Mutations in the Sarcoglycan Genes in Patients with Myopathy

618

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Februar y 27, 1997

The New England Journal of Medicine

MUTATIONS IN THE SARCOGLYCAN GENES IN PATIENTS WITH MYOPATHY

D

AVID

J. D

UGGAN

, B.S., J. R

AFAEL

G

OROSPE

, M.D., P

H

.D., M

ARINA

F

ANIN

, M.S., E

RIC

P. H

OFFMAN

, P

H

.D.,

AND

C

ORRADO

A

NGELINI

, M.D.

A

BSTRACT

Background

Some patients with autosomal re-cessive limb-girdle muscular dystrophy have mu-tations in the genes coding for the sarcoglycan pro-teins (

a

-,

b

-,

g

-, and

d

-sarcoglycan). To determine thefrequency of sarcoglycan-gene mutations and the re-lation between the clinical features and genotype,we studied several hundred patients with myopathy.

Methods

Antibody against

a

-sarcoglycan was usedto stain muscle-biopsy specimens from 556 patientswith myopathy and normal dystrophin genes (thegene frequently deleted in X-linked muscular dystro-phy). Patients whose biopsy specimens showed a de-ficiency of

a

-sarcoglycan on immunostaining werestudied for mutations of the

a

-,

b

-, and

g

-sarcoglycangenes with reverse transcription of muscle RNA, anal-ysis involving single-strand conformation polymor-phisms, and sequencing.

Results

Levels of

a

-sarcoglycan were found tobe decreased on immunostaining of muscle-biopsyspecimens from 54 of the 556 patients (10 percent);in 25 of these patients no

a

-sarcoglycan was detect-ed. Screening for sarcoglycan-gene mutations in 50of the 54 patients revealed mutations in 29 patients(58 percent): 17 (34 percent) had mutations in the

a

-sarcoglycan gene, 8 (16 percent) in the

b

-sarcogly-can gene, and 4 (8 percent) in the

g

-sarcoglycan gene.No mutations were found in 21 patients (42 percent).The prevalence of sarcoglycan-gene mutations washighest among patients with severe (Duchenne-like)muscular dystrophy that began in childhood (18 of83 patients, or 22 percent); the prevalence amongpatients with proximal (limb-girdle) muscular dystro-phy with a later onset was 6 percent (11 of 180 pa-tients).

Conclusions

Defects in the genes coding for thesarcoglycan proteins are limited to patients with Du-chenne-like and limb-girdle muscular dystrophy withnormal dystrophin and occur in 11 percent of suchpatients. (N Engl J Med 1997;336:618-24.)

©1997, Massachusetts Medical Society.

From the Departments of Human Genetics, Molecular Genetics andBiochemistry, Pediatrics, and Neurology, University of Pittsburgh, Pitts-burgh (D.J.D., J.R.G., E.P.H.); and the Regional Neuromuscular Center,Department of Neurology, University of Padua, Padua, Italy (M.F., C.A.).Address reprint requests to Dr. Hoffman at Biomedical Science TowerW1211, Department of Molecular Genetics and Biochemistry, Universityof Pittsburgh School of Medicine, Pittsburgh, PA 15261.

Other authors were E. Pegoraro (University of Pittsburgh, Pittsburgh),S. Noguchi and E. Ozawa (National Center of Neurology and Psychiatry,Tokyo, Japan), W. Pendlebury (University of Vermont, Burlington), A.J.Waclawik (University of Wisconsin–Madison Medical School, Madison),D.A. Duenas (Miami Children’s Hospital, Miami), I. Hausmanowa-Petrusewicz and A. Fidzianska (Polish Academy of Sciences, Warsaw, Po-land), S.C. Bean (Medical Center of Delaware, Newark), J.S. Haller (Alba-ny Medical Center, Albany, N.Y.), J. Bodensteiner (Robert C. Byrd HealthSciences Center, Morgantown, W.V.), C.M. Greco (California Pacific Med-ical Center, San Francisco), A. Pestronk (Washington University, St. Louis),A. Berardinelli (Neurological Institute C.M. Mondino, Pavia, Italy), D.F.Gelinas (California Pacific Medical Center, San Francisco), H. Abram(Nemours Children’s Clinic, Jacksonville, Fla.), and R.W. Kuncl (JohnsHopkins University, Baltimore).

USCLE diseases (myopathies) are mostoften caused by inborn errors result-ing in the degeneration of muscle fi-bers (muscular dystrophies). The mus-

cular dystrophies are a clinically and geneticallyheterogeneous group of disorders. Clinically, manyof the muscular dystrophies present with proximal-limb muscle weakness or wasting and elevated se-rum creatine kinase concentrations. Genetically, thepattern of inheritance can be X-linked recessive (as

M

in Duchenne’s or Becker’s muscular dystrophy), au-tosomal dominant (as in limb-girdle muscular dys-trophy type 1), or autosomal recessive (as in limb-girdle muscular dystrophy type 2). Recently, manyof these phenotypically similar, yet genetically dis-tinct, disorders have been shown to be caused byabnormalities of the plasma membrane of musclefibers. This membrane contains an extensive cyto-skeleton that stabilizes the myofibrillar membraneduring contraction. Part of this cytoskeleton appearsto be responsible for attaching intracellular actin tothe extracellular basal lamina by means of dystrophin(Fig. 1).

1-3

Abnormalities of dystrophin are a common causeof muscular dystrophy,

4,5

and testing for the dystro-phin gene or protein has become part of the routinediagnostic evaluation of patients who present withprogressive proximal muscle weakness, high serumcreatine kinase concentrations, and histopathologicalevidence of a dystrophic process. Patients who haveno dystrophin abnormalities are assumed to have anautosomal recessive muscular dystrophy; most casesare isolated, and large families with multiple affectedpatients are uncommon.

In addition to dystrophin, other proteins, oftenreferred to as dystrophin-associated proteins, con-tribute to the membrane cytoskeleton of myofibers.These proteins are divided into the dystroglycan,sarcoglycan, and syntrophin subcomplexes (Fig. 1).

3,6

Recently, genetic defects of the sarcoglycan complex(sarcoglycanopathies) were found in some patientswith autosomal recessive muscular dystrophy.

7-20

Thegenes and proteins corresponding to these genetic

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Volume 336 Number 9

�

619

defects are

a

-sarcoglycan,

7

b

-sarcoglycan,

9,10

g

-sar-coglycan,

8

and

d

-sarcoglycan.

20

Each of these sar-coglycans is a relatively small transmembrane proteinexpressed either only (

a

,

g

, and

d

) or predominantly(

b

) in striated muscle (skeletal and cardiac).

7-10,20-23

No patients with primary defects of the other dys-trophin-associated proteins (dystroglycans and syn-trophins) have been identified; however, the expres-sion of these proteins is not limited to striatedmuscle.

24,25

Studies with antibodies directed against

a

-,

b

-,

g

-,and

d

-sarcoglycan have revealed deficiencies of allcomponents of the sarcoglycan complex in muscle-biopsy specimens from patients with mutations inany of the sarcoglycan genes.

8-10,17,20

Apparently, mu-tations in a single sarcoglycan gene lead to desta-bilization of the entire complex and secondary de-ficiency of the other sarcoglycan proteins. Thus, agenetic defect of any component of the protein com-plex should be detectable with antibody against anyof the sarcoglycan proteins.

26

Because biopsy speci-mens from patients with dystrophin-gene abnormal-ities reveal secondary deficiencies of the sarcogly-

cans,

27,28

testing for sarcoglycan proteins is informativeonly if dystrophin is normal. Thus, testing of biopsyspecimens from patients with normal dystrophinwith antibody directed against

a

-sarcoglycan shouldidentify most or all patients with mutations of the

a

-,

b

-,

g

-, and

d

-sarcoglycan genes. We used both bi-ochemical and molecular approaches to screen forabnormalities and genetic defects in the sarcoglycanproteins in patients with myopathy.

METHODS

Selection of Patients

We selected muscle-biopsy specimens for

a

-sarcoglycan immu-nostaining from patients with myopathy whose records and biopsyspecimens were available at the University of Padua (322 patients)and University of Pittsburgh (234 patients) and who had normaldystrophin on the basis of immunofluorescence or immunoblotanalysis and histopathological evidence of myopathy. The Italianpatients were from northeastern Italy and were examined in Paduaby a single team. The patients whose biopsy specimens were stud-ied in Pittsburgh were examined in neuromuscular clinics through-out the United States with a standard clinical-assessment protocol(manual muscle testing and muscle biopsy). The Italian patients (ortheir parents) provided written informed consent. The studiesdone in Pittsburgh were approved by the institutional review

Figure 1.

Organization of the Plasma-Membrane Dystrophin Cytoskeleton of Myofibers.Dystrophin is part of a large oligomeric complex tightly associated with several other protein complexes. The dystroglycan complexconsists of

a

-dystroglycan, which associates with the basal-lamina protein merosin, and

b

-dystroglycan, which binds

a

-dystrogly-can and dystrophin. Syntrophin binds to the distal C-terminal region of dystrophin. The sarcoglycan complex consists of four trans-membrane proteins:

a

-,

b

-,

g

-, and

d

-sarcoglycan. The function of the sarcoglycan complex and the nature of the interactions withinthe complex and between it and the other complexes are not clear. The sarcoglycan complex is formed only in striated muscle,and its subunits preferentially associate with each other, suggesting that the complex may function as a single unit.

dg

SyntrophinActin

Dystrophin

Intracellular

Plasma membrane

Merosin

Sarcoglycans Dystroglycans

Extracellular

a

bba


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Februar y 27, 1997


board, but did not require informed consent because the biopsyspecimens were “preexisting pathological specimens” obtained fordiagnostic purposes (to rule out a dystrophin abnormality).

The patients were divided into three groups on the basis of aclinical examination (Padua) or clinical summaries (Pittsburgh):those with congenital muscular dystrophy, those with a dystro-phinopathy-like disorder, and those with other neuromusculardisorders (Table 1). Congenital muscular dystrophy was definedas the presence of weakness or hypotonia at birth, contracturesbefore six months of age, and dystrophy on muscle biopsy. Thiscategory included patients with and those without central nerv-ous system involvement. A dystrophinopathy-like disorder was de-fined on the basis of the presence of progressive proximal muscleweakness beginning after six months of age, high serum creatinekinase concentrations, and dystrophy on muscle biopsy. Otherneuromuscular disorders were defined on the basis of findings ofmuscle weakness or wasting, hypotonia, calf hypertrophy, or highserum creatine kinase concentrations and included the followingdiagnoses: inflammatory myopathy (9 patients), metabolic myop-athy (9), congenital structural myopathy (10), myoglobinuria (14),distal myopathy (2), congenital hypotonia (2), familial myopathywith high serum creatine kinase concentrations (5), isolated highserum creatine kinase concentrations (23), isolated myopathy withhigh serum creatine kinase concentrations or cramps (40), andother muscular dystrophies (78). An additional 32 patients fromthe Pittsburgh referral center had inadequate clinical informationfor precise classification of the neuromuscular disorder. All the pa-tients in the group with other neuromuscular disorders had nor-mal results of

a

-sarcoglycan immunostaining.The patients with a dystrophinopathy-like disorder were subdi-

vided on the basis of their age at presentation into those withDuchenne-like muscular dystrophy (also known as severe child-hood autosomal recessive muscular dystrophy) if they presented ator before the age of 10 years and those with limb-girdle musculardystrophy (similar to Becker’s muscular dystrophy) if they present-ed after 10 years of age. All patients with Duchenne-like musculardystrophy had Gowers’ sign (rising from the floor by pushingone’s hands against the shins, knees, and then thighs) before losingthe ability to walk, and all patients with either type of musculardystrophy had high serum creatine kinase concentrations.

Biochemical Analysis

Frozen sections (4 to 6

m

m) of muscle obtained by biopsy werestained with

a

-sarcoglycan antibody (NCL-50DAG, NovocastraLaboratories, Newcastle, United Kingdom) as previously de-

scribed.

15

The sections were scored as being completely or par-tially deficient in

a

-sarcoglycan relative to results in muscle tissueobtained and processed in parallel from subjects with no histo-logic evidence of muscle disease, as previously described.

15

Theidentification of partial deficiencies of

a

-sarcoglycan was subjec-tive, but interobserver reliability was increased by the use of onlytwo observers for all biopsy specimens (one each in Padua andPittsburgh), who were unaware of the clinical diagnosis.

Molecular Analyses

RNA Extraction and Reverse Transcription

Total RNA was isolated from the muscle-biopsy specimens ofpatients with decreased or no

a

-sarcoglycan staining and reversetranscribed as previously described.

15

Polymerase Chain Reaction

Overlapping polymerase-chain-reaction (PCR) products for thecomplete coding sequences of complementary DNA for the

a

-,

b

-, and

g

-sarcoglycan genes were generated. The

a

-sarcoglycanPCR products, primers, and conditions have been described pre-viously.

14,15

The

b

-sarcoglycan PCR products were generated withtwo primer pairs in addition to those previously described

9

:primer pairs 5 (nucleotides

�

40 to 267; 5

�

ACAGTCGGGCG-GGGAGCT3

�

and 5

�

CACGGCCCAAATAACAAGTG3

�

) and 6(nucleotides 773 to 982; 5

�

ATGGATCTGTATGGTCAGC3

�

and5

�

CATGTTGGTGACCTCTGGG3

�

), designed on the basis of the

b

-sarcoglycan messenger RNA sequence

9

with use of the Primerprogram (Daly MJ, et al.: unpublished data). PCR conditions wereas described previously,

9,15

except that an annealing temperature of60°C and 5 percent dimethylsulfoxide were used for primer pair5. The

g

-sarcoglycan PCR products, primers, and conditions havebeen described previously.

8,17

Analysis Involving Single-Strand Conformation Polymorphisms

Radiolabeled PCR product was mixed with an equal volume ofstop solution, and the samples were denatured and analyzed withthree single-strand conformation polymorphism (SSCP) gels asdescribed previously.

15,29

DNA Sequencing

Bands showing an electrophoretic shift in mobility on the basisof SSCP analysis were directly sequenced with the original PCR

*Other neuromuscular disorders included inflammatory myopathy (9 patients), metabolic myopathy (9), congenitalstructural myopathy (10), myoglobinuria (14), distal myopathy (2), congenital hypotonia (2), familial myopathy withhigh serum creatine kinase concentrations (5), isolated high serum creatine kinase concentrations (23), isolated myopathywith high serum creatine kinase concentrations or cramps (40), and other muscular dystrophies (78). In the case of 32patients, inadequate clinical information was available for precise classification of the disorder.

T

ABLE

1.

C

LINICAL

D

IAGNOSIS

, R

ESULTS

OF IMMUNOSTAINING FOR a-SARCOGLYCAN, AND FREQUENCY OF SARCOGLYCAN-GENE MUTATIONS IN 556 PATIENTS WITH MYOPATHY.

CLINICAL DIAGNOSIS IMMUNOSTAINING RESULTS TYPE OF SARCOGLYCAN-GENE MUTATION

NO. OF

PATIENTS

COMPLETE

DEFICIENCY

PARTIAL

DEFICIENCY a b g NONE

NOT

TESTED

no. of patients (%)

Congenital muscular dystrophy 69 — 2 — — — 1 1

Dystrophinopathy-like disorderDuchenne-like muscular dystrophyLimb-girdle muscular dystrophy

83180

1411

1512

8 (10)9 (5)

8 (10)—

2 (2)2 (1)

11 (13)9 (5)

—3

Other neuromuscular disorders* 224 — — — — — — —

Total 556 25 29 17 8 4 21 4



Volume 336 Number 9 � 621

primers and the prism Ready Reaction Dyedeoxy Terminator Cy-cle Sequencing Kit (Applied Biosystems, Foster City, Calif.) ac-cording to the manufacturer’s recommendations.15

Confirmation of Nucleotide Changes

Nucleotide changes and an autosomal recessive pattern of inher-itance were confirmed in the genomic DNA of the patients andtheir parents with exon-specific PCR primers.18,19 When the iden-tified sequence variant destroyed or created a restriction-enzymesite, the appropriate PCR product was digested with a specific re-striction endonuclease. When the identified sequence variant didnot change a restriction-enzyme site, amplification-refractory mu-tation tests were designed.30 Insertions, duplications, and deletionswere analyzed by size fractionation with denaturing polyacryla-mide-gel electrophoresis.

DNA Analysis

Genomic DNA was isolated from peripheral-blood lympho-cytes of the patients and their parents or the patients’ muscle-biopsy specimens as described previously.31,32 Approximately 100ng of DNA was used for PCR amplification.

RESULTS

Muscle-biopsy specimens from 556 patients withmyopathy and normal dystrophin were available forimmunostaining for a-sarcoglycan (Fig. 2). The clin-ical classifications of these patients and the stainingresults are shown in Table 1. Deficiency of a-sar-coglycan was evident in biopsy specimens from 54patients (10 percent). The deficiency was completein 25 patients (4.5 percent) and partial in 29 pa-tients (5.2 percent). Fifty-two of the 54 patientswith a-sarcoglycan deficiency had clinical manifesta-tions of either Duchenne-like or limb-girdle muscu-lar dystrophy (a dystrophinopathy-like disorder). Twoof the 69 patients with congenital muscular dystro-phy had a partial deficiency of a-sarcoglycan. Theprevalence of a-sarcoglycan deficiency in the threegroups of patients was similar in the Italian and Amer-ican patients.

RNA was isolated from the muscle-biopsy spec-imens of 50 of the 54 patients with abnormala-sarcoglycan immunostaining and screened for mu-tations in the a-, b-, and g-sarcoglycan genes. Mu-tations were detected in 29 (58 percent) of the pa-tients (Tables 1, 2, and 3): 18 of 29 patients withDuchenne-like muscular dystrophy (62 percent) and11 of 19 patients with limb-girdle muscular dystro-phy (58 percent). The homozygous or compoundheterozygous status of the 25 patients who had mu-tations of both sarcoglycan-gene alleles was con-firmed by tests on genomic DNA from all the pa-tients and, when available, both their parents (17 of25 patients); in each case their parents were hetero-zygous for one of the two identified mutant alleles.In four patients (Patients 6, 18, 39, and 40), onlyone mutant allele was identified. In Patient 6, onlymutant RNA (T371C) was present in the muscle-biopsy specimen, whereas analysis of genomic DNArevealed the patient to be heterozygous (one wild-type and one mutant allele) at this nucleotide posi-

Figure 2. a-Sarcoglycan Immunostaining of Muscle.Transverse sections of muscle-biopsy specimens were stainedwith an antibody directed against a-sarcoglycan plus a Cy-3–con-jugated antibody. Muscle from a normal subject shows uniformstaining of the periphery of each muscle fiber (Panel A, �70).Muscle from Patient 7 shows a marked reduction in the inten-sity and uniformity of staining (Panel B, �70).

A

B

*Four of the 54 patients with a-sarcoglycan deficiency were not tested:1 with a complete deficiency of a-sarcoglycan and limb-girdle musculardystrophy, 2 with a partial deficiency and limb-girdle muscular dystrophy,and 1 with a partial deficiency and congenital muscular dystrophy.

TABLE 2. SARCOGLYCAN-GENE MUTATIONS IDENTIFIED IN 50 PATIENTS WITH a-SARCOGLYCAN DEFICIENCY.*

IMMUNOSTAINING

RESULT

NO. OF

PATIENTS

TYPE OF SARCOGLYCAN-GENE MUTATION

a b g NONE

no. of patients (%)

Completedeficiency

24 14 (58) 5 (21) 0 5 (21)

Partial deficiency 26 3 (12) 3 (12) 4 (15) 16 (62)


622 � Februar y 27, 1997


*LGMD denotes limb-girdle muscular dystrophy, DMD-like Duchenne-like muscular dystrophy, C complete, NA notavailable, ND not determined, and P partial.

†The following patients have been described previously: Patient 8 (Fanin et al.16); Patients 441 and 442 (Duggan etal.15); Patient 43 (Bönnemann et al.9); and Patients 7, 19, 26, and 44 (McNally et al.17).

‡Unless otherwise noted, patients for whom only one mutation is listed are homozygous for that mutation. The nu-cleotides and amino acids are numbered according to the translational reading frames deduced on the basis of data fromMcNally et al.,23 Bönnemann et al.,9 and Noguchi et al.8

§This patient was the product of a consanguineous union.

¶Single-strand conformation polymorphism analysis showed an aberrant band of exon 2 that could not be sequenced.

�Mutations were identified with the use of genomic DNA.

TABLE 3. SARCOGLYCAN-GENE MUTATIONS IN THE PATIENTS WITH ABNORMAL RESULTS OF a-SARCOGLYCAN IMMUNOSTAINING.*

PATIENT NO.AND TYPE

OF MUTATION†CLINICAL

DIAGNOSIS

CURRENT AGE (YR)

LOSS OF ABILITY

TO WALK

(AGE AT TIME)

TYPE OF

a-SARCOGLYCAN

DEFICIENCY

NUCLEOTIDE

CHANGE‡

AMINO ACID SUBSTITUTION

OR OTHER CONSEQUENCE

OF MUTATION‡

Mutation in the a-sarcoglycan gene

3 LGMD 11 No C C229T Arg77Cys4 LGMD NA No C G101A

C229TArg34HisArg77Cys

5 DMD-like 9 No C C229TT371C

Arg77CysIle124Thr

6 LGMD 9 No C T371CND

Ile124ThrND

8 DMD-like 11 Yes (10 yr) C T472T Leu158Phe11 DMD-like 10 No C G586A Val197Ile12 DMD-like NA Yes (12 yr) C T308C

T518CIle103ThrLeu173Pro

21 LGMD 31 No C C292TC614A

Arg98CysPro205His

25 LGMD 17 No P T92CC850T

Leu31ProArg284Cys

33§ LGMD 41 No P C850T Arg284Cys39 DMD-like 15 Yes (12 yr) C C229T

ND¶Arg77CysND

40 LGMD 13 Yes (13 yr) C Duplication of347–351

ND

Frame shift

ND42 DMD-like 14 No P C614A

C683APro205HisPro228Gln

441 DMD-like 12 No C C229TA290G

Arg77CysAsp97Gly

442 LGMD 18 No C G101A Arg34His443 DMD-like 13 No C C229T

C278TArg77CysAla93Val

510 LGMD 12 No C C229T Arg77Cys

Mutation in the b-sarcoglycan gene

2 DMD-like 8 No C C31TA(�2 exon 3)G

Gln11StopInsertion

9§ DMD-like 14 Yes (11 yr) C C31G Gln11Glu10§ DMD-like 21 Yes (15 yr) C C341T Ser114Phe18 DMD-like 13 No P G416A

NDGly139StopND

29 DMD-like 11 No C Duplication of 376–383

Frame shift

Deletion of 699–702

Frame shift

31§ DMD-like 20 Yes (10 yr) P Duplication of 376–383

Frame shift

43 DMD-like 4 No C Duplication of 376–383

T552G

Frame shift

Tyr184Stop487� DMD-like 16 Yes (14 yr) P A544G Tyr182Ala

Mutation in the g-sarcoglycan gene

7 DMD-like 12 No P Deletion of TG at 793–794

Frame shift

19§ LGMD 10 No P Deletion of Tat 519

Frame shift

26 LGMD 23 Yes (17 yr) P Deletion of TCat 801–802

Frame shift

44 DMD-like 20 Yes (14 yr) P Insertion of T at 87–88

Frame shift



Volume 336 Number 9 � 623

tion. This patient probably had a second undetectedmutation that prevented the expression of the wild-type allele, allowing only the mutant allele to be de-tected in the RNA of the muscle. Because all pa-tients with a primary sarcoglycanopathy are assumedto have an autosomal recessive pattern of inheritancewith two mutant alleles, Patients 18, 39, and 40were presumed to have a second, unidentified muta-tion. One hundred normal chromosomes were ex-amined for all novel mutations, and all tested nega-tive. Thus, among the 50 patients with a deficiencyof a-sarcoglycan on immunostaining, mutations inthe a-sarcoglycan gene were detected in 17 (34 per-cent), mutations in the b-sarcoglycan gene weredetected in 8 (16 percent), and mutations in theg-sarcoglycan gene were detected in 4 (8 percent).

The mean (�SD) age at presentation or the diag-nosis of myopathy in the patients with mutations inthe a-sarcoglycan gene was 6�4 years; it was 3�2and 6�4 years, respectively, in the patients withmutations in the b-sarcoglycan and g-sarcoglycangenes. All patients presented with clinical findingsthat included calf hypertrophy, Gowers’ sign, and adeficit in motor skills. The mean serum creatine ki-nase concentrations in the patients with mutationsin the a-sarcoglycan, b-sarcoglycan, and g-sarcogly-can genes were 13,732�6196, 15,226�12,528, and16,900�8224 IU per liter, respectively (normal,�200).

DISCUSSION

Mutations of any component of the sarcoglycancomplex result in the secondary loss of the othercomponents of the complex. Thus, we hypothesizedthat we could identify patients who were likely tohave mutations in the a-, b-, or g-sarcoglycan geneby immunostaining for a-sarcoglycan. Among the556 patients with normal dystrophin whom westudied, 54 had a deficiency of a-sarcoglycan on im-munostaining. Molecular studies were performed in50 of these 54 patients and revealed that 58 percenthad mutations of the a-, b-, or g-sarcoglycan gene.It is therefore clear that a mutation in these genesdoes not account for all cases of a-sarcoglycan defi-ciency, even after adjustment for the secondary defi-ciency of the sarcoglycan complex in Duchenne’sand Becker’s muscular dystrophies.

Some patients with primary sarcoglycan defici-ency (that involving a mutation) might have beenmissed by our use of a-sarcoglycan immunostainingas the initial screening test. However, the availabledata suggest that we should have identified most ifnot all patients with sarcoglycan-gene mutationsusing this approach.8-10 Previous studies have iden-tified two unrelated families with mutations in theb-sarcoglycan gene and four unrelated families withmutations in the g-sarcoglycan gene; all patients hada-sarcoglycan deficiency in muscle, even though this

deficiency was not a criterion for the identificationof the study patients. Moreover, the patients withmutations in the b- or g-sarcoglycan gene in thisstudy all had marked reductions in the levels of theseproteins on immunostaining (data not shown).

We may not have detected all sarcoglycan muta-tions in the biopsy specimens tested, but we thinkthat this possibility is unlikely. In autosomal reces-sive disorders there are mutations in both alleles ofthe particular gene, and if the mutation-detectionstrategy lacks sensitivity, then only one mutationwill be detected in some patients (they will appearto be heterozygous). We found only 4 of 29 patientsto be heterozygous in this study, suggesting that thesensitivity of our mutation analysis was approximate-ly 93 percent (54 of 58 possible mutant alleles de-tected) and that our ability to identify patients withat least one mutant allele exceeded 99 percent.

The large number of patients studied allowed usto estimate the frequencies of sarcoglycan-gene mu-tations in patients with myopathies and normaldystrophin (Table 1). We found a deficiency ofa-sarcoglycan protein in 10 percent of patients withnormal dystrophin. If we include only patients withDuchenne-like or limb-girdle muscular dystrophy inthe analysis, this figure rises to 20 percent. In thepatients with a-sarcoglycan deficiency on immuno-staining, a complete deficiency (absence of staining)was more specific for mutations in the a-sarcoglycangene than for mutations in the b- or g-sarcoglycangene (Table 2). On the other hand, a partial defi-ciency of a-sarcoglycan on immunostaining was notspecific for mutations of any one of the three genes,and the majority (61 percent) of patients with a par-tial deficiency had no mutations.

We, like others, found that patients with a primarysarcoglycanopathy are clinically indistinguishable fromthose with either Duchenne’s or Becker’s musculardystrophy.7-17 Cognitive function was normal in allpatients with sarcoglycan-gene mutations. Althoughsarcoglycans are normally present in both skeletaland cardiac muscle, there were no electrocardio-graphic or echocardiographic abnormalities in theseven patients tested. There were differences in theclinical severity, in that all patients with mutations inthe b-sarcoglycan gene had severe dystrophy, where-as half those with mutations in the a- or g-sarcogly-can gene had milder disease.

Many genes are known to cause muscular dystro-phy, including the genes for dystrophin (Duchenne’sor Becker’s muscular dystrophy), calpain III (limb-girdle muscular dystrophy type 2A), merosin (con-genital muscular dystrophy with merosin deficiency),and a-, b-, g-, and d-sarcoglycan (limb-girdle mus-cular dystrophy types 2D, 2E, 2C, and 2F, respective-ly). Molecular genetic analyses can now identify thedisease-causing mutations in all these genes.

The muscular dystrophies characterized by pro-


624 � Februar y 27, 1997


gressive muscle weakness of the pelvic and shouldergirdle (Duchenne’s, Becker’s, and limb-girdle muscu-lar dystrophies) are a clinically and genetically hetero-geneous group of diseases. The elucidation of the ge-netic defect leading to the dystrophy has implicationsfor the diagnosis and care of affected patients (andtheir families). Because defects in several genes cancause phenotypically similar clinical and biochemicalmanifestations, a definitive diagnosis can only bemade by testing of candidate genes or proteins.

Supported by grants from the National Institutes of Health (NS-28403[to Dr. Hoffman]), the Muscular Dystrophy Association (to Dr. Hoffman),and Telethon–Italy (695 [to Ms. Fanin] and 552 [to Dr. Angelini]). Dr.Hoffman is an Established Investigator of the American Heart Association.

REFERENCES

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Mutations in the Sarcoglycan Genes in Patients with Myopathy

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