Translational Research in the Dystrophinopathies

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DESCRIPTION (provided by applicant): Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD) are devastating disorders. Both are associated with mutations in the dystrophin gene, a huge gene with 79 exons spread over 2.4 million bases of genomic sequence. Deletions of large portions of the gene account for around 60% of all dystrophin mutations. The remainder consist of point mutations (primarily premature stop codon mutations), small deletions resulting in shift of the reading frame, and (in less than 5%) duplications. Dystrophin gene deletion testing is commercially and readily available, but point mutation testing is not. Recent studies in the mdx mouse, a model for DMD due to a premature stop codon mutation, have demonstrated the ability of aminoglycosides to increase the expression of dystrophin protein via induction of increased read-through. Recently, we and others have demonstrated some rules for the specificity of this effect, and a growing body of data suggests that aminoglycoside therapy may prove beneficial in some patients. We have developed the methodology to rapidly, robustly, and economically perform direct sequence analysis of the entire coding and regulatory regions of the dystrophin gene, greatly expediting the characterization of mutations in non-deleted dystrophinopathy patients. Using this methodology, we propose to characterize the mutations responsible for DMD and BMD in a large cohort of patients, from whom a standardized and thorough phenotypic characterization, will be obtained. Phenotype/genotype information will be compiled in a pilot dystrophinopathy registry database. Correlation of the phenotype to the sequence context of specific individual mutations will generate hypotheses of aminoglycoside-induced read-through efficiency in specific sequence contexts, which will be tested in an in vitro dual-luciferase transfection assay. This same assay will be used to systematically study other pharmaceutical compounds, which may cause read-through of premature stop codon or frameshift mutations, and to study other potential mechanisms for modifying intrinsic frame shifting and read-through. Finally, we propose to develop a dual-GFP transgenic mouse, which will allow in vivo characterization of tissue-specific variation in aminoglycoside-induced read-through. Although we do not propose to perform an aminoglycoside treatment trial at present, this proposed study will identify a cohort of patients who may be candidates for any future trials here or at other institutions, and may provide a rationale to suggest that individual compounds or dosages may need to be tailored to specific sequence variations in all future trials.