ABSTRACT: Down syndrome (DS), resulting from trisomy 21 (T21), has many associated defects, a hallmark being the 100- fold higher risk of Hirschsprung disease (HSCR or congenital colonic aganglionosis). The mechanism of this association is elusive, but a critical clue is that a common RET enhancer variant, associated with non- syndromic HSCR, is also associated with DS cases with HSCR but not DS cases without. These data suggest that the association is from increased dosage of chromosome 21 (HSA21) genes dysregulating the RET-EDNRB gene regulatory network (GRN) to increase HSCR risk, a mechanism likely explaining other DS traits. One specific HSA21 candidate is SOD1, a putative negative regulator of RET, that can further reduce RET expression in DS cases with RET deficiency enhancer alleles. Further, aneuploidy-specific protein imbalance can also affect cell proliferation and HSCR. Here, we propose three aims investigating human genetics, functional genomics in engineered pluripotent stem cells (iPSC) and mouse models of HSA21 genes and RET, to understand the mechanisms by which HSA21 gene-specific and T21-specific genetic effects lead to HSCR- associated DS.