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).

In 76 muscle specimens (normal controls, 9; Duchenne dystrophy, 11; scleroderma, 11; dermatomyositis, 13; polymyositis, 15; inclusion body myositis, 17), mononuclear cells were analyzed at perivascular, perimysial, and endomysial sites of accumulation. Monoclonal antibodies reactive for B cells, T cells, T cell subsets, killer (K) or natural killer (NK) cells, and the Ia antigen were used for cell typing. Macrophages were identified by the acid phosphatase reaction. Few extravascular mononuclear cells occurred in normal muscle. In all inflammatory myopathies, a mixed exudate of T cells, B cells, and macrophages was present. Mature K/NK cells were rare in all diseases. In dermatomyositis, polymyositis, and inclusion body myositis, there was a positive gradient for T cells, T8+ cells, and activated T cells and a negative gradient for B cells and T4+ cells between perivascular and endomysial sites. In scleroderma the predominant perimysial exudate consisted mostly of T cells and macrophages. The percentage of B cells at all sites, and the T4+/T cell ratio in the endomysium, were significantly higher in dermatomyositis than in the other diseases. In polymyositis and inclusion body myositis, the endomysial exudate contained a large number of T cells, T8+ cells, and activated T cells but only sparse B cells. T cells accompanied by macrophages focally surrounded and invaded nonnecrotic fibers in polymyositis and inclusion body myositis. Rare fibers in Duchenne dystrophy and a very few fibers in dermatomyositis and scleroderma were similarly affected. We infer that (1) T-B, T-T, and T-macrophage cooperativities are likely to exist in muscle in different myopathies; (2) T cell-mediated fiber injury plays a role in polymyositis and inclusion body myositis; (3) T cell-mediated fiber injury can also occur in inherited diseases, such as Duchenne dystrophy; and (4) a local humoral response may occur in muscle in dermatomyositis and possibly in polymyositis and inclusion body myositis.

The basal lamina of muscle fibers plays a crucial role in the development and function of skeletal muscle. An important laminin receptor in muscle is integrin alpha7beta1D. Integrin beta1 is expressed throughout the body, while integrin alpha7 is more muscle-specific. To address the role of integrin alpha7 in human muscle disease, we determined alpha7 protein expression in muscle biopsies from 117 patients with unclassified congenital myopathy and congenital muscular dystrophy by immunocytochemistry. We found three unrelated patients with integrin alpha7 deficiency and normal laminin alpha2 chain expression. To determine if any of these three patients had mutations of the integrin alpha7 gene, ITGA7, we cloned and sequenced the full-length human ITGA7 cDNA, and screened the patients for mutations. One patient had splice mutations on both alleles; one causing a 21-bp insertion in the conserved cysteine-rich region, and the other causing a 98-bp deletion. A second patient was a compound heterozygote for the same 98-bp deletion, and had a 1-bp frame-shift deletion on the other allele. A third showed marked deficiency of ITGA7 mRNA. Clinically, these patients showed congenital myopathy with delayed motor milestones. Our results demonstrate that mutations in ITGA7 are involved in a form of congenital myopathy.

Using the gene targeting technique, we have generated a new mouse model of congenital muscular dystrophy (CMD), a null mutant for the laminin oc2 chain. These homozygous mice, designated dy 3K ldy 3K , are characterized by growth retardation and severe muscular dystrophic symptoms and die by 5 weeks of age. Light microscopy revealed that muscle fiber degeneration in these mice begins no later than postnatal day 9. In degenerating muscles, considerable amounts of TUNEL positive nuclei were detected as well as DNA laddering, suggesting increased apoptotic cell death was involved in the process of muscle fiber degeneration.

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