Functional characterization of genes and genetic variants associated with hypoplastic heart syndrome in Drosophila - PROJECT SUMMARY Hypoplastic left heart syndrome (HLHS) and hypoplastic right heart syndrome (HRHS), collectively referred to as hypoplastic heart syndrome (HHS), are severe congenital heart diseases that significantly impact newborns, with HLHS affecting approximately 1 in 5,000 births. While advances in surgery have improved survival rates, HHS still often requires multiple surgeries or heart transplants. The potential for gene therapy in HHS hinges on the accurate identification and validation of causative genetic mutations. Despite extensive sequencing efforts that have identified large numbers of variants in HHS patients, the causal roles of these variants remain unclear. To address this, we have developed a cost-effective, high-throughput Drosophila “gene replacement” approach to validate genetic variants associated with HHS. Using a bioinformatics pipeline, we screened millions of genetic variants with potential links to cardiac developmental defects and identified 386 conserved gene variants associated with a hypoplastic heart phenotype that are most suitable to be modeled in Drosophila. These 386 variants were classified into three categories: loss-of-function (LOF), gain-of-function (GOF), and variants of uncertain effect (VUE). In this proposal, we aim to apply specific strategies for each group of variants to provide essential in vivo functional validation and confirm their causal relationship with HHS. Furthermore, we have identified 18 patients carrying multiple high-confidence variants, suggesting a polygenic basis for HHS. We will use the gene-replacement approach to test genetic interactions among these variants within the same patients, enabling the development of novel polygenic HHS models and revealing underlying molecular mechanisms. Ultimately, this project will establish an efficient platform for variant screening and validation, generate a comprehensive collection of Drosophila precision disease models for HHS, and provide insights into the molecular mechanisms underlying HHS. These findings will contribute to the future development of targeted therapies for HHS.
