Hirschsprung disease (HSCR) is a complex genetic disorder that gives rise to absence of intrinsic ganglia in the distal intestine and consequently produces gastrointestinal dysmotility. Mutations in any one of several genes can give rise to the intestinal aganglionosis that is the hallmark of this disease. Incomplete penetrance and variable expressivity of the neural crest (NC) defects exhibited by family members carrying identical gene mutations suggests that multiple genes modulate HSCR severity. Mouse models of HSCR have been extremely valuable for identifying genes that participate in pathogenesis of enteric nervous system (ENS) defects. Genetic and embryonic analysis of the mouse mutant Sox10Dom has defined a role for this transcription factor in development of the ENS. Our analysis of Sox10Dom mice in F1 hybrid and inbred strains demonstrates that genetic background impacts severity of intestinal aganglionosis. The phenotypic variation we observe mimics that seen in human HSCR sibs and suggests that modifier loci influence development of Sox10 derivatives in mouse and man. In the proposed experiments we will test the hypothesis that gene interactions between loci impact ENS development. Congenic lines of Sox10Dom mice will be used to define the timing and effect of genetic background on the enteric NC. Specifically we will evaluate survival, proliferation, and migration of enteric NC in embryos from Sox10Dom congenic lines. To identify the genes responsible for the phenotypic variation in the Sox10Dom HSCR model we will evaluate association between alleles at candidate modifier loci and severity of intestinal aganglionosis in Sox10Dom animals maintained on an F1 hybrid background. Potential gene interactions will be validated in vivo by examining severity of aganglionosis in crosses between Sox10Dom and mutants at candidate modifier loci. Our preliminary analysis has already identified a highly significant interaction between Sox10 and EdnrB and we have identified Sox10 consensus binding sites in EdnrB flanking regions. To investigate the biological interaction between Sox10 and EdnrB we will identify sequence variants at the EdnrB locus. The functional effects of polymorphisms on levels of EdnrB mRNA and function will be evaluated in neural tube (NT) and enteric ganglia cultures from our congenic lines. These experiments are part of a unified strategy to define the mechanisms of gene interaction that participate in development and pathogenesis of the ENS.
