ABSTRACT: Hirschsprung disease (HSCR), or congenital aganglionosis, results from defects in enteric neural crest cell precursor (ENCC) development in a sex-biased manner. Through human genetic studies we have identified and/or analyzed 35 genes and loci with multiple pathogenic alleles that explain ~62% of the population attributable risk, with the greatest effects from mutations within the RET-EDNRB gene regulatory network (GRN). In this proposal, we use our gene discoveries to conduct genomic, cellular and developmental analyses in patients and mouse models, and investigate how genetic defects in RET and EDNRB signaling, and other genetic defects, produce the cellular changes across development that can lead to aganglionosis. We propose studies to improve HSCR risk prediction from patient genome sequence, advance the mechanistic understanding of HSCR at the genetic and cellular levels, and identify the rate-limiting genetic steps suitable for therapeutics. Our goals are to provide a genetic paradigm for uncovering the pathophysiology of a sex- biased complex multifactorial disorder.