A multi-enhancerRETregulatory code is disrupted in Hirschsprung disease

The major genetic risk factors for Hirschsprung disease (HSCR) are three common polymorphisms within cis regulatory elements (CREs) of the RET receptor tyrosine kinase gene that reduce its gene expression during enteric nervous system (ENS) development. These variants have synergistic effects on RET gene expression and additionally dysregulate other ENS and HSCR genes in the RET-EDNRB gene regulatory network (GRN). Here, we use siRNA, ChIP and CRISPR/Cas9 deletion analyses in the SK-N-SH cell line to ask, how many HSCR-associated risk variants reside in CREs and affect RET gene expression? We demonstrate that 31 HSCR-associated variants reside in candidate RET CREs, ten with differential allele-specific in vitro enhancer activity and seven affecting RET gene expression; of these, five bind the transcription factors PAX3, RARB, GATA2 and SOX10. These and our prior results demonstrate that common sequence variants in at least 10 RET enhancers affect HSCR risk, extending the known RET-EDNRB GRN to reveal an extensive regulatory code modulating disease risk even at a single gene.