Variability in proliferative and migratory defects in Hirschsprung disease-associated RET pathogenic variants.

Hirschsprung disease (HSCR) exhibits extensive genetic heterogeneity, with 72% of cases involving pathogenic variants in 10 genes forming a gene regulatory network (GRN) essential for enteric nervous system (ENS) development. The receptor tyrosine kinase gene RET is the most significant contributor, implicated in 12%-50% of individuals depending on the phenotype. RET plays a critical role in ENS precursor proliferation and migration, and defects in these processes lead to HSCR. However, the functional impact of RET pathogenic variants and their mechanisms of disease remain poorly understood. To address this, we investigated proliferative and migratory phenotypes in a RET-dependent neural crest-derived cell line harboring one of five missense (c.166C>A [p.Leu56Met]; c.532G>C [p.Glu178Gln]; c.2372A>T [p.Tyr791Phe]; c.2765C>A [p.Ser922Tyr]; or c.2994T>A [p.Phe998Leu]) or three nonsense (c.612C>A, c.2308C>T, or c.2943C>G) heterozygous pathogenic RET variants. Using cDNA- and CRISPR-based prime reverse insertion mechanism engineering (PRIME) editing coupled with quantitative proliferation and migration assays, we observed significant losses in proliferation and migration in three missense (c.612C>A [p.Tyr204