The applicant's long-term objectives are to define the biochemical and genetic basis for human inherited neuromuscular disorders. In line with his previous work on Duchenne and Becker muscular dystrophies, the applicant proposes to apply molecular genetic, biochemical and cell biological techniques towards this goal, using diagnostic muscle biopsies, DNA, and clinical data as his primary materials. In this proposal, the applicant describes plans to continue to develop dystrophin testing as a means for biochemical diagnosis of myopathies. Dystrophin testing appears capable of detecting all Duchenne/Becker dystrophy patients even before the onset of obvious clinical symptoms, and the results are clearly prognostic. DNA testing, on the other hand, is only capable of detecting two-thirds of Duchenne/Becker patients, and the results are much less prognostic of the expected clinical severity. The proposed biochemical studies will delineate the range of clinical phenotypes possible from dystrophin abnormalities by investigating dystrophin abnormalities in limb-girdle and congenital myopathy patients. An important result of the continued patient testing will be the establishment of an extensive patient database of true limb-girdle and congenital myopathy patients. This database will be used as starting material for investigations directed towards identifying underlying biochemical defects in patients not having dystrophin abnormalities. The experimental approach to be used for identifying the protein defects in these patients will be the candidate gene/protein approach. Specifically, we have recently developed experimental methods by which we can clone human genes and produce large quantities of the corresponding human protein from any published vertebrate sequence with only a few hours of work. Antibodies raised against the produced proteins have proven to highly sensitive and specifically directed against the human protein. In this proposal we describe our application of these techniques towards two human proteins, the chromosome-6 dystrophin-related protein, and the skeletal muscle sodium channel. The cloned human genes and the corresponding antibodies will be used to investigate the possible involvement of these proteins in neuromuscular disease.
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