Functional study of non-coding variants in Hirschsprung disease using human pluripotent stem cell-based model

Abstract

Hirschsprung disease (HSCR) is a complex multigenic disorder which is caused by incomplete colonization of enteric neural crest cells (ENCCs) in the gut. The aganglionic gut of HSCR patients is functionally obstructed due to uncoordinated muscular peristalsis. Previously, our whole genome sequencing study has identified thousands of HSCR-associated genetic variants residing in the non-coding regions of the human genome. Intriguingly, many of these variants are spanning throughout the non-coding regions of the RET gene and they are highly associated with HSCR susceptibility. rs2435357 (C>T), in particular, has been suggested to increase the risks of HSCR by >4-fold, especially in Chinese patients. In this study, we established an experimental paradigm to interconnect the HSCR associated non-coding variants with the disease phenotypes and severity using human pluripotent stem cell (hiPSC)-based model. By CRISPR-Cas9 genome editing technology, C allele of rs2435357 was changed to T allele in the control hiPSCs or vice versa in the patient-derived hiPSCs. The hiPSCs were directed to ENCC lineage through inhibition of BMP and TGFβ pathways, followed by activation of the WNT pathway and caudalized with retinoic acid. The ENCCs would then be differentiated into neurons by addition of various neurotrophic factors. We examined the RET expression in different developmental stages of cells and found that rs2435357 (C>T) alone is not sufficient to alter RET expression level in either ENCCs or their neuronal derivatives. Based on in silico analysis, we have identified more HSCR susceptible variants in the putative enhancers of RET gene. In further study, multiplex approach will be used to examine the accumulative effect of multiple variants in the development of enteric neurons.