A multi-enhancer RET regulatory code is disrupted in Hirschsprung disease

In Hirschsprung disease (HSCR; congenital colonic aganglionosis), three GWAS-identified common variants residing in three distinct enhancers of the RET receptor tyrosine kinase gene reduce its gene expression and are causal disease risk variants. Further, their combined effects significantly dysregulate the expression of other functionally-related genes defining the RET gene regulatory network (GRN). In this study, we asked how many variants in how many distinct RET enhancers affect HSCR risk by reducing RET gene expression? We demonstrate that 22 additional HSCR-associated polymorphisms, both independent and associated, reside within multiple candidate RET enhancers and among which 7 display differential allelic enhancer activities. Of these 7 RET enhancers, two bind PAX3 and extend the known RET-EDNRB GRN. Therefore, sequence variants within a minimum of 10 RET enhancers affect HSCR risk, revealing a diverse regulatory code modulating complex disease risk even at a single locus.