Project Summary Large numbers of gene variants were identified from genomic sequencing of Congenital Heart Disease patients, but lack of functional verification in heart development precludes assigning “disease gene” status. Genetic control of heart development is conserved from Drosophila to humans, thus investigations in flies can illuminate gene functions in human heart development and disease. We developed a gene validation system in Drosophila to screen large numbers of genes for roles in heart development, and quantitative analysis tools to assess multiple phenotypic parameters. We also developed novel strategies to test patient-¬derived genetic mutations in flies for in vivo evidence linking specific gene variants to disease. We observed that many histone modifying genes mutated in disease patients have roles in fly heart development. We propose studies designed to elucidate ancestral roles of histone-¬modifying genes in heart development and disease, and to generate personalized fly heart disease models for specific gene variants. Using the high-throughput Drosophila gene validation screen for essential roles in heart development, we will test candidate disease genes identified from publicly available datasets and collaborators’ unpublished datasets. We will also screen Drosophila genes encoding enzymes for histone methylation/demethylation and acetylation/deacetylation for roles in heart development. Histone modifying genes validated by screening will be phenotyped using multiple quantifiable morphological and functional readouts. We will identify histone modifications that are most important for heart development. Genes will be prioritized based on multiple criteria, and for highest priority genes we will examine the transcriptional profile of heart tissue from flies in which the gene of interest was silenced in cardiac cells. We will identify conserved targets of histone modification effects by comparing our results to data from murine models and patient tissue samples. We will generate transgenic and knock-in fly models to provide in vivo functional evidence for involvement of high priority gene variants in congenital heart disease. In pReplacement, we will express wild type or mutant transgene versions of a given human disease gene in the fly heart while simultaneously silencing the endogenous fly homolog. We will also generate “knock-in” Drosophila models using CRISPR/Cas9-mediated gene editing. In this pCRISPR approach, the endogenous Drosophila homolog is precisely modified to encode a protein with amino acid changes identical to those encoded in the patient-¬derived mutant allele. We will also use Drosophila to model polygenic disease based on selected patients...