Yoshihide Sunada

β–sarcoglycan: characterization and role in limb–girdle muscular dystrophy linked to 4q12

β–sarcoglycan, a 43 kDa dystrophin–associated glycoprotein, is an integral component of the dystrophin–glycoprotein complex. We have cloned human β–sarcoglycan cDNA and mapped the β–sarcoglycan gene to chromosome 4q12. Pericentromeric markers and an intragenic polymorphic CA repeat cosegregated perfectly with autosomal recessive limb–girdle muscular dystrophy in several Amish families. A Thr–to–Arg missense mutation was identified within the β–sarcoglycan gene that leads to a dramatically reduced expression of β–sarcoglycan in the sarcolemma and a concomitant loss of adhalin and 35 DAG, which may represent a disruption of a functional subcomplex within the dystrophin–glycoprotein complex. Thus, the β–sarcoglycan gene is the fifth locus identified (LGMD2E) that is involved in autosomal recessive limb–girdle muscular dystrophy.

Mutational diversity and hot spots in the alpha-sarcoglycan gene in autosomal recessive muscular dystrophy (LGMD2D).

Sarcoglycanopathies are a genetically heterogeneous group of autosomal recessive muscular dystrophies in which the primary defect may reside in any of the genes coding for the different partners of the sarcolemmal sarcoglycan (SG) complex: the alpha-SG (LGMD2D at 17q21.2), the beta-SG (LGMD2E at 4q12), the gamma-SG (LGMD2C at 13q12), and the delta-SG (LGMD2F at 5q33). We report a series of 20 new unrelated families with 14 different mutations in the alpha-SG gene. Along with the mutations that we previously reported this brings our cohort of patients with alpha-sarcoglycanopathy to a total of 31 unrelated patients, carrying 25 different mutations. The missense mutations reside in the extracellular domain of the protein. Five of 15 missense mutations, carried by unrelated subjects on different haplotype backgrounds and of widespread geographical origins, account for 58% of the mutated chromosomes, with a striking prevalence of the R77C substitution (32%). The severity of the disease varies strikingly and correlates at least in part with the amount of residual protein and the type of mutation. The recurrent R284C substitution is associated with a benign disease course.

Characterization of δ-sarcoglycan, a novel component of the oligomeric sarcoglycan complex involved in limb-girdle muscular dystrophy

The sarcoglycan complex is known to be involved in limb-girdle muscular dystrophy (LGMD) and is composed of at least three proteins: α-, β-, and γ-sarcoglycan. δ-Sarcoglycan has now been identified as a second 35-kDa sarcolemmal transmembrane glycoprotein that shares high homology with γ-sarcoglycan and is expressed mainly in skeletal and cardiac muscle. Biochemical analysis has demonstrated that γ- and δ-sarcoglycan are separate entities within the sarcoglycan complex and that all four sarcoglycans exist in the complex on a stoichiometrically equal basis. Immunohistochemical analysis of skeletal muscle biopsies from patients with LGMD2C, LGMD2D, and LGMD2E demonstrated a reduction of the entire sarcoglycan complex in these muscular dystrophies. Furthermore, we have mapped the human δ-sarcoglycan gene to chromosome 5q33-q34 in a region overlapping the recently linked autosomal recessive LGMD2F locus.

Absence of γ-sarcoglycan (35 DAG) in autosomal recessive muscular dystrophy linked to chromosome 13q12

We have partially sequenced rabbit skeletal muscle γ‐sarcoglycan an integral component of the dystrophin‐glycoprotein complex. Specific antibodies were produced against a γ‐sarcoglycan peptide and used to examine the expression of γ‐sarcoglycan in skeletal muscle of patients with severe childhood autosomal muscular dystrophy linked to chromosome 13q12 (SCARMD). We show by immunofluorescence and Western blotting that in skeletal muscle from these patients γ‐sarcoglycan is completely absent and α‐ and β‐sarcoglycan are greatly reduced in abundance, whereas other components of the DGC are preserved. In addition, we show that in normal muscle α‐, β‐, and γ‐sarcoglycan constitute a tightly associated sarcolemma complex which can not be disrupted by SDS treatment.

β-Sarcoglycan: genomic analysis and identification of a novel missense mutation in the LGMD2E Amish isolate

The sarcoglycan complex is involved in the etiology of four autosomal recessive limb-girdle muscular dystrophies (LGMD2C-F). A missense mutation (T151R) in the beta-sarcoglycan gene on chromosome 4q12 has been shown to cause a mild form of LGMD2E in 11 families from a Southern Indiana Amish community sharing a common haplotype. We now report that two sibs from another Amish family with mild LGMD2E are compound heterozygotes for chromosome 4q12 markers. In order to characterize the genetic defect in this new family, we determined the genomic organization of the beta-sarcoglycan gene. A second missense mutation (R91C) has now been identified in this LGMD2E Amish family. This mutation is also present in the homozygous state in another family of probable Amish ancestry. Finally, analysis of all the components of the dystrophin-glycoprotein complex was performed for the first time on a biopsy from a patient homozygous for the beta-sarcoglycan mutation (T151R). Interestingly, in addition to the loss of the entire sarcoglycan complex, we detected a reduction of alpha-dystroglycan which suggests a role for the sarcoglycan complex in stabilizing alpha-dystroglycan at the sarcolemma.

Identification of muscle-specific calpain and β-sarcoglycan genes in progressive autosomal recessive muscular dystrophies

The autosomal recessive forms of limb-girdle muscular dystrophies are encoded by at least five distinct genes. The work performed towards the identification of two of these is summarized in this report. This success illustrates the growing importance of genetics in modern nosology.

A syntrophin gene maps to mouse Chromosome 8 and is not the myodystrophy gene

A complex of proteins and glycoproteins spans the muscle cell membrane and anchors the intracellular cytoskeletal proteins dystrophin and actin to the extracellular matrix. This dystrophinassociated complex is critical in maintaining the integrity of the muscle cell; disruption of its components causes various forms of muscular dystrophy (reviewed in Campbell 1995). Absence of dystrophin (Xp21.2) leads to Duchenne muscular dystrophy, and truncated or dysfunctional dystrophin causes Becket muscular dystrophy. Adhalin (17q) is absent in severe childhood autosomal recessive muscular dystrophy (SCARMD) and other muscular dystrophies with variable manifestations (Piccolo et al. 1995; Hayashi et al. 1995). Merosin (6q22-23) deficiency has been demonstrated in some cases of congenital muscular dystrophy. Other components of this complex are obvious candidates for dystrophies where the mutation is as yet unknown. Syntrophin is a 59-kDa protein triplet that associates with the carboxy-terminus of dystrophin and the related protein utrophin. Syntrophin cDNA clones have been isolated from Torpedo and mouse (Adams et al. 1993), rabbit (Yang et al. 1994), and human (Ahn et al. 1994). Syntrophins can be characterized as acidic or basic (Yamamoto et al. 1993), and this distinction underlies the nomenclature. The acidic mouse isoform, tx-syntrophin (SntaI), has a human homolog on Chr 20ql 1 (Campbell 1995). The basic isoform, [3-syntrophin (Sntb2), is homologous to a human expressed sequence tag that maps to 16q23 (EST25263; unpublished, Genbank accession no. T29784). A third basic human isoform maps to 8@3-24 and has no known mouse homolog. Sntb2 was likely to map to mouse Chr 8 on the basis of the location of its human homolog at 16q23 and the existence of a known region of synteny (Ceci, 1994). A mouse neuromuscular mutant, myodystrophy (myd), also maps to mouse Chr 8, near the region of synteny with 16q23. Myodystrophy is a spontaneously arising autosomal recessive mutation that is characterized by dystrophic muscle pathology and elevated serum CK. Affected animals have abnormal hindlimb posture when they are held suspended. The disorder is progressive and results in premature death (Lane et al. 1976; Mathews et al. 1995). Sntb2 was an attractive candidate gene for myd because of the homologous chromosomal location and the association with dystrophirdutrophin. There is no disease process cm-rently known to result from mutation of this gene in either human or mouse. We investigated Sntb2 in myd mice through both genetic mapping and protein analysis. As shown in Fig. 1, Sntb2 maps to mouse Chr 8 distal to D8Mit211 and proximal to D8Mitllt. This places the gene within an interval of 4.82 -+ 1.66 cM. The distance between these two flanking markers in the MIT map, a cross between different subspecies, is 5.5 cM (Release 9, 3/1/95; Dietrich et al. 1994). There were no recombinants with D8Mit12, which lies between these two