Genomic screening for [beta]-sarcoglycan gene mutations: missense mutations may cause severe limb-girdle muscular dystrophy type 2E (LGMD 2E)
Genomic screening for [beta]-sarcoglycan gene mutations: missense mutations may cause severe limb-girdle muscular dystrophy type 2E (LGMD 2E)Carsten G. Bönnemann1,2, M. Rita Passos-Bueno3, Elizabeth M. McNally1, Mariz Vainzof2, Eloísa de Sá Moreira3, Suely K. Marie4, Rita C. M. Pavanello3, Satoru Noguchi5, Eijiro Ozawa5, Mayana Zatz3 and Louis M. Kunkel1,*
1Howard Hughes Medical Institute and Division of Genetics, and 2Department of Neurology, Children's Hospital, and Harvard Medical School, Boston, MA 02115, USA, 3Departamento di Biologia, Instituto de Biosciências, and 4Departamento de Neurologia, Faculdade de Medicina, Universidade de São Paulo, CEP 05508-900 São Paulo, Brazil and 5National Institute of Neuroscience, Kodaira, Tokyo 187, Japan
Received July 15, 1996;Revised and Accepted September 17, 1996
Autosomal recessive limb-girdle muscular dystrophies (LGMDs) are genetically heterogeneous. A subgroup of these disorders is caused by mutations in the dystrophin-associated sarcoglycan complex. Truncating mutations in the 43 kDa [beta]-sarcoglycan gene (LGMD 2E) were originally identified in a sporadic case of Duchenne-like muscular dystrophy, and a common missense mutation (T151R) was identified independently in Indiana Amish pedigrees with a milder form of LGMD. To facilitate mutational analysis of larger numbers of patients directly from genomic DNA, as opposed to reverse transcribed RNA from muscle biopsies, we have determined the genomic structure of the [beta]-sarcoglycan gene. The open reading frame of the [beta]-sarcoglycan coding region extends over six exons. Primers were designed for PCR amplification of single exons from genomic DNA and subsequent single strand conformation polymorphism (SSCP) analysis. We screened 15 patients from the Brazilian LGMD patient population, 13 of whom followed a severe course. Most of the patients had been assessed previously for deficiency of [alpha]-sarcoglycan immunofluorescence on muscle biopsy sections as a marker for disease of the sarcoglycan complex. Novel mutations in two familial and two sporadic cases of severe childhood-onset LGMD were identified. Only one of these patients carried a truncating mutation (homozygous 2 bp deletion, FS164TER), while the other three carried missense mutations (homozygous R91P, homozygous M100K, heterozygous recessive L108R; only one allele could be identified in this family). All three missense mutations occurred in exon 3, coding for the immediate extracellular domain. Complete absence for all three of the known sarcoglycans was noted by immunohistochemistry on muscle biopsy sections of the patients.
Limb-girdle muscular dystrophies (LGMDs) are a genetically heterogeneous group of disorders, characterized clinically by predominantly proximal muscle weakness of variable severity and dystrophic changes on muscle biopsy. In addition to the X-linked recessive Duchenne and Becker muscular dystrophies caused by mutations in the gene locus encoding dystrophin (1 -3 ), a number of autosomal genes have been implicated recently in LGMD. Autosomal dominant and recessive forms of LGMD are known to exist. The phenotypic spectrum ranges from severe LGMDs of childhood, resembling Duchenne muscular dystrophy, to presentations with slower rate of progression or later age of onset. The autosomal recessive group [LGMD type 2 in the nomenclature of Bushby and Beckmann (4 )] in particular shows a remarkable degree of genetic heterogeneity. Included in this group are muscular dystrophies that are caused by mutations in the genes coding for the components of the sarcoglycan complex, [alpha]-, [beta]- and [gamma]-sarcoglycan, located on chromosomes 17q21, 4q12 and 13q12 and referred to as LGMD 2D, 2E and 2C, respectively (5 -12 ). Very recently, a fourth sarcoglycan-deficient LGMD has been mapped to chromosome 5q33-34 (LGMD 2F) (13 ). Type 2A appears to be different from the types of LGMD that affect the sarcoglycan complex; it is associated with mutations in the gene for calpain 3 located on chromosome 15 (14 ). Type 2B shows genetic linkage to chromosome 2 (15 ); the disease-causing gene for this disorder is still unknown.
The sarcoglycan proteins were identified as part of a complex of proteins which co-purifies with dystrophin from skeletal muscle membrane preparations (16 ,17 ). Dystrophin, a large rod-like subsarcolemmal protein, appears to provide a link from actin within the cell to the extracellular matrix component laminin-2 via the dystrophin-associated proteins [alpha]- and [beta]-dystroglycan (18 -22 ). The known sarcoglycans, proteins of mol. wts of 50 ([alpha], formerly known as adhalin), 43 ([beta]), and 35 kDa ([gamma]), can be separated from the other dystrophin-associated proteins using n-octyl [beta]-D-glucoside (23 ), and thus may constitute a complex independent from dystroglycan (24 ). A second 35 kDa sarcoglycan, [delta]-sarcoglycan, has been identified recently (25 ) and the gene mapped to the same genomic region (5q33-34) as LGMD 2F (13 ), making it a likely candidate for this disorder. By immunohistochemistry, the sarcoglycan proteins have been shown to be collectively deficient in heterogeneous cases of LGMD (10 -12 ,26 ). This evidence indicates the functional importance of the complex as a unit, but its normal biological function is presently unclear.
The [beta]-sarcoglycan gene on chromosome 4q12 has been isolated recently (10 ,11 ). Mutations identified so far have included a frameshifting and a nonsense mutation in a young girl presenting with a Duchenne-like muscular dystrophy (10 ) as well as a homozygous missense mutation (T151R) in Amish families in Southern Indiana (11 ), in whom the disorder follows a generally milder course. Screening for [beta]-sarcoglycan mutations has, until now, only been possible on cDNA obtained by reverse transcription of RNA isolated from muscle biopsies. To facilitate mutational analysis through use of much more commonly available genomic DNA sources, we defined the exon-intron structure for the [beta]-sarcoglycan gene across the coding region as well as the promoter sequence. A set of primer pairs was designed to allow the PCR amplification of single exons from genomic DNA for mutation detection by single strand conformation polymorphism (SSCP) analysis. The genomic screening assay for [beta]-sarcoglycan mutations was then used to study a population of Brazilian patients mostly with a severe LGMD of childhood. Most of the patients were shown to be [alpha]-sarcoglycan deficient but dystrophin positive by immunohistochemistry on muscle biopsy sections. Among the 15 patients studied, two familial and two sporadic cases were found to bear primary mutations in [beta]-sarcoglycan. Interestingly, three of these novel mutations were missense changes underlying the severe phenotype of LGMD.
The six exons encoding [beta]-sarcoglycan span ~13.5 kb of genomic DNA (Fig. 1 ). Coding exon sizes range from 33 bp for exon 1 to 210 bp for exon 2 (Table 1 ). Exon-intron borders were determined by direct sequencing using genomic phage as a template for intron 5 and by direct sequencing of the borders of intron-spanning PCR products for introns 2-4. The border of intron 1 to exon 2 was determined using PCR against a linker adapter ligated library of uncloned human genomic DNA. The nested primers on exon 2 were designed to yield a product that would extend exon 2 upstream into intron 1. The intronic end of this PCR product was used as a starting point for a second PCR directed further upstream against the linker adapter ligated library of uncloned human genomic DNA and also as a template for a probe to isolate a genomic phage containing exon 1. The border of exon 1 to intron 1 was determined by sequencing a subclone of this genomic phage. Intron sizes were estimated by intron-spanning PCR followed by agarose gel electrophoresis.
The subclone containing exon 1 also contained 1146 bp of sequence upstream of the translational initiation codon (GenBank accession no. U63796). The main transcriptional start is estimated to occur 40 bp upstream of the translational initiation codon, based on our previously reported 5' RACE experiments (10 ), and sequence similarity to the transcriptional initiator consensus sequence (27 ). The genomic region 5' to the predicted transcriptional start of [beta]-sarcoglycan does not appear to have a TATA or CAAT box . A number of potential SP1 sites, as well as a number of other putative transcription factor binding sites, could be identified. These include four putative E boxes (CANNTG) at positions -406, -669, -767 and -898 relative to the assumed transcriptional start site. The entire genomic region around exon 1 was found to be highly CG rich. Sequence homology searches of the EMBL database using the genomic sequences isolated for the 5' region of the [beta]-sarcoglycan gene (GenBank accession no. U63796) identified sequence HS48D12F (accession no. Z65591). HS48D12F is a 287 base long sequence of the end (forward read) of a clone 48d12 that had been isolated using a CpG island-enriching affinity column strategy (28 ) in an effort to establish a human CpG island library. The sequence is identical to our clone no.U63796 from base pair 134 in our sequence onwards (973 bp upstream of the assumed transcriptional start site). The reverse read HS48D12R (accession no. Z65592) from the other end of the same CpG island clone 48d12 was found to be identical to the upstream end of the second PCR performed against the linker adapter ligated library of uncloned human genomic DNA. This sequence lies still within intron 1. Thus, the CpG island clone 48d12 is predicted to contain the [beta]-sarcoglycan gene promoter and exon 1.
Fifteen patients mostly with severe forms of LGMD were selected for mutation analysis of the [beta]-sarcoglycan gene. Thirteen of the 15 patients had developed symptoms of muscular dystrophy prior to the age of 8 years and lost the ability to ambulate in their second decade. Two patients had a milder course with only mild weakness evident at 10 and 15 years of age respectively. All patients showed a pattern of weakness that predominantly affected the proximal limb girdle. Sections of muscle biopsy were available for 13 patients and showed that dystrophin was present and [alpha]-sarcoglycan immunofluorescence was absent or severely deficient. Genomic DNA was isolated from peripheral blood of each patient and analyzed by PCR using primers flanking the individual exons (Table 1 ). PCR products were subjected to SSCP analysis on Mutation Detection Enhancement (MDETM) gels both with and without the addition of glycerol. Abnormally migrating conformers were excised from the gel, reamplified and directly sequenced. Sequencing of the reamplified PCR product revealed one microdeletion and three missense mutations in the [beta]-sarcoglycan gene (Fig. 2 ). All nucleotide positions in the following are given according to the numbering in (10 ), accession no. U31116, with the first base of the first methionine codon as position 1. The three missense mutations were all found in exon 3 (Fig. 2 ). Patient 1-a was found to be homozygous for a G -> C transversion at nucleotide position 272, predicting an arginine -> proline substitution at amino acid position 91 (R91P). Patient 2-a was found to have a T -> G transversion at nucleotide position 323, predicting a leucine -> arginine substitution at amino acid position 108 (L108R). The patient was heterozygous for the mutation; no second allele different from normal was detected. Patient 3 was found to be homozygous for a T -> A transversion at nucleotide position 299, predicting a methionine -> lysine substitution at amino acid position 100 (M100K). Patient 4 was found to be homozygous for a 2 bp deletion in exon 4 (delAG, nucleotide positions 465 and 466), which causes a frameshift leading to a premature stop at amino acid position 164 after eight missense amino acids (FS164TER). This predicts a truncation of the protein ablating the terminal two-thirds of the extracellular portion of [beta]-sarcoglycan, including all three glycosylation sites. None of the missense mutations were detected on 168 control chromosomes assayed by SSCP analysis. The mutated amino acids R91, M100 and L108 are evolutionarily conserved in the rabbit (10 ), hamster (29 ) and mouse (Bönnemann and Kunkel, unpublished), indicating potential functional importance of these residues.
Patient 1-a had two affected brothers (1-b and 1-c) and four unaffected siblings. The segregation of the G -> C change was shown in this family using an `amplification refractory mutation system' (ARMS) assay (30 ) (Fig. 3 a). In this family, both parents were heterozygous for the mutation, while all three affected siblings were homozygous. Although the parents are not closely related, they both originate from the same small village in Brazil and may thus be distantly related. Patient 2-a had an affected brother (2-b) who died at the age of 16 years from muscular dystrophy; DNA was not available from him. By SSCP analysis, the T -> G transversion was found to be inherited from the father (Fig. 3 b). Neither the mother nor any of the three unaffected siblings carried the change. Patient 3 is the offspring of a first cousin marriage, and has one unaffected brother. DNA from the other family members was not available. Patient 4 is the only affected child among five siblings. No DNA was available for analysis from the other family members. The parents also originated from the same small village in Brazil.
An affinity-purified polyclonal antibody against a full-length [beta]-sarcoglycan-GST fusion protein was generated for immunohistochemistry. An anti-[gamma]-sarcoglycan polyclonal antibody was generated similarly (E.M. McNally and L.M. Kunkel, unpublished). Immunohistochemical analysis of four of the patients (1-a, 1-b, 2-a and 4) was performed. In patient 1-b complete absence of [alpha]-, [beta]- as well as [gamma]-sarcoglycan was found (Fig. 4 ). Patients 1-a and 4 showed similar findings (not shown). In patient 2-a, only [alpha]- and [gamma]-sarcoglycan could be studied and were found to be absent (not shown). This loss was irrespective of the primary mutation in [beta]-sarcoglycan. Dystrophin was present at the membrane in all the samples (data not shown).
[beta]-Sarcoglycan is encoded by six exons on chromosome 4q12. The [beta]-sarcoglycan gene promoter region, exon 1 and the adjacent intron 1 sequences have a very high CG content, suggesting the presence of a CpG island around the 5' end of the gene. This finding is supported by the identification in database searches of a genomic clone with sequence identity that had been isolated using a strategy to enrich for unmethylated CpG islands (28 ). Based on our previously reported 5' RACE experiments (10 ), as well as surrounding consensus sequence, we estimate the transcriptional start site for the [beta]-sarcoglycan mRNA to lie ~40 bases upstream from the translational initiation codon. However, alternative transcriptional initiation sites in the vicinity cannot be excluded. The promoter contains a number of SP1 sites characteristic of CG-rich TATA-less eukaryotic promoters (31 ). There are four putative E boxes of the consensus sequence CANNTG contained within the analyzed 5' regulatory sequence. These elements are frequently found in muscle-specific enhancers and can be recognized potentially by helix-loop-helix proteins of the MyoD family (32 ). Several other putative transcription factor binding sites were identified in addition (not shown); however, detailed functional promoter analysis will be necessary to delineate promotor elements relevant for [beta]-sarcoglycan gene regulation.
Based on the intron sequences, primers were designed to allow each exon to be subjected to SSCP analysis. We have applied this genomic screening assay for [beta]-sarcoglycan gene mutations to a population of muscular dystrophy patients in Brazil presenting mainly with a severe LGMD of childhood. Previous studies in this population have clearly demonstrated the genetic heterogeneity of LGMD (9 ,33 -35 ). The existence of one or more additional genetic loci responsible for autosomal recessive LGMD had already been predicted based on analysis and exclusion of the then known loci on chromosomes 15q (type 2A), 2p (type 2B), 13q (type 2C) and 17q (type 2 D) for a subset of the familial cases (36 ). These additional loci have now been identified as LGMD 2E (reported here), and a newly discovered sarcoglycan-deficient LGMD 2F locus on chromosome 5q33-34 (13 ). There is evidence for at least one additional locus for sarcoglycan-positive LGMD (13 ).
For those patients for whom muscle biopsy material was available, we have narrowed our sample further by only including patients with positive staining for dystrophin and deficiency of [alpha]-sarcoglycan as judged by immunohistochemistry. The two patients for whom no more biopsy material was available were included as well. The [alpha]-sarcoglycan antibody has already been used extensively for immunohistochemistry to characterize types of muscular dystrophy (33 ,34 ,37 -39 ). This screening tool is useful under the assumption that [beta]-sarcoglycan mutations will be reflected by [alpha]-sarcoglycan deficiency. Deficiency of the three known sarcoglycans has been observed in the [beta]-sarcoglycan mutations identified so far (10 ,11 ), and this combined secondary deficiency may be a more general marker of diseases of the sarcoglycan complex (12 ,24 ,26 ,40 ). Although [alpha]-sarcoglycan deficiency thus probably is a reasonably good indicator of a sarcoglycan disorder, a screen not based on the ascertainment of [alpha]-sarcoglycan deficiency will be needed in the future to investigate the possibility of [beta]-sarcoglycan gene mutations that do not necessarily cause a secondary deficiency of [alpha]-sarcoglycan on immunohistochemistry.
In our screen of 15 such selected patients, nine of which were familial cases, we identified four index cases with novel [beta]-sarcoglycan mutations. Two families had multiple affected members, and two had only one affected child. Seven patients were affected in total. The phenotype of all seven patients was that of a severe LGMD with onset before age 7, loss of independent ambulation in the early teens, weakness in the pelvic girdle greater than in the shoulder girdle, sparing of the extraocular muscles, no cognitive impairment and elevation of serum creatine kinase activities. The disease severity in these patients resembles that of Duchenne muscular dystrophy, justifying the designation as severe LGMD of childhood. One of these mutations was a homozygous 2 bp deletion, causing a frameshift; the other three were missense mutations.
The functional consequences of missense mutations are difficult to predict. They depend on the amino acid change they are predicted to cause, and their precise location within the protein. In addition, there may be effects on the processing, transport and stability of the mutant protein (41 ). Nonetheless, the ascertainment of two homozygous missense mutations in our sample, rather than compound heterozygotes, may be helpful in trying to understand potential structure-function correlations in [beta]-sarcoglycan. [beta]-Sarcoglycan is predicted to be a type II transmembrane protein with the C-terminal site providing the extracellular domain (10 ,11 ). The Arg 91 -> Pro substitution in patients 1-a, 1-b and 1-c lies just behind the transmembrane stretch, and would be predicted to cause a change in the secondary structure of the protein by introducing a sharp bend, probably rendering its proper insertion into the membrane and orderly assembly of the complex impossible. The methionine to lysine change at position 100 in patient 3 also occurs in this immediate extracellular domain. For one patient (2-a), only one mutant allele was found. Detection of the other mutant allele might have escaped the sensitivity of the SSCP assay, or the mutation could conceivably also lie in the 5' regulatory regions that were not assayed for, potentially affecting transcription of one allele. An additional possibility would be a larger deletion on the other allele, but sparing exon 3. The mutation is predicted to change an aliphatic amino acid, leucine, to a charged one, arginine. In the three patients with [beta]-sarcoglycan mutations analyzed by immunohistochemistry, we observed complete absence of all the three sarcoglycans from the membrane on muscle biopsy sections, irrespective of their primary mutation in [beta]-sarcoglycan. Thus, [beta]-sarcoglycan may possibly have a critical role for assembly and/or maintenance of the sarcoglycan complex. Such a role could render the entire complex more `vulnerable' to missense changes in [beta]-sarcoglycan. The impairment of assembly and/or maintenance of the complex would then lead to the rapid proteolytic degradation of all the sarcoglycan proteins with resulting negative immunostaining for the complex. Interestingly, all three missense mutations occurred in the immediate extracellular domain. The clustering of the mutations in this domain in patients with a severe phenotype may point towards this region as critical for interactions within the complex. These findings have to be evaluated in comparison with the more extensively studied primary [alpha]-sarcoglycan disorder in which the more commonly found missense mutations in general seem to show a trend towards a milder phenotype (5 -9 ). The one [beta]-sarcoglycan missense mutation in exon 4 described by (11 ) in the Amish LGMD 2E families (T151R) caused a milder phenotype as well, although with considerable variability towards a more severe course. Whether [beta]-sarcoglycan missense mutations on the whole, or exon 3 mutations in particular, will indeed show a tendency towards a more severe phenotype cannot yet be concluded from this study, given our selection bias for such a severe phenotype and the still small numbers of patients identified.
There were no appreciable clinical differences between patients with mutations in the [beta]-sarcoglycan gene compared with those in whom no mutations in this gene could be found. It has to be concluded that in patients presenting with LGMD and immunohistochemical evidence of a disease of the sarcoglycan complex, genetic analysis of all the potential genes known to be involved in such a phenotype will be necessary. The availability of screening tools using genomic DNA should be very helpful to that end. It remains to be seen whether careful immunohistochemical and possibly Western blot analysis of all sarcoglycans will be able to predict the most likely gene locus affected in a given case.
Most exon-intron borders were obtained by direct PCR amplification across introns 2, 3 and 4 using exonic primers and the long range polymerase ExTaq (Takara, Kyoto, Japan). PCR products were isolated on a low melt agarose gel, excised from the gel and purified using the Wizard PCR purification system (Promega, Madison, WI). Aliquots of the purified products were sequenced directly using the primers used in the amplification. Exon-intron borders were identified at the point of divergence of the sequence from the known cDNA sequence [GenBank accession no. U31116 (10 )] and by virtue of their conformation with splice donor and acceptor consensus sequences. For intron 5, the borders were determined by direct sequencing using the phage [lambda]EMBL4-3 as a template (10 ) with primers located on exon 5 and on exon 6. All sequencing in this study was done on an ABI automated sequencer using Taq cycle sequencing. Sequences of the exon-intron borders are deposited under GenBank accession nos U63796-U63801.
To determine the border between intron 1 and exon 2, a library of uncloned, linker-adapted genomic DNA fragments (Promoter finder kit, Clontech, Palo Alto, CA) was used according to the manufacturers instructions. Using nested exonic primers under amplification conditions according to the manufacturer, a 3 kb PCR product was generated and subcloned into the TA cloning vector (Invitrogen, San Diego, CA). The intron border was determined by sequencing the ends of the subclone. Sequencing of the opposite end of the insert yielded sequence within intron 1. To obtain a genomic clone extending 5' to contain exon 1, a 32P-labeled probe was generated by PCR from the end of the subclone that located to within intron 1. This probe was used to hybridize a human fibroblast genomic phage library (Invitrogen, San Diego, CA) according to standard procedures (42 ). Positively hybridizing clones were isolated and characterized by restriction analysis. To examine whether these phage clones contained sequences from exon 1, a [[gamma]-32P]dATP kinase-labeled (42 ) oligonucleotide designed from the 5'-untranslated region of the [beta]-sarcoglycan cDNA was prepared. A 5.5 kb BamHI-HindIII restriction fragment was identified and subcloned into pBluescript and sequenced to identify the border of exon 1 to intron 1 as well as the genomic sequence upstream of the translational start (GenBank accession no. U63796). Trans-intron PCR was performed as outlined before, using exonic primers to estimate intron sizes.
The 15 patients were ascertained at the Centro de Miopatias of the University of São Paulo and selected according to the following criteria: predominantly proximal muscle weakness, elevated serum creatine kinase measurements in the serum, family history not compatible with X-linked inheritance and normal dystrophin on Western blot or immunohistochemical analysis. Histological analysis of the muscle biopsies was consistent with a muscular dystrophy indistinguishable from the histology seen in Duchenne and Becker muscular dystrophies. With the exception of two cases who could not be assessed (there was no more muscle biopsy available, both patients were female and both subsequently were discovered to carry mutations in [beta]- and [gamma]-sarcoglycan, respectively) all others were deficient for [alpha]-sarcoglycan as judged by immunohistochemical analysis on muscle biopsy sections. Thirteen of the 15 patients followed a severe course with onset of clinical signs and symptoms before 8 years of age and loss of ambulation in their early teens. Two patients aged 10 and 15 years respectively had a milder course. Among the 15 unrelated index cases which were screened, six were isolated cases and nine belonged to families with multiple affected patients. In eight of the 15 families, the parents of the proband were first or second degree relatives, while in the remaining seven distant consanguinity could not be excluded because both parents came from the same small village. Informed consent was obtained from the participating families, and genomic DNA was extracted from peripheral blood according to (43 ).
A total of 90 ng of genomic DNA was amplified in a 10 [mu]l reaction as described in (10 ). Primer pairs used are given in Table 1 . Cycling conditions included 2.5 min initial denaturation at 94oC, followed by 35 cycles of 1 min annealing at 55oC, 30 s extension at 72oC, 40 s denaturation at 94oC. For amplification of exon 1, the reaction mix contained 8% dimethylsulfoxide (DMSO) and 1 U of Taq polymerase and the annealing temperature was raised to 62oC. Gel analysis was performed as described (10 ). Briefly, samples were denatured and analyzed under non-denaturing gel condition by electrophoresis in 0.5* MDETM (FMC Bioproducts, Rockland, ME)/0.6*BE and 5-10% glycerol/0.5* MDETM/0.6* TBE at either 600 V or 7 W constant at room temperature for 10-14 h. Gels were dried and exposed to film for 4-18 h at -80oC. Aberrant conformers were excised from the gel, eluted and reamplified for direct sequencing using an ABI automatic sequencer as described (10 ). All mutations were confirmed by direct sequencing of independently generated PCR products from genomic DNA. Sequence analysis was performed using the Sequencher sequence analysis package (Gene Codes, Ann Arbor, MI).
The inheritance of the mutation in family 1 (G272 -> C) was confirmed using ARMS essentially as described in (44 ) and (45 ). A wild-type-specific primer (5' act cca tac tat cac agc cat ttg gtc caa tac 3') or a mutation-specific primer (5' act cca tac tat cac agc cat ttg gtc caa tag 3') were used in conjunction with a common primer located in intron 2 (5' tgg tga taa tat ttt cta ctt gtt ttc caa tta c 3'). Control primers were those given in (44 ,45 ) for a 360 bp fragment from exon III of the [alpha]1-antitrypsin gene. Amplification conditions included denaturation at 94oC for 1 min, annealing at 60oC for 1 min, and extension at 72oC for 1 min for 36 cycles. The reaction was started by manual hotstart with the addition of enzyme to the reaction mixture after 2.5 min incubation of the reaction mixture without enzyme at 95oC. Products were separated by electrophoresis in 3% agarose and visualized with ethidium bromide for gel photography.
To generate the template for a glutathione-S-transferase fusion protein, a linker-adapted full-length [beta]-sarcoglycan cDNA (10 ) was generated by PCR and ligated into pGEX-4T-1 (Pharmacia, Upsala, Sweden). The fusion protein was expressed in Escherichia coli. After lysis and centrifugation of the bacterial culture, the protein was found to be insoluble. An aliquot of the resuspended pellet was separated on a polyacrylamide curtain gel, stained with 4 M Na acetate, and a gel slice containing the overexpressed protein excised. The gel slice was homogenized in liquid nitrogen, resuspended in water and injected into New Zealand white rabbits. Serum was tested for specific antibodies by Western blot analysis and by immunohistochemistry on muscle sections. Serum was affinity purified over a column of GST-[beta]-sarcoglycan fusion protein coupled to N-hydroxysuccimide ester-activated agarose (Bio-Rad, Hercules, CA). Immunohistochemistry was performed essentially as described (10 ). Briefly, 7 [mu]m sections cut from snap-frozen muscle biopsies were stained with antibodies against dystrophin (antibody generously provided by Jeffrey Chamberlain), [alpha]-sarcoglycan (monoclonal antibody generously provided by Louise Anderson), [beta]-sarcoglycan, as described here, and [gamma]-sarcoglycan (polyclonal antibody raised against a [gamma]-sarcoglycan-GST fusion protein, E.M. McNally and L.M. Kunkel, unpublished). Sections were reacted with goat anti-rabbit or anti-mouse antibody conjugated with Cy3 (Jackson Immunochemicals, West Grove, PA) and visualized and photographed on a Zeiss Axiophot microscope.
The authors wish to thank Dick Bennett and Gigi Bang for their expert assistance with the sequencing, and the members of the Kunkel laboratory for their insightful review of the manuscript. The invaluable help of the following individuals is gratefully acknowledged: Marta Canovas, Antonia Cerqueira, Simone Campiotti, João Ricardo de Oliveira and Constancia Urbani. We are also grateful to Dr Ivo Pavanello for performing the muscle biopsies. E.M.M. is supported by NIH NL 03448-01. The project described was supported by grant number 5 RO1 NS 23740-09 of the NINDS to L.M.K.. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NINDS. L.M.K. is an investigator of the Howard Hughes Medical Institute. Support for this project was also provided by FAPESP, PADCT, CNPq, the International Atomic Energy Agency and the MDA to M.Z. Additional support was provided by Research Grant (5A-1) for Nervous and Mental Disorders from the Ministry of Health and Welfare, Japan to E.O.
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A mutated [delta]-sarcoglycan gene has now been found in families with LGMD 2F (V. Nigro et al., Nature Genet., 14, 195-198.)
*To whom correspondence should be addressed
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