A multi-enhancer <i>RET</i> regulatory code is disrupted in Hirschsprung disease

Abstract

The major genetic risk factors for Hirschsprung disease (HSCR) are three common polymorphisms within <i>cis</i>-regulatory elements (CREs) of the receptor tyrosine kinase gene <i>RET</i>, which reduce its expression during enteric nervous system (ENS) development. These risk variants attenuate binding of the transcription factors RARB, GATA2, and SOX10 to their cognate CREs, reduce <i>RET</i> gene expression, and dysregulate other ENS and HSCR genes in the <i>RET-EDNRB</i> 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 additional HSCR-associated risk variants reside in <i>RET</i> CREs that affect its gene expression. We identify 22 HSCR-associated variants in candidate <i>RET</i> CREs, of which seven have differential allele-specific in vitro enhancer activity, and four of these seven affect <i>RET</i> gene expression; of these, two enhancers are bound by the transcription factor PAX3. We also show that deleting multiple variant-containing enhancers leads to synergistic effects on <i>RET</i> gene expression. These, coupled with our prior results, show that common sequence variants in at least 10 <i>RET</i> enhancers affect HSCR risk, seven with experimental evidence of affecting <i>RET</i> gene expression, extending the known <i>RET-EDNRB</i> GRN to reveal an extensive regulatory code modulating disease risk at a single gene.