The genetic basis of neuromuscular disorders

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T he molecular defects underpinning neuromuscular diseases can lie in the innervating nerve. at the contact zone between nerve and muscle referred to as the neuromuscular junction (NMJ), or in the muscle itself. Recent advances in molecular genetics have revealed the gene locations and gene products for most of the common inherited muscle diisest. Although the molecular basis of many of these d&eases is single base mutation in the coding region of the corresponding gene, there are seven1 e.xamples where snher different types of mutation are observed (Table 1). In this article, we review the CutTent .stztus of research into the molecular genetics of 3 selection of neuromuscular diseases, to illustrate the variety of mutation types associated with these disorders.

DuchenneandBeckermusculardysttvphies:an enormous target for mutations

Duchenne mwscular dystrophy (DMD). which affec% 1 in 33Cltl mak%, is the commonest and most severe mwscular dystrophyl. The disease is caused by the mutation of 3 gene encoding the large cyqoskeletaal protein, dystrophin, which normally bind.. to aczin and to a complex of proteins at the muscle membnne (Fig. I: reviewed in Refs 3, 4). One notable feature of DMD is the high spontaneous mutation mte, which is estimated to be 1 X IO-’ per genrrJtioni, almost two orders of magnitude’ higher thJn for other X-linked disorder&. The expktnalion for this phenomenon lies. in pan, in the enormous size of this gene. The 79 exons and vari- ous promoters of the dystrophin gene span more than 2.7Mh on the short arm of the X chromosome. How- ever. this large target for muation is not the only unusual feature of the genetics of DMD. When DMD prttients lvere imtially screened for mutation, researchers were surprised to find tht the major type of mutation was deletion of pan or all of the gene. D&tions are seen in more thJn 6CWu of patients and range in size from a few kik)bases to SeVerJl megJbJws. In DMD patients. deletions generally disrupt the reading frJnw :md no dystrophin is produced. If the de1etion.s do not disrupt the reading frJme of the transcript. a milder phenotype results: Becker muscuktr dystrophy (RhlD)-.

Interestingly, deletions arc found throughour the gene ;il!hough d&ions in oni) the 3’ end of tile ge:rr.e :we very rare. The end-points of the deletion\ occur

The genetic basis of neuromuscuhr disorders RALPHNAWRDlZtU,D~J.BIAgEAl’lD KAY E DAVIES

In the last dccadc, our kmwkdge c@man diseasegenes basbeeflgrowilgm~asamsldt~tbe~~of resoums and tecbm&pes f&r mapptng and seqmewctag tbe burrcan g- New d&easegews am mw mported atmJstweetdy.nJfsn?vkwilbrstnrtesbofotbe ide@~alianofgenesi~inRemn?mnscnlar disorders has led to the char- of sot on& novel genes but also ofa variety qfd@mat tyjws of gewttc mutattoli. These obsm wbtcb tactude bigb dektiorc

~qmcte~HrrrlaMcta-rcpcPl~q=-Pgerotnie d#tkatiom and trtplcr repeat expansiomq barn fkilitat~d tbe ideM@xtion of similar typs of mutation in otbergeaettc disorders.

preferentially in two introns, but are not clustered within the intron itselfs.9. The rea.son for this clustering of deletions remains a mysrery. If the deletions are caused by misalignment of homologous sequences. an equal number of duplications would be expected. However, although duplications exist, they are not observed at a very high frequency’“. Sequencing of a deletion iunc- tion fmgment from a DMD patient reveJ]ed that the proxtma! hreakp6nr lies within the sequence of a IrJnspoxson-like element belonging to the THE-l family”. THE-l sequences have also been reponed in the intro” 7 deletion hot spot of the DMD gene”. The significance of trJnspo.son-like elements in promoting deletions in DMD patients remains to be determined becwse the analysis of another deletion-junction fragment did not reveal such .sequencest+. A high frequent? of recombination has also been reported across the dystrophin gene w?th hot spots coinciding with the deletion hot spots’*. One possible explanation is that the chromatin .ztructure in Xp21 associJted wrth the expression of this enormous gene plays a role in the wcurrence of deletion or recombination hot :qots.

Poini mutationa and smaii deirrions ishoner &n one rxon) of the dystrophin gene account for virtually

x-nuked-ive Duchenne and Becker muscular

dystrophy

Autosomdrcctessive Limb girdle muscular dystrophy Fascioscapulohumecl dystrophy Spinal muscular atrophy

Autawmai&mtnant Myotonic dystrophy Charcoc-Marie-Tmh disease

DM!YBMD Xp21 Large deletions Dystmphin

i.GMD2.4 FSHD SMA

15q15 4q35 5al3

Digenic rr.utations’ Calpain- protease Deletions of tandem rePeat Deletions? SMN, NAlP Gene conversions?

DM 19q13 Triplet repear expansion CMTlA 17p11 Genomic duplication

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6q22-23 CMD

Extracellular matrix

Sarcolemma

Cytoplasm Chromosome 18

FIGURE 1. Yodel of dystrophin and ib a.ssociatcul proteins at the mu.& membrane. In normal skeletal muscle. the N-terminal domain of d!*trophm binds cyto\keletal sctin. The cental pan of the molecule consish of repealed spectrin-like sequences. At its C-terminus. dyblrophin 15 asxxi:wd wth 3 protein complex consisting of .xveml s&complexes. dystroglycan, the 25 kDa dystlophin-associated prorem (?iDAP). the wcoglycan complex and dystrobrevin*. The extracellular glycoprotein a-dystroglycan hinds to laminin-2. thereby effectively hnking the a&t-bawd cytoskeleton ~(3 dystrophin to the extracellular matnx. Mutatmns in memben of the ~rcoglycan complex have recently been idrntificd in autosomal recessive muscular dyatrophieb. Boxes indicate the chromosomal location of the human genes and. in wmc in~tzces. inherited diseases aswciated wth mutations in the retpwtiw genes.

all the remaining mutations in patients with DMD and RMD analysed so far’+:‘. In DMD patients, most of the point mutations lead to premature translational termi- nanon resulting in the production of little or no dys- rrophin. either because of tcmnscript or protein instability. Conversely, missense mutations account for most of the point mutattons in BMD patients. This observation supports the hypothesis that mutations in the dystrophin gene that leave the reading fmme intact cdu~e a mi!d Ecckcr phcno?;rz’. Ho~c~<c:, tbe:e are examples of single amino acid substitutions giving rise to a DMD phenotype’-.

Limbgirdle muscular dystrophy In normal muscle the C-terminal region of dys-

rrophin binds to P-dystroglycan, a member of the dystrophin glycoprotein complex WCX). The DGC spans the plasma membrane, effectively linking the muscle qtoskeleton, via the N-terminal act&binding domain of dystrophin, to the extracellular matrix (Fig. 1). In the absence of dystrophm. this structure is lost3.4. This model predicts that mutations in other components of the complex might lead to other forms of muscular dystrophy. Indeed, recent reports have confirmed that some limb-girdle muscular dystrophies show mutations in member of the sarcoglycan component of the DGC (Fig. 1; reviewed in Ref. 18). Mutations have been observed in all three sarcoglycans. In addition, mutations

in rhe gene encoding the a2 chain of laminin-2 have been found in some patients with congenital muscular dystrophy (CMD). No mutations have yet hen reported for Q- and P-dystroglycan. This might reflect the wider expression of the dystroglycan gene and its involvement in eplthelial differentiation”‘. Such mutations might, thus, be lethal. It is noteworthy that a similar complex of proteins is also present at the NMJ complexed with the dystrophin-related protein. utrophin. However, defects in neuromuscular trans- mission have not heen reported in patients with mutations in genes encoding components of the DGC. Screening for mutations in the syntrophin and dystrobrevin 8enes have not yet Isc?n carried out in an exharlstive manner.

Limb-girdle muscular dystrophies are not all caused by mutations in genes encoding components of the DGC. LGMD2A maps to chromosome 15 and the disease gene encodes a muscle-specific calpain- protease’o. An unexpectedly high number of different mutations co-.segregate with the disease in LGMD2A families. Si different mutations are found in patients in the small inbred population on the ‘La Reunion’ island in the Indian Ocean. This heterogeneity is unexpected for a small population and a digenic model of inheritance was suggested for the disease. Calpain- protease mutations can lx common in a population, but are by themselves insufftcient to cause the disease. Only when


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mutations occur in another. 3s yet unidentified, locus is LGMDZA manifested”. in this model, the hypothetical second locus is believed to contain the founder gene, the common mcebtor haplotype of small inbred popu- lations. such 3s the ‘La Reunion’ families. More studies 3re required to test this model.

The analysis of the calpain- protease gene in LGMDM also provides tin e.umple of synonymous codon mutxions in m&t. A silent third base muration in glycine (CCC to GGT) at codon 624 could be consid- ered zt ncutnl polymorphism. However, the tmnscrip- tional xxtlysis of the gene revealed that this sequence change cxws abemtnt splicing by the introduction of B novel donor splice site. Thii result surges I that the asses.;ment of apparently neutnl polymorphisms in humln disease might best be done at the RNA, as well 3s the DSA, level Y. herever possible.

Myotonic dystrophy trimchide repeat expansion In adults, myotonic dystrophy (DM) is the most fre-

quent genetic neuromuscular disorder affe&ng 1 in 8500 individuals”. Mildly affected DM pxients czm suffer from cx;lr;Icttr while multisystemic symptoms (muscular dystrophy. myotonkt. endocrine dysfunction and men- 1;tl rerartkttion) occur in more were :tnd exly onset UWS. DM is an autowmal dominant dise:w shotving ‘anticipation‘. This term ~3s introduced to describe the incwtsing severity of genetic diheabrb over sever4 gen- emtions or the onset of the dihe:tx 3t 3n earlier age in the ofl+ing than in the pxents~x. It is now widely recog nixed that such inheritance patterns are espktined by the progrebhive esp;msion of triplet reptut sequences in sue- ccx,ive generations. DM wts one of the first disorders in \vhich this phenometwn was reported. The disease is cw.sed by the expansion of 3 TG trinucleotide repeat on chromosome 19 (reviewtxl in Ref. 29). The repelt is highly polymorphic in the normal population, mnging in copy number from 5 to 35 CTG repezs. and there is an approximate correlation between the number of

Charcot-Mari~Tooth dii type lAt genomic duplication

Charrnt-bl:trittTc,oth (C&IT) disease is 3 petipheml neuropathy charxterized by progressive wasting of the distal muscles of the limbs. It affeca approximately 1 in 500 individuals. CMT i.s usually inherited in an autosomal dominant fit&ion. although one X-lied dominnnt fomi (CSlTsI ) has been repotted. The gene for the most common form of CMT disease. CMTlA. has been Itral- ixd to chromosome 17. However. unlike otiler forms of CMT, the moht common mechanism of mutation is not a point mutation but, mther. genomic duplication.

The vast majority of patients with CMTlA have genomic duplications on 17~. which include l.SMb of DNA and span 3 genetic disrance of 6&l (reviewed in Ref. 32). Surprisingly. the duplication event is .xen in sporadic and familial cases. ivlolecuktr analysis of the breakpoints in patients has established that they cluster within 3 region of repetitive sequences that promote un- cyual crossing over and, hence, duplication. These low copy repe-JLS, known 35 CMTlA-REP, span 354Okb. Such a mechanism should also give rise to deletion of this region of 17~. Indeed this is found to be the case, and

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the ciiical phenotype in these patients with deletions Is hereditary neuropathy with liibility to pressure paIs&.

The duplication of 1.5Mb of 17p raises questions regarding how many gerti are Involved in the manifes- tation of the clinical phenotype. A clue comes from the study of the trembkr mutation in the mouse, which is characterized by a hypomyelinathtg neuropathy and Is proposed as the animal model of CMTlA. This disorder is dominantly inherited and caused by a point mutation In the gene encoding peripheral myelin protein-22 (Pmp221 on mouse chromosome 11. The human homo- logue maps to the duplicated region on 17p and CMTlA patients have been found with point mutations In thii gene. Thus, although several genes must be involved in the duplication and deletion of 17p, the critical gene appears to be PMP22.

te/ (CTG)n z

59 DMPK DMAHP + is.?-- CpG island

Direction of transcription - -

--

Although gene dosage caused by DNA rearrange- ment is responsible for the dominant form of CMTlA, the exact relationship between gene dosage and phenotype remains to be determined. In addition, recent findings show that a single base pair change in the PMP22 gene can be inherited as a recessive mutations3.

FIGUSE 2. The myotonic dystrophy (DM) iocus on chromosome 19~~13. DM is caused by the expansion of GIG repeats wirhm the 3’ untranslated region (3’ tJTR) of the myotonic dystrophy protein kinase CDMPK) gene. Whether or not DMpKis the disease gene and how the CTG repeat expansion in the 3’ UTR affects its gene expression is controversial. However, the expression of two other genes, located upstream OXfAHP) and downstream (59) of the DMpKgene, are affected by increasing CTG repeat numbers as well. Therefore, more than one gene could be involved in the mechanism of DM.

It will be interesting in the future to determine whether other genetic disorders are caused by large genomic duplications. Conversely, there might be few regions of the genome where extra copies of several genes are tolerated and, hence, compatible with survival. Although single base pair changes in the gene encoding proteolipid protein (PLP) are the most common mutations causing X-linked Pelizaeus-Merzbacher disease, a long-range genomic duplication event, which includes the PLP gene, has recently been reported in a patient%. The precise extent of the duplication has yet to be elucidated, but it is tempting to speculate that the mechanism generating the duplication might be very similar to that operating in CMTIA.

inverted duplication and that this region contains two almost identical copies of a gene called the survival motor neuron (SMN) gene (Fig. 3). The two copies of the gene differ by five nucleotides, none of which changes the amino acid sequence of the correspondktg protein. The two copies of the SMN gene can be distinguished using single-strand-ccnformation polymorphism analysis. PCR products of exons 7 and 8 migrate diierentiy In acrylamide gels owing to differences in their structures on renaturation due to the single base pair differences. The telomeric copy of the SMNgene is deleted in more than 90% of patients. The same deletion frequency is observed in types I, II and 111 patients. The centromeric copy of the SMNgene is present in ali patients and deleted in 5% of normal individuals. ‘Ihe lack of patients deleted for both copies of the SMNgene suggests that this might result in a very severe or !etha! phenotype.

Genomic duplication and iastabiity at the spiaal muscular atrophy locus

Autosomal recessive spinal muscular atrophy (SMA) is a disease affecting the lower motor neu:on, which maps to chromosome 5q13 (Refs 35-37). Although it is a leading cause of infant mortality, nothing is known about whether the primary defect lies in the muscle or nerve. The disease can be classified Into three types according to the clinical severity. Type 1 (Werdnig- HO~~UM disease) is the severe infantile form with onset at bii and death usually within two years. The patients never sit or walk. Type II patients sit, but never walk unaided and survival depends on the invo!vement of the respiratory muscles. Type III (Kugelberg- Welander disease) patients can have onset in adolescence or later and can have a normal life span. All three types map to chromosome 5q13.

Evidence strongly suggesting that the SMNgene is the gene causing SMA or that mutations in the gene play a major role in the disease, is the identifkation of single base mutations and gene conversion events between the centromeric and telometic copies of the gene in SMA patients without deletionss9~*4 However, even in these families there is no correlation between the severity of the disease and the mutation. More recently, deletions in the SMNgene have been reported

Genomic region is I-2Mb

-- +-----m SUN NAIP p44

I I Critical region

deleted in patients

Four candidate genes have been identified, none of which alone iiiIRIls all the criteria of the caus- ative ones. MeIki and co-worker@ showed that the disease locus lies within a region containing an

tel

Ftarar~ 3. The genomic organization of spinal mwcular atrophy WA) candidate region. Note that only the telomeric versions of WA! NMPand ,044are deleted in patients. eNAlP is a pseudogene. XSGJ is not shown because the posaioo of the gene within the regjon has not been established.

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in asymptomatic sibs of haploidentical affected patient@.‘Q. This suggests the presence of an additional gene that mod&s the SMA phenotype either at 5q13 or elsewhere in the genome. The phenotype of mice lack- ing the SMNgene are awaited with interest.

The second candidate gene, encoding the neuronal apoptosis inhibitor protein fNAIP1, is present in mul- tiple copies in 5q13 and is also dtleted In patients43. However, deletions of NAIP are seen much more fre- quently in type I patients (45-600/o) compared with type II or typ III patients (lo-20%). The third candidate gene, XsG3, shows a stretch of homology to one of the exons of the NAPgene and has a similar deletion profile”. The precise relationship benveen XSG.3 and N&F has not been determined. The AWP gene might be a modifyiig gene as deletion of the NAIP coding sequence alone, without the deletion of the SMNgene, has not been observed in SMA patients. Although a few per cent of normal carders of the disease are also homozygously deleted for the NM&’ gene, this result is diicult to evaluate when there are so many copies of exons of the gene in 5ql3. A recent study demonstrat- ing the role of NAIP in apoptosis suggests that it might play a role in motor neuron surviva14s.

A fourth gene that has been identified in the region is the p44 gene. This gene is deleted at a frequency similar to that observed for the NAlPgene, but no complete cDNA has yet been reported46. A schematic represen- tation of the genes mapping to the SMA candidate region is given in Fig. 3.

All the deletion assays in SMA patients are based on differences in single exons of the candidate genes which are themselves present at variable copy number across rhe region. Moreover, the genomic maps across the region are not consistent probably reflecting vari- ation between different genomes%. This genomic poly- . morphtsm mtght tnrk the rea! genets events un__ derly- ing the disease, which might involve meiotic instability of the region and copy number of genes. The instability of the region is also indicated by the fact that new mutations are not a rare event in this disorderi’.*“. A high frequency of mutation should be rare for an autosomal recessive disease. Such events suggest that germline mosaicism might be present.

conchtsion One of the first triumphs of positional cloning was

the isolation of the dystrophii gene in DMD. The identification of the genetic basis of other muscle disorders that followed has bcen a fascinating story. As discussed in this review, the identification of unstable repeat sequences in FSHD and genomic duplications in SMA might well reveal further novel types of mutation in the genome. ‘Ihe chalienge for the future will be to correlate genotype with phenotype and to develop effective treatments.

Acknowledgements We thank Keith Johnson for Fi8. 2. and Veronica van

Heynin8cn and Sue Malcolm for helpful discussions. We apologize to the many workers on the individual diseases whose work we were unable to quote in the interests of space. More specialist reviews have been given wherever possible. We thank the MRC and the DFG for fundin

1 Kaplan, J-C. and Fontaine, B. (1996) Neummusc. Diwrd. 6, l-10

2 Emery. A.E.H. (19931 DucbenneMurcubrQrstn@y (vol. 2). Oxford University Press

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The genetic basis of neuromuscular disorders