Abstract

The activation of the RET signaling pathway during embryogenesis is a crucial prerequisite for a directional migration of enteric nervous system progenitor cells. Loss-of-function germline mutations of the RET proto-oncogene are reported in familial and sporadic cases of Hirschsprung disease (HSCR) with a variable frequency. Furthermore, variants of several RET polymorphisms are over- or under-represented in HSCR populations. Specifically, the c.135A RETvariant has been previously shown as strongly associated with the HSCR phenotype. We have reported a HSCR-phenotype modifying effect of the RETc. 135G > A polymorphism due to a within-gene interaction in patients harboring RET germline mutations, yet the function of the c.l35G> A variant is unknown.

The basic RET promoter region was investigated by DNA sequencing approach in 80 HSCR patients. Identified polymorphisms were genotyped in the HSCR and in a control population and haplotypes were reconstructed. The dual-luciferase assay was used to evaluate the activity of different RET promoter haplotypes. We demonstrate that variants of two RET promoter polymorphisms −5G > A and −1C > A from the transcription start site are associated with HSCR. Furthermore, the −5G > A polymorphism is in strong linkage disequilibrium with the c.l35G> A polymorphism. The promoter haplotype −5/ −1AC associated with HSCR has a significantly lower activity in an in-vitro dual-luciferase expression assay compared to those haplotypes identified in the majority of normal controls.

These data suggest a role for RET haplotypes containing the −5A promoter variant in the etiology of HSCR, in a dose-dependant fashion.