Project Summary/Abstract Congenital Heart Disease (CHD) is the most prevalent birth defect affecting 1% of live births and is one the top causes of infant mortality worldwide. Heterotaxy (HTX) is a severe form of CHD resulting from abnormal left- right (LR) patterning during embryonic development. Heterotaxy is highly genetic, yet the complex genetic underpinnings of this potentially life-threatening disorder remain poorly understood. Whole exome sequencing of two siblings born with heterotaxy symptoms identified a novel CHD gene Trinucleotide Repeat Containing Protein 18, TNRC18. TNRC18 is understudied with only one, recent study investigating its function. A preliminary loss-of-function (LOF) screen in the high-throughput human disease model Xenopus resulted in malformation of the cardiac outflow tract and LR patterning defects, recapitulating the patient phenotype. Thus, the overall goal of this proposal is to elucidate the molecular mechanism by which abnormal TNRC18 leads defects in LR patterning and CHD/HTX. The first aim will use LOF experiments to determine the earliest role of TNRC18 in LR patterning cascade by visualizing global pitx2c expression and dand5 expression in the left-right organizer. Determination of spatiotemporal tnrc18 expression in early development in will focus subsequent mechanistic analysis. The second aim will explore the functional significance of TNRC18 patient variants and whether the BAH domain known to have significance for chromatin modification also impacts development. Together, these experiments will elucidate the role of tnrc18 in left-right patterning, cardiac development, and CHD pathogenesis. This will aid future patient diagnosis and outcomes as their treatment can be targeted to genotype in addition to CHD phenotype. This application also outlines my (the applicant’s) training plan in advanced coursework, research mentorship, new techniques, and professional skills including written and oral data presentation. This training plan will equip me with the skills to pursue a career as an independent physician-scientist.