Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy.

Miyoshi myopathy (MM) is an adult onset, recessive inherited distal muscular dystrophy that we have mapped to human chromosome 2p13. We recently constructed a 3-Mb P1-derived artificial chromosome (PAC) contig spanning the MM candidate region. This clarified the order of genetic markers across the MM locus, provided five new polymorphic markers within it and narrowed the locus to approximately 2 Mb. Five skeletal muscle expressed sequence tags (ESTs) map in this region. We report that one of these is located in a novel, full-length 6.9-kb muscle cDNA, and we designate the corresponding protein 'dysferlin'. We describe nine mutations in the dysferlin gene in nine families; five are predicted to prevent dysferlin expression. Identical mutations in the dysferlin gene can produce more than one myopathy phenotype (MM, limb girdle dystrophy, distal myopathy with anterior tibial onset).

Generation of a 3-Mb PAC contig spanning the Miyoshi myopathy/limb-girdle muscular dystrophy (MM/LGMD2B) locus on chromosome 2p13.

Miyoshi myopathy (MM) and limb-girdle muscular dystrophy subtype 2B (LGMD2B) map to the same region on chromosome 2p13. To facilitate the cloning of the defective gene causing these two diseases, we used a combination of chromosome walking and expressed sequence tag (EST) screening and identified 864 P1-derived artificial chromosomes (PACs) whose inserts map to the MM/LGMD2B candidate region and surrounding areas. Among them, 139 are from a chromosome 2-specific PAC library and 725 are from a total genomic PAC library. A 3-Mb contig spanning the candidate region for MM/LGMD2B was assembled. This contig contains 200 PACs, 10 known genetic markers, 5 new polymorphic markers, 57 sequence tagged sites (STSs) generated from PAC end fragments, and 4 random STSs. In addition, we mapped 24 ESTs to this contig and excluded 37 ESTs from the contig, thus eliminating them as candidate MM/LGMD2B genes. The high-resolution, sequence-ready PAC contig for the MM/LGMD2B region provides a backbone for the identification of the disease gene(s) and for clarification of the relationship between the two diseases.

Genetic fine mapping of the Miyoshi myopathy locus and exclusion of eight candidate genes.

Miyoshi myopathy (MM) is an early adult-onset, autosomal recessive disorder characterized by weakness and muscular atrophy starting in the distal muscles. The disease locus has been previously mapped by linkage analysis to chromosome 2p using the microsatellite marker D2S291. Initial haplotype analysis of markers in families from three different origins (North American, Japanese, and Tunisian) suggested that the MM gene is located in a 4-cM region flanked by markers D2S292 on the telomeric side and D2S286 on the centromeric side. To delineate critical recombination events revealing a more refined localization of the MM gene, we have determined the pattern of segregation of 12 marker loci in two consanguineous families of Tunisian origin. In this study we have: (1) detected recombination events with the disease locus in one family, placing the MM gene most likely between markers D2S443 (CHLC.GGAA4D07.1876) and D2S2109; (2) generated a yeast artificial chromosome contig that spans approximately 3.8 megabases and extends from marker D2S358 to marker D2S286; (3) physically mapped 21 polymorphic markers, 5 genes, 3 STSs, and 1 EST within this contig; (4) detected and mapped a new polymorphism within this interval, allowing us to further reduce the MM locus to a 360-kilobase segment; (5) mapped the gene for the cytoskeletal protein beta-adducin within the MM candidate region, failing to find a consistent pattern of mutation of this gene in our MM patients; (6) excluded seven other candidate myopathy genes from the Miyoshi locus.