Progress during past year: Disease mechanisms in zaspopathy, a prototype myofibrillar myopathy: During past year, we generated transgenic zebrafish expressing WT and mutant GFP-tagged ZASP in skeletal muscle in collaboration with Dr. Allan Beggs (Harvard Medical School, Boston). In regards to knock in project in mice, we completed ES-cell screening using Southern blotting, PCR-based assays, and Sanger sequencing and identified 3/80 ES cell clones containing A165V mutation, which are best suited for generating chimera. Only in the last week we have received first set of mouse tails of chimera for genotyping from Dr. Sharan's laboratory (NCI). Despite rigorous attempts to reproduce A147T mutation, we could not generate knock in construct using recombineering method. At present, we have begun using CRISPR/Cas9 to generate A147T knock-in mice in collaborations with Dr. Sharan (NCI). We have expanded our studies of ZASP in skeletal muscle to nervous system. We have determined ZASP expression in neurons and glia in brain and spinal cord in mice and human. This was prompted by exome-identification of ZASP R268C mutation in a patient with typical ALS (referred by UDP/NHGRI). We have collaborated with Bao Tran (NIC, Frederick) to examine spectrum of alternatively spliced ZASP isoforms in brain using PacBio system. We have already identified novel shorter ZASP isoforms and are now completing the analysis of the longer ZASP isoforms. Patient-derived iPSc cell lines have been made and currently we are checking expression of the mutant ZASP isoform at RNA and protein levels. We are making knock in zebrafish model of R268C to model human motor neuron disease. In addition, to determine the structure of ZASP-actin complex (collaboration with Wingfield and Steven laboratories), we have now completed initial characterization of recombinant tagged- and untagged versions of ZASP proteins (different isoforms, with and without mutations). We see a potential effect of mutation on folding of ZASP. We have measured actin monomer/dimer-binding affinity of ZASP proteins using isothermal titration calorimetry and surface plasmon resonance. We are currently performing F-actin co-sedimentation assays with these proteins. 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, which are proving to be difficult. We have begun studies to examine ZASP-actin protein complex with cryo-electron microscope. We are using yeast two-hybrid assay to determine binding site of ZASP-actin within exons 6 and 11. Patient studies: We completed MRI analysis of the NIH Duchenne muscular dystrophy (DMD) Imaging study. We have completed one manuscript highlighting IDEAL-CPMG muscle fat fraction (AFF) and muscle water T2 as useful biomarkers of disease activity and treatment efficacy in DMD. Importantly AFF of all examined thigh muscles correlated with clinical outcome measure of six-minute walked distance (Mankodi A, et al. Manuscript in Submission process). Other manuscripts highlighting MRI biomarkers of exercise effects in the lower leg muscles as well as myocardium and skeletal muscles of the upper extremity in DMD are under preparation. We enrolled 17 subjects into the Myotonic Dystrophy Biomarker study (protocol 14-N-0132)and 15 subjects have completed 3-month follow up evaluations and two subjects completed final evaluation at 1 year. We identified an interesting effect of divalent ions modulating symptom severity of a rare skeletal muscle sodium channelopathy manifesting as periodic paralysis and paramyotonia congenital (manuscript in press). 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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