Variants in RET associated with Hirschsprung's disease affect binding of transcription factors and gene expression.

BACKGROUND & AIMS: Two noncoding variations in RET-the T allele of the single nucleotide polymorphism (SNP) rs2435357 (Enh1:C>T) and the A allele of the SNP rs2506004 (Enh2:C>A)-are associated with Hirschsprung's disease. These SNPs are in strong linkage disequilibrium and located in an enhancer element in intron 1 of the RET gene. The T allele of the Enh1 variant results in reduced expression of RET, compared with the C allele, because the T allele disrupts binding to the transcription factor SOX10. We studied whether the A allele of Enh2 (Enh2-A) also affects RET gene expression.

METHODS: We evaluated the function of Enh1 and Enh2 using luciferase reporter assays with constructs that contained each allele, separately or in combination. We performed in silico analysis to identify transcription activators or repressors that bind to Enh2-C.

RESULTS: The Enh1-T and the Enh2-A alleles reduced expression of the luciferase reporter gene. In silico analysis identified the sequence of Enh2-C and its surrounding sequence (ACGTG) as a potential binding site for the NXF-ARNT2 and SIM2-ARNT2 transcription factor heterodimers. The affinity of NXF-ARNT2 for Enh2-C was confirmed by electrophoresis mobility shift and supershift assays. Transfection of neuroblastoma cell lines with NXF-ARNT2 or SIM2-ARNT2 increased and decreased expression of RET, respectively.

CONCLUSIONS: More than one SNP on an associated haplotype can influence gene expression and ultimately disease phenotype. Binding of the transcription factors NXF, ARNT2, and SIM2 to RET depend on the RET polymorphism of Enh2 and affect RET expression and the development of Hirschsprung's disease.