The long term goals of this proposal are to identify and characterize new skeletal muscle sarcomeric genes and proteins and to understand how abnormalities of these proteins contribute to the pathophysiology of human neuromuscular diseases. This competitive renewal application describes complementary approaches to address these issues from both a basic science and reverse genetics perspective. The first involves continued characterization of six novel genes identified in a yeast two-hybrid screen using skeletal muscle a-actinins as """"""""bait."""""""" Extensive characterization of one of these, now called """"""""myozenin,"""""""" reveals that it binds a-actinin in a number of in vitro biochemical assays and that it co-localizes at sarcomeric Z lines. Indirect immunofluorescence analysis of muscle from patients with nemaline myopathy (NM) reveals that myozenin is abnormally localized within the nemaline rods. Additional biochemical and yeast two-hybrid studies on myozenin and the other new proteins will provide new insights into Z line and thin filament structure and function. A complementary approach to learning about these structures is to use reverse genetics to identify and characterize the genes mutated in patients with NM, a clinically and genetically heterogeneous group of inherited neuromuscular diseases distinguished by variably progressive skeletal muscle weakness and characteristic rod bodies in muscles of affected individuals. The applicant's laboratory has identified NM mutations in three sarcomeric genes (a-tropomyosin, nebulin and actin) to date, and has genetic data that one or more additional NM genes must exist. Each new sarcomeric gene identified above, as well as a group of known sarcomeric candidate genes, will be tested for mutations in NM. Finally, it is not clear how mutations in each of the known NM genes relate to the extremely variable clinical and pathological phenotypes seen in this disease. To address this question of pathogenesis, global gene expression profiles of NM skeletal muscle from human patients and transgenic mouse models of NM will be developed and interesting new skeletal muscle genes whose expression is perturbed will be studied. Success in this project will enable accurate diagnostic and prognostic testing for all NM patients as well as shed new light on the structure and functions of normal and abnormal Z lines and thin filaments in skeletal muscle.
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