JMed Genet 1997;34:973-977

Genetic epidemiology of muscular dystrophiesresulting from sarcoglycan gene mutations

M Fanin, D J Duggan, M L Mostacciuolo, F Martinello, M P Freda, G Soraru_,C P Trevisan, E P Hoffman, C Angelini

AbstractBackground-The autosomal recessivelimb-girdle muscular dystrophies(LGMDs) are a group of geneticallyheterogeneous muscle diseases charac-terised by progressive proximal limbmuscle weakness. Six different loci havebeen mapped and pathogenetic mutationsin the genes encoding the sarcoglycancomplex components (a-, p-, y-, and6-sarcoglycan) have been documented.LGMD patients affected with primary"sarcoglycanopathies" are classified asLGMD2D, 2E, 2C, and 2F, respectively.Methods-A geographical area in northeast Italy (2 319 147 inhabitants) was se-lected for a genetic epidemiological studyon primary sarcoglycanopathies. Withinthe period 1982 to 1996, all patients livingin this region and diagnosed with muscu-lar dystrophy were seen at our centre.Immunohistochemical and immunoblotscreening for a-sarcoglycan protein defi-ciency was performed on all muscle biop-sies from patients with a progressivemuscular dystrophy ofunknown aetiologyand normal dystrophin. Sarcoglycan mu-tation analyses were conducted on allpatient muscle biopsies shown to havecomplete or partial absence ofa-sarcoglycan immunostaining or a de-creased quantity of a-sarcoglycan proteinon immunoblotting.Results-Two hundred and four patientmuscle biopsies were screened fora-sarcoglycan protein deficiency and 18biopsies showed a deficiency. Pathoge-netic mutations involving one gene forsarcoglycan complex components wereidentified in 13 patients: a-sarcoglycan inseven, ,-sarcoglycan in two, y-sarcoglycanin four, and none in the 5-sarcoglycangene. The overall prevalence of primarysarcoglycanopathies, as of 31 December1996, was estimated to be 5.6 x 10-6 inhab-itants.Conclusion-The prevalence rate esti-mated in this study is the first to beobtained after biochemical and mole-cular genetic screening for sarcoglycandefects.(7Med Genet 1997;34:973-977)

Keywords: autosomal recessive limb-girdle musculardystrophy; Duchenne-like muscular dystrophy; sar-coglycan complex

The autosomal recessive limb-girdle musculardystrophies (LGMDs) are a clinically hetero-geneous group of diseases characterised byprogressive muscle weakness of the pelvic andshoulder girdles. Several genes are involved inthe aetiology of autosomal recessive LGMDsand a new classification has been proposed'according to the molecular basis (table 1):LGMD2A is mapped on chromosome 15q15,2LGMD2B on 2pl2,3 LGMD2C on 13ql2,4LGMD2D on 17ql2,5 LGMD2E on 4q12,6 7and LGMD2F on 5q33.V Apart fromLGMD2B, all other genes have been clonedand their muscle specific protein productsidentified: calpain-3 is involved in LGMD2A,9a-sarcoglycan (50 kDa dystrophin associatedglycoprotein DAG, adhalin) in LGMD2D,'°3-sarcoglycan (43 kDa DAG) in LGMD2E,6y-sarcoglycan (35 kDa DAG) in LGMD2C,"and 6-sarcoglycan (35 kDa DAG) inLGMD2F. 12The a-, 3-, y- and 6-sarcoglycan proteins

form the sarcoglycan (SG) complex, one com-ponent ofthe dystrophin-glycoprotein complexon the skeletal muscle membrane.'3 " Pathoge-netic mutations in each gene for SG complexcomponents determine a group of diseasescalled "sarcoglycanopathies". Because the en-tire SG complex is disrupted, all sarcoglycano-pathies are characterised by biochemical defi-ciency of all subunits independently of anyprimary gene defect.4 16

After the first description of primary sar-coglycanopathy in a French family,'" manypatients from different countries have beenreported4 7 11 `-` in whom a variety of genemutations and heterogeneous clinical pheno-types were found.

Genetic epidemiologal studies of musculardystrophies resulting from SG gene defectshave been inhibited by several factors: thesediseases are rare, the methodology used todetect small mutations was made available onlyrecently, and, even more important, the collec-tion and screening of all the cases in a definedgeographical area is time consuming.The present study was undertaken to

estimate the prevalence of autosomal recessiveLGMD resulting from SG genes mutations(LGMD2C, 2D, 2E, 2F) in a restricted area ofnorth east Italy.

Materials and methodsMETHODS OF ASCERTAINMENTThe present epidemiological survey was donein north east Italy and included three neigh-bouring administrative districts (Padova,

RegionalNeuromuscularCentre, Department ofNeurology, UniversityofPadova, ViaGiustiniani 5, 35128Padova, ItalyM FaninF MartinelloM P FredaG SorariiC P TrevisanC Angelini

Departments ofMolecular Geneticsand Biochemistry,Pediatrics, Neurology,and Human Genetics,University ofPittsburgh, USAD J DugganE P Hoffman

Section ofHumanGenetics, Departmentof Biology, UniversityofPadova, ItalyM L Mostacciuolo

Correspondence to:Dr Fanin.

Received 7 March 1997Revised version accepted forpublication 9 July 1997

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Table 1 Classification of autosomal recessive limb-girdle muscular dystrophies (LGMDs)

Nomenclature Chromosomal localisation Deficient protein

LGMD2A 15q15.1 Calpain-3 or CANP3LGMD2B 2pl3-pl6 UnknownLGMD2C 13q12 35 kDa DAG or y-sarcoglycanLGMD2D 17q12-q21.33 50 kDa DAG or a-sarcoglycanLGMD2E 4q12 43 kDa DAG or P-sarcoglycanLGMD2F 5q33-34 35 kDa DAG or 6-sarcoglycan

CANP3: calcium activated neutral protease-3. kDa: kiloDaltons. DAG: dystrophin associatedglycoprotein.

Treviso, Vicenza). This area is 7341 km with2 319 147 inhabitants at 31 December 1996.This study was based on the collection of casesaffected with muscular dystrophy, diagnosed atthe Regional Neuromuscular Centre, Univer-sity of Padova within the period 1982 to 1996.The diagnostic procedures always includedclinical examination, serum creatine kinase(CK) determination, electromyography, andmuscle biopsy, obtained after written consent.The ascertainment of all cases of muscular

dystrophy in this area was ensured by thefollowing: (1) the Regional NeuromuscularCentre in Padova is the referral unit for musclediseases in this area; since 1975 it has regularlycollected clinical records and muscle biopsiesfrom muscular dystrophy patients from all thehospitals in this area and from most specialistsin the field (neurologists, paediatricians, ge-neticists); (2) the Regional Centre for GeneticCounselling on neuromuscular disorders hasoperated in this area regularly from 1973 andrepresents a referral centre in this region.

SELECTION CRITERIAOf all muscle biopsies processed in our centrein the period 1982-1996, 515 patients receiveda diagnosis of "progressive muscular dystro-phy". They all had a clinical history and mus-cle weakness compatible with muscular dystro-phy, muscle biopsy findings corresponding todystrophic changes, and increased CK levels.The first exclusion criterion involved the

patients who had been diagnosed on a molecu-lar basis as having Duchenne muscular dystro-phy (144), Becker muscular dystrophy (135),merosin deficient congenital muscular dystro-phy (11), and symptomatic carriers of dys-trophinopathy (12). Patients in whom an auto-somal dominant pattern of inheritance wasreported were also excluded (9). In theremaining 204 muscular dystrophy patients, inwhom the primary genetic defect was unknownand dystrophin was normal, biochemical (im-munohistochemical and immunoblotting)screening for a-SG was carried out.

BIOCHEMICAL METHODSMuscle biopsy cryosections were immuno-stained for a-SG by overnight incubation atroom temperature with 1:20 diluted mono-clonal antibodies (Novocastra, Newcastle,UK). Indirect immunofluoresence visualisa-tion was obtained by the avidin-biotin system,as described elsewhere.7 Immunoblotting fora-SG was performed using the same antibodyas described elsewhere.28

MOLECULAR METHODSAnalysis of the genes coding for a-SG, P-SG,y-SG, and 6-SG was performed by directsequencing of abnormal SSCP (single strandconformation polymorphism) conformers ob-tained from reverse transcription (RT-PCR) ofmuscle biopsy RNA or exon specific PCRamplification of genomic DNA, as describedelsewhere.22 25 28 30

The size of the PCR products is such that thesensitivity of our SSCP analysis is estimated tobe 90 to 95%31; in addition we run more thanone SSCP procedure and therefore this in-creases the probability of detecting a mutation.As we were testing patients with autosomal

recessive inheritance, where two gene muta-tions should be detected, the large majority ofmutation positive patients would have beendetected as homozygotes or heterozygotes(>99%).

ResultsSCREENING FOR PROTEIN AND GENE DEFECTS INTHE SARCOGLYCAN COMPLEXA biochemical a-SG defect was found in 18patients, 13 of whom (72%) showed apathogenetic mutation in the a-SG, P-SG, ory-SG genes. Three mutation positive patientswere identified because they are sibs of theindex patients undergoing a muscle biopsy (fig1). A mutation in the a-SG gene (LGMD2D)was found in seven patients, in the 3-SG gene(LGMD2E) in two patients, and in the y-SGgene (LGMD2C) in four patients (table 2).

Detailed molecular data on patients 1-3 and8-11 have been reported previously.7 23 28 29 Inthe remaining six patients (three pairs of sibs)the molecular data showed the following.Patients 4 and 5 were heterozygous for an a-SGG- A (nucleotide 739) base change resultingin a valine to methionine amino acid substitu-tion. Patients 6 and 7 were homozygous for ana-SG C-*T (nucleotide 850) base changeresulting in an arginine to cysteine amino acidsubstitution. Patients 12 and 13 were hetero-zygous for both y-SG T insertion (nucleotide

Figure 1 The geographical area selected in this study(thick border) includes three administrative districts(Padova, Vicenza, Treviso) of the Veneto region in northeast Italy. The places where the affected patients live isshown by triangles (a-SG), squares (/B-SG), or circles(y-SG).

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Genetic epidemiology ofprimary sarcoglycanopathies

Table 2 Familial and clinical data in patients affected with primary sarcoglycanopathy in three districts of Veneto region

Patient Affected Parental Age atNo District Sex relatives consanguinity diagnosis (y) Clinical phenotype SG gene defect Ref

1 Padova M + 9 Duchenne-like a(LGMD2D) 292 Padova F - + 44 Limb-girdle a(LGMD2D) 28t3 Treviso M - 16 Limb-girdle a(LGMD2D) 28t4 § Padova F + - 36 Limb-girdle a(LGMD2D)5 § Padova M + - 31 Limb-girdle a(LGMD2D)6 ¶ Treviso F + - 40 Limb-girdle a(LGMD2D)7 Treviso M + - 36 Asymptomatic* a(LGMD2D)8 Padova M - - 9 Duchenne-like ,B(LGMD2E) 28t9 Vicenza F - - 3 Duchenne-like P3(LGMD2E) 710 Vicenza F - - 19 Duchenne-like y(LGMD2C) 23t11 Vicenza M - - 23 Limb-girdle y(LGMD2C) 23t1211 Vicenza M + - 16 Duchenne-like y(LGMD2G)13 Vicenza F + - 13 Duchenne-like y(LGMD2C)

SG: sarcoglycan; LGMD: limb-girdle muscular dystrophy. *Kyphoscoliosis and high serum creatine kinase level. tPatients 2, 3, and8 are referred to as patients 33, 25 and 29 in Duggan et al."' tPatients 10 and 11 are referred to as patients 44 and 26 in McNally etal.23 §, $ 11 :sibs.

87-88) and a two base pair (TC) deletion(nucleotide 801).

In all except two patients, mutations on bothalleles (either as homozygotes or as compoundheterozygotes) were confirmed against parentalgenomic DNA; the parents were found to carryone of the two identified mutant alleles in aheterozygous state. In the remaining two cases(patients 4 and 5) only one mutant allele wasidentified by SSCP and confirmed in theirmother.

PRIMARY a-SARCOGLYCANOPATHY (LGMD2D),|3-SARCOGLYCANOPATHY (LGMD2E), ANDY-SARCOGLYCANOPATHY (LGMD2C)Seven patients from five different familieswere shown to suffer from a primarya-sarcoglycanopathy. The clinical severity ofthese patients ranged from a severe Duchenne-like muscular dystrophy to a very mild LGMDphenotype.An autosomal recessive pattern of inherit-

ance was reported in four of the five families:patient 2 was born to unaffected secondcousins, while patients 3-7 (including two setsof affected sibs of both genders) were born tohealthy, non-consanguineous parents. Patient1 had an affected cousin who was not availablefor study. The prevalence ofa-sarcoglycanopathy (LGMD2D) was esti-mated to be 3.02 x 106 (7/2 319 147) inhabit-ants (table 3).Two unrelated patients (8 and 9)were shown

to have a primary f-sarcoglycanopathy. Bothpatients had a progressive, severe DMD-likephenotype. Both family histories were negativefor neuromuscular disorders and consanguin-ity. The prevalence of f-sarcoglycanopathy(LGMD2E) was estimated to be 0.86 10-6(2/2 319 147) inhabitants (table 3).

Table 3 Estimate of the prevalence ofprimarysarcoglycanopathies in three districts of Veneto region

No of Prevalence*Sarcoglycan gene defect affected cases (x 1(6)a-sarcoglycan (LGMD2D) 7 3.02f1-sarcoglycan (LGMD2E) 2 0.86y-sarcoglycan (LGMD2C) 4 1.72Total primary 13 5.60

sarcoglycanopathies

*'Te prevalence rate was calculated at 31 December 1996(population 2 319 147). LGMD: limb-girdle muscular dystro-phy.

Four patients (two unrelated patients andtwo sibs) were shown to have a primaryy-sarcoglycanopathy (Nos 10-13). Three ofthe four patients suffered from a severeDMD-like muscular dystrophy (Nos 10, 12,and 13) and one from milder LGMD (No 1 1).Consanguinity was not reported in any ofthese patients. The prevalence ofPy-sarcoglycanopathy (LGMD2G) was esti-mated to be 1.72 x 10-6 (4/2 319 147) inhabit-ants (table 3).

RELATIVE PROPORTION OF PRIMARYSARCOGLYCANOPATHIES AMONG PATIENTS WITHDMD-LIKE AND LGMD PHENOTYPESThe overall prevalence of a-SG, P-SG, y-SG,and 6-SG gene defects was estimated to be5.60 x 10-6 (13/2 319 147) subjects (table 3).

In the selected area, 11 patients werediagnosed on their clinical phenotype asDMD-like muscular dystrophy (prevalence 4.7x 10-6) and six of them (54.5%) were affectedwith primary sarcoglycanopathy. In the samearea, 40 patients were diagnosed on their clini-cal phenotype and biopsy findings as having anLGMD phenotype (prevalence 17.2 x 10-6)and seven of them (17.5%) were affected withprimary sarcoglycanopathy.

DiscussionPrimary sarcoglycanopathies have been de-scribed in families with a broad range of clini-cal presentations.6 7 1 1 7-30 Many cases havebeen previously described under the nameSCARMD (severe childhood autosomal reces-sive muscular dystrophy) or DMD-like muscu-lar dystrophy. However, the term SCARMD,used to describe the severity of the disease, isnot always accurate as a description of themyopathy associated with primary sarcogly-canopathy, because the phenotype may bemilder with juvenile or adult onset. Thus, thegenetic and clinical heterogeneity of this groupof disorders has made the current LGMDnomenclature useful.'

Since identification, correct classification,and prevalence estimates of the sarcoglycano-pathies cannot be based on clinical criteria, themolecular characterisation of muscular dystro-phies with normal dystrophin represented thebasis ofthis study. Our neuromuscular centre isworking in an area where full ascertainment

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Fanin et al

and epidemiological statistics are possible. Webelieve that all the affected cases came to ournotice since muscular dystrophy patients livingin this area are referred here to receive eitherdiagnosis or genetic counselling. All the casessuspected to be affected with muscular dystro-phy usually undergo a muscle biopsy to obtainclinical and molecular definition. Muscle biop-sies from patients shown to have a progressivemuscular dystrophy of unknown aetiology(normal dystrophin) have been screened fora-SG.Data acquired to date suggest that a-SG

immunostaining can be considered a markerof SG complex integrity, being absent orhighly reduced in every primarysarcoglycanopathy.6 1 15 16 Thus, we believethat we have detected all SG deficient patientsusing biochemical a-SG studies as the primaryscreening tool. A possible source of inaccuracyin the prevalence estimate is if we were unableto detect all mutations in the biopsies tested bythe SSCP method. Since the rate of mutationdetection using SSCP in our study is estimatedto be 90-95%, the stated prevalence rates couldincrease by a maximum of 10%.

In the area selected in this study, primaryLGMD2D is twice as frequent as LGMD2Cand four times more frequent than LGMD2E;primary LGMD2F was not found. Fivepatients with a-SG deficiency did not showmutations of the a-, 3-, y-, or 6-SG genes underinvestigation, suggesting that the observeda-SG deficiency was secondary to an as yetunidentified primary protein defect.The overall prevalence of primary sarcogly-

canopathies is estimated to be 5.6 per million,a value that is lower than that estimated forother recessive neuromuscular diseases in thesame area: 11 per million for spinal muscularatrophy types II and III32 and 6.8 per million forcongenital muscular dystrophy.33 Parental con-sanguinity observed in this and otherstudies'0 13 confirms that the sarcoglycanopa-thies are rare recessive disorders.Comparison between published genetic epi-

demiological data and those obtained in thepresent study is difficult for several reasons.First, the estimates currently available for theprevalence of LGMD were based only on theclinical phenotype and precede any of themolecular characterisation of this group of dis-orders. Furthermore, because of the wide clini-cal heterogeneity, primary sarcoglycanopathypatients might have been previously includedin the estimates of either X linked andautosomal recessive DMD-like muscular dys-trophy or LGMD phenotypes.Most of the prevalence rates reported34 for

LGMD lie between 7 and 40 x 10-6, while foran autosomal recessive DMD-like musculardystrophy they are less than 5 x 1 06. Two stud-ies dealt with the relative proportions of thevarious defined gene localisations in LGMD.Stec et aP5 estimated that the proportion ofpatients suffering from autosomal DMD-likemuscular dystrophy that could be the result ofsarcoglycan genes mutations is 11.8% of all thepatients with a DMD phenotype. However, thefrequency reported in this study is speculative,

since molecular studies on SG genes were notperformed.

Passos-Bueno et a16 reported that amongnine autosomal recessive LGMD families theystudied, 22% mapped to chromosome 1 7q(primary a-SG). Since this study was per-formed with families in whom only linkageanalysis could be done, this represents arelatively small minority of affected patientsand the data cannot be extrapolated from a fewfamilies to the whole population.The only population study on a-SG deficient

patients was reported by Hayashi et al37 whoanalysed 243 patients' biopsies showing nor-mal dystrophin and identified five cases withimmunohistochemical a-SG defects. The fre-quency ofa-SG protein deficiency in Japan wasestimated to be 1.0-2.1 x 10-6. However, noscreening for mutations was done on thesepatients, so not knowing how many of thesepatients represent primary defects of a-, 3-, y-,or 6-SG makes this number a rough estimate.

In a previous epidemiological study38 survey-ing the same geographical area, the prevalencerates for DMD-like muscular dystrophy andautosomal recessive LGMD based on clinicalphenotypes were calculated to be 3.7 x 106 and17.3 x 10-6, respectively. These values are veryclose to those found in the present study (4.3and 17.2 x 10-6, respectively). The reliability ofour results is therefore proven by the consist-ency of the data with those derived from a pre-vious study surveying the same area and havingthe same clinical and epidemiological ascer-tainment methods.We found that the relative proportion of

patients with defects in the SG genes amongthose who received the clinical diagnosis ofDMD-like muscular dystrophy was 54.5%(6/1 1) and 17.5% (7/40) for LGMD cases. In acumulative series of 556 myopathic patientsscreened for an a-SG defect,28 22% of DMD-like muscular dystrophy and 6% of LGMDpatients were affected with primary sarcogly-canopathies. The difference between the twostudies probably represents a difference inpatient diagnosis (DMD-like versus LGMD),or the ethnic diversity of the two patientcohorts (a more heterogeneous North Ameri-can population versus a more homogeneousItalian population). Alternatively, the differ-ence may reflect variation in the frequency ofsarcoglycan gene mutations in various worldpopulations. In the study conducted by Hay-ashi et al,37 they found approximately 5% ofJapanese patients with a DMD/BMD-likediagnosis to have an a-SG protein deficiency.Although this percentage reflects the biochemi-cal frequency of a-SG deficiency, the molecu-lar frequencies cannot exceed this number(assuming all mutation positive patients show abiochemical deficiency). Thus, we see that inthree different world populations, the fre-quency of sarcoglycan gene mutations in a par-ticular diagnostic category differs. Therefore,the prevalence rates of the individual sarcogly-canopathies and the frequencies of the sar-coglycanopathies in the various patient popula-tions may need to be determined for all thedifferent world populations.

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Genetic epidemiology ofprimary sarcoglycanopathies

The possibility that additional SG mutationpositive cases may be detected among LGMDor mild myopathic patients showing a nearlynormal biochemical a-SG defect cannot becompletely excluded.The present study has addressed two issues:

(1) the estimate of the prevalence rate of eachprimary sarcoglycanopathy, assessed by bothbiochemical and molecular screening; and (2)the relative proportion of patients with primarysarcoglycanopathies among those who receivedthe clinical diagnosis of DMD-like or LGMDmuscular dystrophy.

Because of the extensive clinical heterogene-ity in LGMD patients with normal dystrophin,the correct classification of these patients bymolecular analysis should be pursued becauseof its usefulness in the differentiation betweenautosomal recessive and dominant and Xlinked recessive inheritance, an issue that iscrucial for genetic counselling.

The financial support of Telethon (grants 695 to MF and C19to CA) is gratefully acknowledged.

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mutations.dystrophies resulting from sarcoglycan gene Genetic epidemiology of muscular

Sorarù, C P Trevisan, E P Hoffman and C AngeliniM Fanin, D J Duggan, M L Mostacciuolo, F Martinello, M P Freda, G

doi: 10.1136/jmg.34.12.9731997 34: 973-977 J Med Genet 

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