The Seventh Form of Autosomal Recessive Limb-Girdle Muscular Dystrophy Is Mapped to 17q11-12

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1 Am. J. Hum. Genet. 61: , 1997 The Seventh Form of Autosomal Recessive Limb-Girdle Muscular Dystrophy Is Mapped to 17q11-12 Eloisa S. Moreira, Mariz Vainzof, Sueli K. Marie, Andrea L. Sertié, Mayana Zatz, and Maria R. Passos-Bueno Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, São Paulo Summary Introduction The group of autosomal recessive (AR) muscular dystrophies Autosomal recessive (AR) limb-girdle muscular dystrophies includes, among others, two main clinical entities, (LGMDs) represent a heterogeneous group of dis- the limb-girdle muscular dystrophies (LGMDs) and the eases, characterized by a primary and progressive muscle distal muscular dystrophies. The former are characterspectrum degeneration of the pelvic and shoulder girdles. A wide ized mainly by muscle wasting of the upper and lower of clinical disability is observed, ranging from limbs, with a wide range of clinical severity. This clinical very mild to severe forms (Walton and Gardner-Medwin heterogeneity has been demonstrated at the molecular 1988; Bushby and Beckmann 1995). level, since the genes for six AR forms have been cloned Within the last few years, six genes that are responsi- and/or have been mapped to 15q15.1 (LGMD2A), ble for the AR LGMDs have been mapped and/or 2p12-16 (LGMD2B), 13q12 (LGMD2C), 17q12- cloned, confirming their clinical heterogeneity at the moq21.33 (LGMD2D), 4q12 (LGMD2E), and 5q33-34 lecular level. These six different forms have been named (LGMD2F). The AR distal muscular dystrophies origias follows: LGMD2A at 15q15.1 (Beckmann et al. 1991; chronologically, according to their gene identification, nally included two subgroups, Miyoshi myopathy, characterized mainly by extremely elevated serum creatine Richard et al. 1995), LGMD2B at 2p12-16 (Bashir et kinase (CK) activity and by a dystrophic muscle pattern, al. 1994), LGMD2C at 13q12 (Othmane et al. 1992; and Nonaka myopathy, which is distinct from the others Noguchi et al. 1995), LGMD2D at 17q12-q21.33 (Rob- because of the normal to slightly elevated serum CK erds et al. 1993, 1994; McNally et al. 1994), LGMD2E levels and a myopathic muscle pattern with rimmed vac- at 4q12 (Bönnemann et al. 1995; Lim et al. 1995), and uoles. With regard to our unclassified AR LGMD fami- LGMD2F at 5q33-34 (Nigro et al. 1996a, 1996b; lies, analysis of the affected sibs from one of them (famgene, all the other LGMD genes have been cloned. The Passos-Bueno et al. 1996b). Except for the LGMD2B ily LG61) revealed some clinical and laboratory findings (early involvement of the distal muscles, mildly elevated gene responsible for LGMD2A encodes calpain 3, or serum CK levels, and rimmed vacuoles in muscle biop- CANP3, a muscle-specific protease (Richard et al. sies) that usually are not observed in the analysis of 1995), whereas the genes that cause LGMD2C patients with LGMD2A LGMD2F. In the present intein complex (DGC) (Roberds et al. 1993; McNally et LGMD2F encode proteins of the dystrophin-glycopro- vestigation, through a genomewide search in family LG61, we demonstrated linkage of the allele causing this al. 1994; Bönnemann et al. 1995; Lim et al. 1995; Noform of muscular dystrophy to a 3-cM region on 17q11- guchi et al. 1995; Richard et al. 1995; Nigro et al. 12. We suggest that this form, which, interestingly, clini- 1996b). cally resembles AR Kugelberg-Welander disease, should The DGC is a large complex formed by dystrophin be classified as LGMD2G. In addition, our results indi- and its associated proteins (DAPs) and glycoproteins cate the existence of still another locus causing severe (DAGs) (Campbell 1995; Ozawa et al. 1995; Worton LGMD. 1995). The DAPs (a-, b1-, and b2-syntrophin and dystrobrevin) are arranged in the syntrophin complex (Adams et al. 1993; Ahn et al. 1994; Peter et al. 1994; Yang et al. 1994; Sadoulet-Puccio et al. 1996). The DAGs are distributed between two subgroups, the dystroglycan Received February 20, 1997; accepted for publication May 2, complex (a- and b-dystroglycan) (Ibraghimov-Beskrov- Address for correspondence and reprints: Dr. Maria R. Passos- naya et al. 1993; Yoshida et al. 1994) and the sarcogly- Bueno, Departamento de Biologia, Instituto Biociências, Universidade can complex (a-, b-, g-, and d-sarcoglycan and 25DAG) de São Paulo, Rua do Matao, 277, CEP São Paulo, Brazil. (Campbell 1995; Ozawa et al. 1995). The DGC estab- passos@usp.br 1997 by The American Society of Human Genetics. All rights reserved. lishes a connection between the citoskeleton (through /97/ $02.00 actin) and the extracellular matrix (through laminin), 151

2 152 Am. J. Hum. Genet. 61: , 1997 protecting muscle fibers from mechanical damage during a rapid progression, with the proximal muscles of the contractions (Campbell 1995). It recently has been demonstrated legs, the small hand muscles, and the arm and neck that pathogenic mutations in the a-, b-, g-, muscles being involved at an early stage; (3) normal or and d-sarcoglycan genes are responsible for LGMD2D, mildly elevated serum CK levels; and (4) a myopathic LGMD2E, LGMD2C, and LGMD2F, respectively muscle pattern (with no apparent fiber necrosis and regeneration) (Roberds et al. 1994; Bönnemann et al. 1995; Lim et al. and the presence of rimmed vacuoles as a 1995; Noguchi et al. 1995; Nigro et al. 1996a). LGMD prominent feature (Nonaka et al. 1981; Isaacs et al. caused by a defect in any of the four components of the 1988; Sunohara et al. 1989; Yamanouchi et al. 1994; sarcoglycan complex has been classified as sarcoglyca- Murakami et al. 1995). The distinction between DMRV nopathy. and hereditary inclusion body myopathy (HIBM), which LGMD patients who have mutations in either the are both the result of AR inheritance, is very important LGMD2A locus or the LGMD2B locus have, in the majority (Nonaka et al. 1981; Argov and Yarom 1984). HIBM of cases, a relatively mild phenotype that is, they patients present with progressive muscular weakness, are able to walk unassisted after the age of 16 years normal or mildly increased serum CK levels, and a myo- (Beckmann et al. 1991; Bashir et al. 1994; Passos-Bueno pathic muscle pattern with rimmed vacuoles. However, et al. 1995a, 1996a; Richard et al. 1995). On the other the distinguishing features of this disorder, as compared hand, patients with any of the four sarcoglycanopathies with those of other myopathies, are the sparing of the usually have a severe phenotype associated with total or quadriceps and the age at onset, which is usually 20 partial deficiency of a-sarcoglycan in the muscle (Rob- 40 years of age (Argov and Yarom 1984; Mitrani-Rosenbaum erds et al. 1994; Bönnemann et al. 1995, 1996; Lim et al. et al. 1996). The gene that causes HIBM, 1995; Noguchi et al. 1995; Passos-Bueno et al. 1995b; which apparently has been reported only among individuals McNally et al. 1996; Nigro et al. 1996a; Vainzof et al. of Persian Jewish descent, recently was mapped to 1996). 9p1-q1 (Mitrani-Rosenbaum et al. 1996). AR muscular dystrophy patients with involvement of We have identified 26 families with AR LGMD (21 both the shoulder and pelvic girdles but with atrophy with mild forms and 5 with severe forms), on the basis beginning in the hands or in the feet have been reported of clinical evolution, high serum CK levels, and the anal- in previous studies (Nonaka et al. 1981; Argov and ysis of muscle biopsies (including the assessment of dystrophin Yarom 1984; Markesbery and Criggs 1986; Miyoshi et and of the four sarcoglycans). Linkage analysis al. 1986; Isaacs et al. 1988; Walton and Gardner-Med- of these 26 families has demonstrated the existence of win 1988; Sunohara et al. 1989; Yamanouchi et al. at least another locus, since 3 families still were unlinked 1994). These families originally had been classified as to any known LGMD loci, as well as to the HIBM locus having one of the two forms of distal muscular dystrophy, (Bönnemann et al. 1996; McNally et al. 1996; Passos- Miyoshi myopathy (MM) or Nonaka myopathy Bueno et al. 1996a, 1996b; E. S. Moreira, M. Zatz, and (also known as distal myopathy with rimmed-vacuole M. R. Passos-Bueno, unpublished data). Analysis of formation [ DMRV ]) (Nonaka et al. 1981; Miyoshi muscle biopsies from these three kindreds, which were et al. 1986). all positive for the sarcoglycan complex, revealed the MM is characterized by (1) very high serum creatine presence of a great number of rimmed vacuoles in one kinase (CK) activity; (2) muscle weakness and atrophy of them (family LG61). Since family LG61 is large most marked in the legs, with relative sparing of the enough for mapping through linkage analysis, we performed arms, neck, and small hand muscles; and (3) a dystrophic a genomewide search in this family. The results muscle pattern, with fiber necrosis and regeneration suggest the existence of another locus for AR LGMD at (Miyoshi et al. 1986; Bejaoui et al. 1995). The MM gene 17q recently was mapped to 2p12-14, to the same region as that of the gene for the LGMD2B form (Bejaoui et al. 1995). Weiler et al. (1996) described a large inbred ab- Subjects and Methods original Canadian kindred with AR muscular dystrophy Subjects also linked to this same chromosome 2p region. Interest- Three AR LGMD families (LG6, LG11, and LG61) ingly, seven individuals presented an LGMD phenotype reported in previous studies (Passos-Bueno et al. 1996a, and two an MM phenotype. These authors demon- 1996b) were included in the present analysis. Diagnosis strated that four LGMD patients and the two MM patients was based on clinical examination and course of the were homozygous for the same haplotype, sup- disease, family history, serum CK levels, and assessment porting the hypothesis that LGMD2B and MM may be of dystrophin through immunocytochemistry/western caused by mutations in the same gene. blotting of muscle biopsies. Serum CK analysis was performed DMRV is characterized by (1) onset in the distal muscles by use of Sigma kits; normal levels are consid- of the legs, in the second or third decade of life; (2) ered to be 10 sigma units (SU)/ml for adults and 20

3 Moreira et al.: Mapping of the LGMD2G Gene to 17q SU/ml for children. Five of the six affected sibs in family Table 1 LG61, as well as the three patients in family LG11, Size and Frequency of the Alleles That Are Segregating with the recently have been clinically and neurologically reexam- Disease Allele, in Families LG11 and LG61 ined by one of us (S.K.M.). Muscle strength was assessed in accordance with the manual muscle test, which is Size based on the Medical Research Council s recommendations. Marker and Allele(s) a (bp) Frequency Muscle biopsies of biceps were performed on at D17S1867: least one individual from each family. Dystrophin and a-sarcoglycan immunofluorescence analyses were per formed as reported in previous studies (Vainzof et al. D17S250: 1991; Passos-Bueno et al. 1995b) D17S1851: Genotyping DNA was extracted from whole blood, according to D17S946: standard protocols (Miller et al. 1988). For the ge nomewide search, DNA samples were analyzed by PCR amplification using microsatellite markers purchased D17S1818: from Research Genetics and from Isogen. For refinement of the candidate area, markers were selected from the D17S1814: study by Dib et al. (1996). Standard protocols were used for PCR reactions. The products were visualized on 6.5% denaturing gels, which were dried and exposed a The allele numbers correspond to those shown in the haplotypes to x-ray films for 2-24 h, as reported in a previous study of figure 2. (Passos-Bueno et al. 1995b). to perform ankle dorsiflexion. Difficulty with walking on the heels appeared prior to difficulty with walking on the toes. Extraocular and facial muscles were spared in all the patients. In five sibs, who recently have been clinically and neurologically reassessed (table 3), the neck muscles are only very mildly affected or not af- fected. In the upper limbs, proximal muscle atrophy is marked, whereas, in the lower limbs, proximal and distal muscle atrophy is evident, which is in accordance with the pattern of muscle weakness observed in both limb girdles (table 3). It is interesting that in patient II-3, who shows the mildest clinical evolution, the proximal muscles of the upper and lower limbs are less affected than the distal muscles (table 3). The tendon reflexes are abolished, without involvement of sensory and cranial nerves or of coordination. In addition, foot-drop is a common feature in all of these five patients. Four of the six affected sibs are already in a wheelchair. No evidence of cardiac disease was detected. CK levels were elevated 3 17-fold in the first stages of the disease, but they have decreased, as the patients have aged, to almost normal levels in those patients already in a wheelchair (table 2). Muscle Pathology The analysis of a muscle biopsy, from patient II-8 of family LG61, showed round-shaped fibers, with a marked variation in fiber size. Necrotic and regenerating fibers were found, with an increased number of central myonuclei. Infiltration of connective tissue also was ob- Linkage Analysis Two-point linkage analysis was performed by use of the computer program MLINK (Lathrop et al. 1984), with an estimated gene frequency of.001 for the disease allele and with equal recombination for both sexes. An equal frequency of the alleles for most of the markers was considered, although we are aware that this could have inflated LOD scores (Terwilliger and Ott 1994). Allele frequencies for the markers mapped to the candi- date region were estimated on the basis of the analysis of chromosomes from normal Caucasian subjects from our population (table 1). The sizes of the alleles were measured through comparison with the DNA se- quence of the bacteriophage M13mp18 (United States Biochemical). Results Pedigree and Clinical Description of Family LG61 Family LG61 is a Caucasian nonconsanguineous kindred of Italian ancestry. The parents (66 and 69 years of age) had a total of eight offspring two of which are unaffected (39 and 44 years of age) and six of which are affected (27 43 years of age). The main clinical findings for all the patients are described in table 2. This family was first seen in our center 20 years ago. The mean age at onset was 12.5 years, which is when the patients showed difficulty with walking, running, and climbing stairs. They report that at the same time or soon after the first signs were noted, they were unable

4 154 Am. J. Hum. Genet. 61: , 1997 Table 2 Main Clinical Findings in the LG61 Patients CK LEVEL (1 NORMAL) CURRENT AGE AGE AT ONSET WHEELCHAIR CONFINEMENT PATIENT (years) (years) (AGE [years]) II Yes (39) II No II Yes (31) II Yes (34) II Yes (31) II No additional candidate regions, where the syntrophin, dystroglycan, and HIBM genes have been mapped (Passos- Bueno et al. 1996a, 1996b; E. S. Moreira, M. Zatz, and M. R. Passos-Bueno, unpublished data). Therefore, a ge- nomewide search was performed for this genealogy. A total of 402 markers (spaced 5 10 cm) were tested in this kindred. Evidence of linkage was observed with the microsatellite marker D17S250 (Z max Å 3.26; u Å.00), which is located at 17q The following additional marker sequences near this locus were genotyped: D17S cm D17S cm D17S cm D17S1850; D17S cm D17S cm D17S933 1 cm D17S cm D17S1851; served. One rimmed vacuole per fiber was detected in a great proportion of the muscle cells (fig. 1A). There was no predominance of type 1 or type 2 fibers and no type grouping (fig. 1B). Dystrophin and a-sarcoglycan showed a positive sarcolemmal staining through immunohistochemical studies. Linkage Analysis In a previous study, family LG61 had been excluded for the six known AR LGMD loci, as well as for eight Table 3 Manual Muscle-Strength Evaluation of the Patients from Family LG61 STRENGTH RATING, FOR EACH PATIENT a MOVEMENT TESTED II-2 II-3 II-5 II-6 II-8 Neck flexion Neck extension Shoulder abduction Shoulder external rotation Elbow flexion Elbow extension Wrist flexion Wrist extension Thumb abduction Hip flexion Hip extension Knee flexion Knee extension Ankle dorsiflexion Ankle plantarflexion Toe flexion Toe extension Figure 1 A, Muscle biopsy, from patient II-8 of family LG61, showing evidence of rimmed vacuoles in affected fibers (haematoxylin and eosin). B, Muscle biopsy, from patient II-8 of family LG61, show- ing no predominance of type 1 or type 2 fibers (ATPase stain at ph 9.4). Bar Å 25 mm. a 0 Å no movement; 1 Å flicker of movement; 2 Å movement of the joint when the effect of gravity is eliminated; 3 Å movement through full range of the joint, against gravity but not against resistance; 4 Å movement of the joint, against gravity and against added resistance; and 5 Å full strength.

5 Moreira et al.: Mapping of the LGMD2G Gene to 17q Figure 2 Haplotypes with markers from the 17q11-12 region in the two LGMD2G families. The haplotype of individual I-1 from family LG11 was deduced from the family s descendants. The haplotype or the portions of it presumed to be ancestrally associated with the disease in the families are boxed; a partially boxed haplotype indicates that it was not possible to establish with certainty the haplotype at risk (owing to lack of informativeness of the parents). An asterisk (*) indicates a marker for which the exact position is unknown; the position estimate was based on the analysis of recombinants found in this genealogy (see text). D17S946 1 cm D17S cm D17S cm The markers from the candidate region also were D17S cm D17S1789; and D17S932 1 cm tested in two other families (LG6 and LG11), which are D17S1861. Although the exact position of D17S250 is unlinked to any known LGMD or candidate loci unknown, we have suggested that this marker may lie (Passos-Bueno et al., 1996a, 1996b; E. S. Moreira, M. between D17S1867 and D17S1814, on the basis of anal- Zatz, and M. R. Passos-Bueno, unpublished data). Positive ysis of recombinants observed in family LG61 (fig. 2). LOD scores were detected in only one family Results of two-point linkage analysis between these (LG11), which was genotyped further for the other 16 microsatellites and the disease gene in family LG61 are markers from the 17q11-12 region (table 4; fig. 2). described in table 4. Confirmation of linkage (LOD In family LG11, although the LOD score was õ3.00, score ú3.00) was obtained by use of two other markers, owing to the small family size, no recombinants were D17S1851 and D17S1818. found within an interval of 5 cm. In addition, homozygosity Two important recombinations were observed in this was observed between the markers D17S250 and genealogy, involving affected individual II-8 and one D17S1814. Interestingly, this haplotype, which was not of his normal sibs, individual II-1. In individual II-8, a found in our control population (data not shown), was recombination between D17S1867 and D17S1851 observed in heterozygosity, in all the affected sibs from placed the gene distal to D17S1867, whereas, in the family LG61 (fig. 2). normal sib (individual II-1), the recombination between D17S1814 and D17S1818 positioned the gene proximal to D17S1814. Therefore, the gene must lie in the interval Discussion between D17S1867 and D17S1814. Homozygosity was In the present report, we were able to demonstrate found only for the 151-bp allele of marker D17S250; linkage to 17q11-12 of another subtype of AR muscular however, this allele is the most common in our popula- dystrophy. Interestingly, this new locus has been tion (fig. 2; table 1). mapped near the a-sarcoglycan gene (Roberds et al.

6 156 Am. J. Hum. Genet. 61: , 1997 Table 4 Results of Two-Point Linkage Analysis between the Disease Gene and 17q11-12 Markers LOD SCORE FOR FAMILY LG11, AT u Å a LOD SCORE FOR FAMILY LG61, AT u Å MARKER D17S D17S D17S D17S D17S D17S D17S D17S D17S D17S D17S D17S D17S D17S D17S D17S D17S a Homogeneity test (Terwilliger and Ott 1994): x 2 Å 1.385; P ú.05. Heterogeneity test (Terwilliger and Ott 1994): x 2 Å 22.87; P õ ); however, it is located at least 9 cm apart from the sarcoglycanopathies. It is important to point out that the a-sarcoglycan gene. the patients noted difficulty with walking on the heels Although family LG61 is not consanguineous, we prior to difficulty with walking on the toes. This observation have found homozygosity with the 151-bp allele of suggests that the tibial anterior muscle was afhave marker D17S250. Since this allele is the most common fected before the gastrocnemius, which is just the opposite in our population, this data does not support evidence of what is described for LGMD2B or MM patients. for linkage disequilibrium. Therefore, on the basis of On the other hand, the observation that the neck muscles, the analysis of recombinants observed in this kindred, as well as the small hand muscles, were only slightly we have estimated that the candidate region for this new or not involved and the finding that the muscle pathology locus spans Ç3 cm. Several genes lie within this area; was typically dystrophic differs from DMRV. In however, their products are not specifically expressed in addition, the patients reported here were confined to the muscle; therefore, they were not considered to be wheelchairs at least 18 years after the first signs, whereas good functional candidate genes for this disease (OMIM DMRV leads to the loss of ambulation within 10 years 1996). after its onset (Murakami et al. 1995). These observa- The phenotypes of the affected patients from this 17q- tions suggest that the family reported here has a different linked family were relatively mild until the third decade muscle disease. of life. However, four of the patients were confined to In addition, one of our still-unlinked LGMD genealogies a wheelchair before 40 years of age, and the youngest (LG11) also may be linked to this locus. The three brother, who is currently 27 years of age, walks with affected sibs from this family also show a relatively mild great difficulty and only with support, suggesting that phenotype, with onset of the disease at the beginning of he will not be able to walk much longer. On the other the second decade of life. Two of them, who are 19 and hand, patient II-3 (tables 2 and 3), who is currently years of age, are still walking without difficulties; years of age, is showing a milder course, illustrating the however, the third one, who is 23 years of age, is still intrafamilial variability in the expression of this disease walking without support but is unable to climb stairs. gene. Although there is severe involvement of the proxi- Although in an earlier stage, the pattern of muscle mal muscles of the upper and lower limbs, the marked involvement in this family seems to be very similar to weakness in the distal muscles of the legs, the moderately that presented by family LG61, in particular with regard increased serum CK levels, and the presence of rimmed to the initial weakness of the tibial anterior muscle (table vacuoles in the muscle biopsy distinguish this form of 5). The patients serum CK levels are only slightly elevated AR muscular dystrophy from LGMD2A, LGMD2B, and (31 SU). Rimmed vacuoles were observed in their

7 Moreira et al.: Mapping of the LGMD2G Gene to 17q Table 5 occurrence of both LGMD and MM phenotypes in one family and by the demonstration that these two pheno- Manual Muscle-Strength Evaluation of the Patients from Family LG11 types might have been caused by mutations in the same gene (Weiler et al. 1996). Another example is the large STRENGTH RATING, inbred Finnish family reported by Udd et al. (1991). In FOR EACH PATIENT a this kindred, two different muscular manifestations, one MOVEMENT TESTED II-2 II-3 II-4 compatible with LGMD and the other with distal myopathy, are segregating (Udd et al. 1991; Udd 1992). Neck flexion On the basis of our findings and of the above-de- Neck extension scribed observations, we propose to classify this new Shoulder abduction form of AR muscular dystrophy as LGMD2G. The fact Shoulder external rotation that this 17q-linked family (LG61), which is of Italian Elbow flexion Elbow extension ancestry, is nonconsanguineous suggests that mutations Wrist flexion in this locus may be relatively common. Therefore, we Wrist extension think that it will be extremely important to test this Thumb abduction new locus in AR muscular dystrophy families who are Hip flexion positive for the sarcoglycan complex independently of Hip extension early distal muscle involvement. Interestingly, there are Knee flexion Knee extension some patients diagnosed as having Kugelberg-Welander Ankle dorsiflexion disease (KW; also known as spinal muscular atrophy Ankle plantarflexion type III ) who have slightly elevated serum CK levels Toe flexion and rimmed vacuoles in their muscle biopsies, thus pre- Toe extension senting with a phenotype resembling that of the LGMD a 0 Å no movement; 1 Å flicker of movement; 2 Å movement of family (LG61) described here (Fukahara et al. 1980; the joint when the effect of gravity is eliminated; 3 Å movement Aubry et al. 1995; Lefebvre et al. 1995; Rodrigues et through full range of the joint, against gravity but not against resis- al. 1996). In most of the KW patients, the disease is due tance; 4 Å movement of the joint, against gravity and against added to mutations in the survival motor-neuron (SMN) gene; resistance; and 5 Å full strength. however, in 5% 10% of these patients, KW seems to be caused by mutations at another locus (Aubry et al. 1995; Lefebvre et al. 1995; Rodrigues et al. 1996; Wang muscle biopsies; however, these vacuoles were very rare. et al. 1996). The possibility that the pathology in these The clinical evolution and the laboratory findings are cases also is caused by mutations in the LGMD2G gene compatible with those of the 17q-linked kindred (LG61) should be considered. reported here. In addition, it is important to point out The identification of the genetic defects responsible that these patients share the same haplotype as those of for the different types of LGMD has been extremely family LG61, which further supports the hypothesis that important for the enhancement of our comprehension the disease in this family also may have been caused by of the underlying pathological mechanisms. Therefore, mutations in this 17q locus. However, definite proof the cloning of the gene that, when mutated, causes will only be possible after the identification of this gene. LGMD2G will be of fundamental importance. The fact that one of our LGMD kindreds (LG6) was excluded from this locus was not surprising, since the patients phenotypes are quite different from those observed Acknowledgments in families LG11 and LG61 (Passos-Bueno et al. We are extremely grateful to Constancia G. Urbani, Flávia 1996b). Although also positive for a-sarcoglycan, the D. Albuquerque, Débora F. Rodrigues, Antonia M. P. Certhree affected sibs from the LG6 genealogy have very queira, Marta Canovas, Simone Campioto, Dr. Rita C. M. high serum CK levels and a severe clinical course typical Pavanello, and Dr. Ivo Pavanello Filho. Also, for their invalu- of Duchenne-like muscular dystrophy. Since this family able collaboration, we would like to express our gratitude is not linked to any of the candidate genes already to Alessandra K. Cardozo, Ana Beatriz A. Peres, Andrea F. Bernardino, Carlos M. Shimokomak, Cleber S. Costa, Denilce known, this result confirms the existence of at least an- R. Sumita, Dulci V. Fonseca, Luciana R. Vasques, Lygia V. other locus causing severe LGMD. Pereira, Paula Iughetti, Roberta Sitnik, Sabine Eggers, Susana The classification of the muscular dystrophies, based Paola R. Paretti, and all the patients and their relatives. This on only clinical and histopathological aspects, has be- work was supported by grants from the Fundação de Amparo come more and more difficult, mainly for the AR sub- à Pesquisa do Estado de São Paulo (FAPESP), the Programa de group, and a new classification based on molecular find- Apoio ao Desenvolvimento Científico e Tecnológico (PADCT), ings seems necessary. This is illustrated clearly by the the Financiadora de Estudos e Projetos (FINEP), the Conselho

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