Screen and functional validation of Pediatric Cardiomyopathy genetic variants in Drosophila

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PROJECT SUMMARY Pediatric cardiomyopathies (PCM) are rare but serious disorders of the cardiac muscle with early childhood onset, limited treatment options, that often culminate in childhood death due to limited effective treatments. A recently published exome sequencing study of 528 patients with various types of PCM, the largest to date, revealed insights into the genetic architecture of PCM and identified 343 variants in 125 genes. The findings affirmed that pediatric CM has different genetic underpinnings than adult-onset CM. It further demonstrated the genetic complexity of PCM; with patients carrying variant in multiple genes, suggesting a polygenic nature, and variants in the same gene associated with different PCM types. Moreover, most of the candidate genes have not been studied in the heart before. To bridge the gap between candidate variant and disease causation, our team has developed an in silico pipeline to identify the candidate suited for modeling in the Drosophila heart, to prioritize them by likelihood to cause a functional defect in the protein, and assign the best modeling approach in fly based on the mutation type. We will then follow this through with our “gene replacement” approach for efficient “gene-level” and “variant-level” functional validations in the fly heart, to rapidly screen the 343 PCM candidate variants. Specifically, we will: 1) Use bioinformatic tools tailored to Drosophila, to screen the candidate PCM genes and variants for their feasibility to be modeled in the fly heart, to determine the best modeling approach for each, and to provide interspecies (fly-human) cardiac phenotypic correlations to be shared with the clinical and research communities. 2) Use our fly heart-specific RNAi gene silencing system to provide “gene- level” validation for all candidate PCM variants, likely to cause loss-of-function, and designated feasible for modeling in the Drosophila heart. We will expand the cardiac phenotype assays for fly to develop a comprehensive assessment that can distinguish between the different PCM types. We will use the findings in the PCM fly models to generate a cardiac phenotype library. 3) Use our Drosophila “gene replacement” approach to provide “variant-level” validation for candidate PCM variants that are gain-of-function or of uncertain effect, as well as to generate precision polygenic models for patients carrying multiple genetic variants. Successful completion of the proposed aims will establish a highly efficient “in silico - in vivo” pipeline to screen and functionally validate genetic variants identified from patients with PCM, provide causal association for hundreds of PCM candidate variants, and establish interspecies cardiac phenotypic correlation from Drosophila to humans. Many of the fly PCM models generated in this project will be “precision disease models” since they carry the specific human patient variants in the fly heart and thus could be used in the future to study disease mechanisms and to test targeted therapies.