMD, respectively; MIM 310200) lent support to the clinicians' notion that the two disorders are allelic (Kingston et al. 1984). Subsequent identification of mutations in the dystrophin gene have confirmed this view (Koenig et al. 1987; Hoffman et al. 1988). Papers in this issue of the Journal describe two further examples where mutations at single loci give rise either to different neuromuscular disorders or to a disorder with marked intrafamilial phenotypic variability. One can be explained by allelic heterogeneity; the other could be explained by the influence of other genetic or environmental factors. Paramyotonia congenita (PMC; MIM 168300) and hyperkalemic periodic paralysis (HYPP; MIM 170500) are rare autosomal dominant disorders which are characterized by episodes of abnormal muscle excitability. The hallmarks of PMC are myotonia and weakness provoked by exercise or exposure to cold, Received February 20, 1992. Address for correspondence and reprints: Kay E. Davies, Ph.D., Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, England. This article represents the opinion of the authors and has not been peer reviewed. 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297/92/5005-0001 $02.00

with a predilection for muscles of the face, tongue, neck, and hand. There is no significant myopathy, but there may be lability of the serum potassium concentration (Walton and Gardner-Medwin 1981). In contrast, HYPP is associated with episodic weakness resulting from depolarization of the muscle cell membrane and increased serum potassium concentration (Engel 1981). This may be associated with myotonia or with a progressive myopathy (Bradley et al. 1990). Some patients with features of PMC also fulfill the diagnostic criteria of HYPP, suggesting that the two disorders are a single nosologic entity. However, careful electromyographic searches have failed to detect myotonia in all cases of HYPP, and potassium loading fails to provoke paralytic attacks in all individuals with PMC (Engel 1981). The distinction between the two conditions has been defended on the basis of these laboratory investigations and on clinical criteria (Harper 1989, p. 64). The gene SCN4A encodes the sodium-channel a-subunit in adult skeletal muscle in man. Unusual sodiumchannel activity in skeletal muscle had been noted in HYPP in 1987 (Lehmann-Horn et al. 1987), and SCN4A has recently been investigated as a candidate gene for HYPP and PMC. The gene has been mapped to human chromosome 17 and shows tight genetic linkage to HYPP (Fontaine et al. 1990; Koch et al. 1991b; Ptacek et al. 1991c). Two different substitutions of highly conserved residues in SCN4A were subsequently documented in two HYPP families (Ptacek et al. 1991a; Rojas et al. 1991). Linkage analyses in families segregating for PMC also show tight linkage to SCN4A, suggesting that allelic heterogeneity at SCN4A is the basis of these two disorders (Koch et al. 1991a; Ptacek et al. 1991b). A third related disorder, acetazolamide-responsive atypical myotonia congenita, is also tightly linked to SCN4A (Ptacek et al. 1992). In this issue of the Journal McClatchey et al. (1992) describe the use of two highly informative dinucleotide repeat polymorphisms in the SCN4A gene to assess the likelihood of HYPP and PMC representing allelic 887

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heterogeneity at SCN4A. Their data confirm the close linkage of these disorders to SCN4A. Only two different haplotypes were associated with HYPP in the 10 families studied, despite their ethnic diversity. Both of these haplotypes are common in the population, suggesting that at least two independent mutations have occurred in this disease. Two HYPP families which have the same disease haplotype demonstrate substitution of the same conserved valine residue as reported by Rojas et al. (1991). In four PMC families the disease locus segregated with two haplotypes which differ from the HYPP haplotypes. These data are consistent with the notion that the difference in phenotype is due to different primary mutations in SCN4A. Therefore, at least four independent mutations have occurred in the SCN4A gene, and these mutations account for at least two distinct phenotypes. The next fascinating step is to define precisely the cellular pathophysiology resulting from a variety of mutations in SCN4A. An understanding of the mechanism underlying the progressive myopathy noted in some HYPP families may have implications for a much more diverse group of muscle diseases. In this issue of the Journal the second paper (McClatchey et al. 1992) that addresses the issue of allelic heterogeneity concerns autosomal recessive proximal spinal muscular atrophy (SMA). This disease affects the anterior horn cells of the spinal cord, with consequent muscle weakness, but nothing is known of the pathogenesis. Affected individuals generally present with one of three forms of the disease (Dubowitz 1978, pp. 146-178). In the most severe type (type 1; Werdnig-Hoffmann disease; MIM 253300) the onset of symptoms is in the first 6 mo of life; the child never sits, and death usually occurs by the age of 2 years. The intermediate form (type II; MIM 253550) has a later onset, but the affected child never walks unaided. In the comparatively mild form of the disease (type III; Kugelberg-Welander disease; MIM 253400) affected individuals maintain independent ambulation. The three types of SMA have been mapped to chromosome 5 (Brzustowicz et al. 1990; Gilliam et al. 1990; Melki et al. 1990a, 1990b; Daniels et al. 1992a, 1992b). In carefully evaluated multiplex pedigrees there is no evidence of either incomplete penetrance or locus heterogeneity (Dubowitz 1991). The variation in phenotype has been attributed to allelic heterogeneity at the SMA locus. Although the clinical features of SMA are usually consistent among the affected individuals in a pedigree, there are several reported pedigrees where wide

Suthers and Davies variation in the severity of muscle weakness was observed. Furthermore, some pedigrees have an unexpectedly high incidence of cases among second-degree relatives. These observations led Becker (1964) to propose a genetic model whereby almost all affected individuals are compound heterozygotes at the SMA locus. He proposed that there are five SMA alleles; a+ is the normal allele, with a frequency of 90%; a* is the rare disease allele; and three modifier allelesa'a",and a"'-share a frequency of 10%. The following genotypes would correspond to the different SMA phenotypes: ata', type I; ata", type II; and a4-a"', type III (a-a* would be extremely rare; individuals homozygous or heterozygous for a+, a', a", or a"' would be normal). If this model is correct, it would have important implications for prenatal diagnosis of the disorder (Muller and Clerget-Darpoux 1991; Daniels et al. 1992a). This proposed genetic complexity is exacerbated by the observation that the segregation ratio in pedigrees with SMA types II or III is significantly less than the value of .25 expected for an autosomal recessive disorder (Baraitser 1982, pp. 180185; Bundey 1985, p. 185). This distortion of the segregation ratio could reflect new mutations, phenocopies, or misdiagnoses. As reported in this issue of the Journal, Muller et al. (1992) tested the Becker model by studying the segregation of chromosome Sq markers in four families where SMA types II and III existed in the same sibship. The data are not consistent with the hypothesis that affected individuals are compound heterozygotes, as all the affected siblings inherited the same alleles from both parents. Therefore other genetic or environmental factors must be responsible for the vari-

ability in phenotype. The concept that allelic heterogeneity at a single locus can account for markedly different phenotypes is not new. For example, such phenotypic variability has been the key to understanding the molecular pathology resulting from mutations at the DMD locus (Monaco et al. 1988). For those seeking candidate genes for a given disease phenotype, the key question is what degree of phenotypic heterogeneity is consistent with allelic heterogeneity at a single locus. The answer may lie in identifying the common thread that links different phenotypes. HYPP and PMC have myotonia and altered serum potassium concentration in common. Different mutations in the COLlA1 gene on chromosome 17 (MIM 120150) result in a variety of mutant phenotypes involving connective tissue, such as bone fragility of mild to severe degree (osteogenesis

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Invited Editorial imperfecta types I-IV) or a combination of hyperelastic skin and lax connective tissue (Ehlers-Danlos syndrome type VII-A). A similar range of phenotypes attends mutations in the COL1A2 gene (MIM 120160) on chromosome 7. The diverse phenotypes of mutations of the COL2A1 gene (MIM 120140) (spondyloepiphyseal dysplasia, osteoarthritis, achondrogenesis-hypochondrogenesis, and Stickler syndrome) primarily involve connective tissue, but the link is a little more tenuous. (The documented mutations of COLlA1, COL1A2, COL2A1, and AAT (and corresponding phenotypes) are tabulated by McKusick (1988); readers are referred to this source for the original references.) An unexpected addition to the range of phenotypes due to mutations in the DMD gene (MIM 310200) was the association of muscle cramps and myalgia in the absence of muscle weakness (Gospe et al. 1989). It would have been even more difficult to predict the common thread linking neonatal jaundice, chronic pulmonary emphysema, and a fatal coagulopathy caused by different mutations in the a-1-antitrypsin gene (MIM 107400). Most examples of allelic heterogeneity involve mutations within the exons of the gene. It is possible that mutations of gene promoters or other nontranslated regulatory elements could account for allelic heterogeneity. The dystrophin gene encodes a number of major isoforms (Bar et al. 1990; Geng et al. 1991; Byers et al. 1991), and tissue-specific promoters have been documented (Barnea et al. 1990). A mutation involving the promoter for the predominant skeletal muscle isoform results in the Becker phenotype (Boyce et al. 1991; Bushby et al. 1991), but the phenotypic consequences of mutations involving the promoters for other major isoforms are not known. But what of intrafamilial phenotypic variability that cannot be attributed to allelic heterogeneity? Some explanations may be biologically trivial, such as the amelioration of the DMD phenotype in a man with Klinefelter syndrome (Suthers et al. 1989). Modifier loci have been postulated to explain the intrafamilial variability noted in numerous conditions including Rh deficiency syndrome (MIM 268150; Race and Sanger 1975, pp. 220-22), Waardenburg syndrome (MIM 193500; Asher and Friedman 1990), and fucosidosis (MIM 230000; Willems et al. 1988). If the primary mutation causes a simple recessive loss of function, a modifier locus may account for intrafamilial variability, as exemplified by the modification of the 1-thalassaemia or sickle cell phenotypes in the presence of heterocellular hereditary persistence of fetal hemoglo-

bin (MIM 142470; Wood et al. 1976). However, more complex mutations may account for a wide variation in phenotype, without the involvement of modifier loci. Autosomal modifier loci have been proposed to account for the bizarre segregation noted in families with the fragile-X syndrome (Steinbach 1986; Israel 1987). However, the unusual nature of the fragile-X mutation is sufficient to account for the range of phenotypes seen (Fu et al. 1991). Allelic heterogeneity can account for a remarkable range of diverse phenotypes in a monogenic disorder. The identification of interfamilial allelic heterogeneity is an important step in analyzing the normal function of the gene and its product. Intrafamilial variation in the mutant phenotype may reflect the action of modifier loci, stochastic or environmental influences, ormost intriguingly - the nature of the mutation itself. From a practical point of view, the process of choosing which candidate genes to evaluate in order to define the mutation underlying a given phenotype is like going fishing: it requires a skillful eye to pick the opportunities, a willingness to cast over a wide area, and luck.

Acknowledgments The authors are supported by the Medical Research Council, The Muscular Dystrophy Group of Great Britain and Northern Ireland, the Muscular Dystrophy Association (USA), and the Nuffield Foundation.

References AsherJH, Friedman TB (1990) Mouse and hamster mutants as models for Waardenburg syndromes in humans. J Med Genet 27:618-626 Bar S, Barnea E, Levy Z, Neuman S, Yaffe D, Nudel U (1990) A novel product of the Duchenne muscular dystrophy gene which greatly differs from the known isoforms in its structure and tissue distribution. Biochem J 272: 557-560 Baraitser M (1982) The genetics of neurological disorders. Oxford University Press, Oxford Barnea E, Zuk D, Simantov R, Nudel U, Yaffe D (1990) Specificity of expression of the muscle and brain dystrophin gene promoters in muscle and brain cells. Neuron 5:881-888 Becker PE (1964) Atrophia musculorum spinalis pseudomyopathica. Z Menschlichen Vererbungsforsch Konstitutionslehre 37:193-220 Boyce FM, Beggs AH, Feener C, Kunkel LM (1991) Dystrophin is transcribed in brain from a distant upstream promoter. Proc Natl Acad Sci USA 88:1276-1280 Bradley WG, Taylor R, Rice DR, Hausmanowa-Petru-

890 zewicz I, Adelman LS, Jenkison M, Jedrzejowska H, et al (1990) Progressive myopathy in hyperkalemic periodic paralysis. Arch Neurol 47:1013-1017 Brzustowicz LM, Lehner T, Castilla LH, Penchaszadeh GK, Wilhelmsen KC, Daniels R, Davies KE, et al (1990) Genetic mapping of chronic childhood-onset spinal muscular atrophy to chromosome Sqll.2-13.3. Nature 344:540541 Bundey S (1985) Genetics and neurology. Churchill-Livingstone, Edinburgh Bushby KMD, Cleghorn NJ, Curtis A, Haggerty ID, Nicholson LVB, Johnson MA, Harris JB, et al (1991) Identification of a mutation in the promoter region of the dystrophin gene in a patient with atypical Becker muscular dystrophy. Hum Genet 88:195-199 Byers TJ, Kunkel LM, Watkins SC (1991) The subcellular distribution of dystrophin in mouse skeletal, cardiac, and smooth muscle. J Cell Biol 115:411-421 Daniels RJ, Suthers GK, Morrison KE, Thomas NH, Francis MJ, Mathew CG, Loughlin S, et al (1992a) Prenatal prediction of spinal muscular atrophy. J Med Genet 29:165170 Daniels RJ, Thomas NH, MacKinnon RN, Lehner T, Ott J, Flint T, Dubowitz V, et al (1992b) Linkage analysis of spinal muscular atrophy. Genomics 12:335-339 Dubowitz V (1978) Muscle disorders in childhood. WB

Saunders, London (1991) Chaos in the classification of the spinal muscular atrophies of childhood. Neuromuscular Dis 1: 77-80

Engel AG (1981) Metabolic and endocrine myopathies. In: Walton JN (ed) Disorders of voluntary muscle, 4th ed. Churchill-Livingston, Edinburgh, pp 481-524 Fontaine B, Khurana TS, Hoffman EP, Bruns GAP, Haines JL, Trofatter JA, Hanson MP, et al (1990) Hyperkalemic periodic paralysis and the adult muscle sodium channel alpha-subunit gene. Science 250:1000-1002 Fu YH, Kuhl DPA, Pizzuti A, Pieretti M, Sutcliffe JS, Richards S, Verkerk AJM, et al (1991) Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell 67:1047-1058 Geng Y, Sicinski P, Gorecki D, Barnard PJ (1991) Developmental and tissue-specific regulation of mouse dystrophin: the embryonic isoform in muscular dystrophy. Neuromuscular Dis 1:125-133 Gilliam TC, Brzustowicz LM, Castilla LH, Lehner T, Penchaszadeh GK, Daniels RJ, Byth BC, et al (1990) Genetic homogeneity between acute and chronic forms of spinal muscular atrophy. Nature 345:823-825 Gospe SM, Lazaro RP, Lava NS, Grootscholten PM, Scott MO, Fischbeck KH (1989) Familial X-linked myalgia and cramps: a nonprogressive myopathy associated with a deletion in the dystrophin gene. Neurology 39:1277-1280 Haines JL, Short MP, Kwiatkowski DJ, Jewell A, Andermann E, Bejjani B, Yang CH, et al (1991) Localization of

Suthers and Davies one gene for tuberous sclerosis within 9q32-9q34, and further evidence for heterogeneity. Am J Hum Genet 49: 764-772 Harper PS (1989) Myotonic dystrophy, 2d ed. WB Saun-

ders, London Hoffman EP, Fishbeck KH, Brown RH, Johnson M, Medori R, Loike JD, Harris JB, et al (1988) Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy. N Engl J Med 318:1363-1368 Israel MH (1987) Autosomal suppressor gene for fragile-X: an hypothesis. Am J Med Genet 26:19-31 Kingston HM, Sarfarazi M, Thomas NST, Harper PS (1984) Localization of the Becker muscular dystrophy gene on the short arm of the X chromosome by linkage to cloned DNA sequences. Hum Genet 67:6-17 Koch MC, Ricker K, Otto M, Grimm T, Bender K, Zoll B, Harper PS, et al (1991a) Linkage data suggesting allelic heterogeneity for paramyotonia congenita and hyperkalaemic periodic paralysis on chromosome 17. Hum Genet 88:71-74 Koch MC, Ricker K, Otto M, Grimm T, Hoffman EP, Rudel R, Bender K, et al (1991b) Confirmation of linkage of hyperkalaemic periodic paralysis to chromosome 17. J Med Genet 28:583-586 Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener C, Kunkel LM (1987) Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 50:509-517 Lehmann-Horn F, Kuther G, Ricker K, Grafe P, Ballanyi K, Rudel R (1987) Adynamia episodica hereditaria with myotonia: a non-inactivating sodium current and the effect of extracellular pH. Muscle Nerve 10:363-374 McKusick VA (1988) OMIM: online Mendelian inheritance in man. Johns Hopkins University Press, Baltimore MelkiJ, Abdelhak S, Sheth P, Bachelot MF, Burlet P, Marcadet A, Aicardi J, et al (1990a) Gene for chronic proximal spinal muscular atrophies maps to chromosome Sq. Nature 344:767-768 Melki J, Sheth P, Abdelhak S, Burlet P, Bachelot M-F, Lathrop GM, Frezal J (1990b) Mapping of acute (type 1) spinal muscular atrophy to chromosome 5ql2-ql4. Lancet 336:271-273 Monaco AP, Bertelson CJ, Liechti-Gallati S, Moser H, Kunkel LM (1988) An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics 2:90-95 Muller B, Clerget-Darpoux F (1991) Becker's model and prenatal diagnosis in proximal spinal muscular atrophy (SMA): a note of caution. Am J Hum Genet 49:238-240 Ptacek LJ, George AL, Griggs RC, Tawil R, Kallen RG, Barchi RL, Robertson M, et al (1991a) Identification of a mutation in the gene causing hyperkalemic periodic paralysis. Cell 67:1021-1027

Invited Editorial Ptacek LJ, Tawil R, Griggs RC, Storvick D, Leppert M (1992) Linkage of atypical myotonia congenita to a sodium channel locus. Neurology 42:431-433 Ptacek LJ, Trimmer JS, Agnew WS, Roberts JW, Petajan JH, Leppert M (199lb) Paramyotonia congenita and hyperkalemic periodic paralysis map to the same sodiumchannel gene locus. Am J Hum Genet 49:851-854 Ptacek LJ, Tyler F, Trimmer JS, Agnew WS, Leppert M (1991c) Analysis in a large hyperkalemic periodic paralysis pedigree supports tight linkage to a sodium channel locus. Am J Hum Genet 49:378-382 Race RR, Sanger R (1975) Blood groups in man, 6th ed. Blackwell, Oxford Rojas CV, Wang J, Schwartz LS, Hoffman EP, Powell BR, Brown RH (1991) A met-to-val mutation in the skeletal muscle Na + channel alpha-subunit in hyperkalaemic periodic paralysis. Nature 354:387-389 Steinbach P (1986) Mental impairment in Martin-Bell syndrome is probably determined by interaction of several genes: simple explanation of phenotypic differences be-

891 tween unaffected and affected males with the same X chromosome. Hum Genet 72:248-252 Suthers GK, Manson JI, Stern LM, Haan EA, Mulley JC (1989) Becker muscular dystrophy (BMD) and Klinefelter's syndrome: a possible cause of variable expression of BMD within a pedigree. J Med Genet 26:251-254 Vance JM (1991) Hereditary motor and sensory neuropathies. J Med Genet 28:1-5 Walton JN, Gardner-Medwin D (1981) Progressive muscular dystrophy and the myotonic disorders. In: Walton JN (ed) Disorders of voluntary muscle, 4th ed. ChurchillLivingston, Edinburgh, pp 481-524 Willems PJ, Garcia CA, de Smedt MCH, Martin-Jimenez R, Darby JK, Duenas DA, Granado-Villar D, et al (1988) Intrafamilial variability in fucosidosis. Clin Genet 34: 7-14 Wood WG, Weatherall DJ, Clegg JB (1976) Interaction of heterocellular hereditary persistence of foetal haemoglobin with beta-thalassaemia and sickle cell anemia. Nature 264:247-249