Investigating the implications of RET variant/mutation in Hirschsprung disease pathogenesis using patient-specific iPSC

Abstract

Hirschsprung (HSCR) disease is a complex congenital disorder and is attributed to failure of enteric neural crest cells (ENCCs) to fully colonize the bowel, leading to bowel obstruction and megacolon. Genetic lesions that affect NCC development (proliferation/survival, migration and differentiation) may lead to HSCR disease. RET/GDNF signaling is crucial for ENCC development and RET is the most predominant susceptibility gene for HSCR. Genetic variant/mutations in coding (CD) and non-coding (NCD) regions of RET are respectively associated with the severe (total colonic aganglionosis, TCA) and mild (short segment, S-HSCR) forms of HSCR. In addition, many of these genetic lesions directly lead to reduced or defective RET/GDNF signaling, suggesting that these variant/mutations may impair NCC development. The goal of the studies was to investigate how these RET CD and NCD variant/mutations affect NCC development. Recently, we have successfully established and characterized two induced pluripotent stem cell (HSCR-iPSC) lines from patients who presented with S-HSCR and TCA, harboring RET NCD and CD variant/mutations respectively. Moreover, we have adapted an efficient protocol for the differentiation of human iPSCs into NCCs. We found that NCCs derived from the HSCR-iPSC line carrying RET NCD showed defects in migration and neuronal differentiation. Importantly, the expression of genes involved in migration, neuronal and glial differentiation was dysregulated, and RET expression was reduced. Besides, early NCC development defects and impaired RET expression were observed in another iPSC line from a TCA-HSCR patient carrying RET CD mutation. These results suggest that RET variant/mutations would affect both differentiation and migration of NCCs in HSCR patients.