ANALYSIS OF DOMINANT MEGACOLON--ANOTHER MODEL FOR HIRSCHSPRUNG DISEASE

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Animals heterozygous for Dominant megacolon (Dom/+) exhibit multiple defects in neural crest development including reduced numbers of melanocytes in the skin and an absence of myenteric ganglion in the colon. A human congenital disorder, Hirschsprung disease also exhibits rectocolic aganglionosis and can be associated with hypopigmentation. Thus Dom/+ mice, as well as the piebald and lethal spotting mutants, serve as mouse models for this disease. Dom arose and has been maintained on a C57BL/6JLe X C3HeB/FeJLe-a/a (B6C3F1) hybrid background, however the severity of the phenotype is dramatically influenced by genetic background. Using continuous backcrossing to different inbred strains of mice, we have found that both spotting and survival are increased in Dom/C3 lines in comparison to Dom/B6 lines. Using inter- and intra-subspecific crosses the Dom interval was narrowed to < 0.001 cM (2 recombinants/1700 meioses). A physcial map has been assembled from the region and Dom has been restricted to an ~150 kb segment. We have used single pass sequencing to identify candidate genes. Analysis of the candidate genes has resulted in the identification of the molecular defect. To evaluate the phenotype of Dom/Dom homozygous mice, the most closely linked markers were applied to genotype intercross progeny. We have found that the DOM gene disrutps expression of two neural crest markers, TRP2 and EDNRB placing the Dom gene early in the neural crest development pathway. Future studies in this area will include in situ hybridization analyses to describe neural crest development in Dom/Dom mutant embryos. Our in vitro neural crest culture system and aggregation chimera analyses will be used to determine if the Dom defect acts intrinsic to the neural crest derived melanocytes. Investigation of the involvement of Dom in Hirschsprung disease will be explored in collaboration with Aravinda Chachravarti (Extramural) throught the use of human pedigree analysis.