Progress during past year: Disease mechanisms in zaspopathy, a prototype myofibrillar myopathy: We completed initial characterization of functional interactions of ZASP with skeletal muscle actin monomers and filaments (published). Our results indicate that ZASP interactions with skeletal muscle actin play a fundamental role in the disease mechanism of zaspopathy. Next steps are to examine the role of ZASP-actin interaction in vivo in disease model systems (knock in mice, transgenic fish, stable inducible muscle cell lines). To this regard, stable inducible muscle cell lines have been generated and are now characterized for the actin phenotype. Transgenic fish lines have been generated and are now bred to expand the lines. Knock in targeting constructs are finally made after rigorous efforts. We have tested and validated screening protocols of knock in recombination. In addition, we have initiated studies to determine the structure of ZASP-actin complex (collaboration with Wingfield and Steven laboratories). It took a significant effort to purify ZASP proteins (His6 tagged and untagged) (collaboration with Wingfield laboratory, NIAMS). We have determined the binding affinities of ZASP to skeletal muscle actin by surface plasmon resonance assay and measured stoichiometry of ZASP-actin interaction. Efforts are underway to crystallize ZASP proteins. We have begun studies to examine ZASP-actin protein complex with cryo-electron microscope. Patient studies: We have initiated annual follow up evaluations of family members of the original zaspopathy pedigree to characterize natural history of the disease. We completed enrollment of subjects (n=33) to the DMD Imaging Biomarker study (protocol ID 11-N-0261). Data analysis is ongoing and is in the final phases. Clinical observations in one of the research subjects were published (see below). We have started enrolling patients into the Myotonic Dystrophy Biomarker study (protocol 14-N-0132). We have identified novel gene defects and known gene mutations in several of our patients including the ones in the Neurogenetics clinic by using NextGen exome analysis. Efforts are underway to characterize the biological effects of the novel variants in cell systems and patient tissues.
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