A common haplotype at the 5′ end of the RET proto-oncogene, overrepresented in Hirschsprung patients, is associated with reduced gene expression

Hirschsprung disease (HSCR) is a complex genetic defect of intestinal innervation mainly ascribed to loss of function mutations of the RET gene. Although RET coding mutations account for only 15% of HSCR sporadic cases, several linkage and association studies still indicate RET as a major HSCR gene, suggesting the existence of noncoding RET variants or common polymorphisms which can act in HSCR pathogenesis. We previously described a predisposing RET haplotype (A-C-A) composed of alleles at three SNPs (-1 bp and -5 bp from the RET transcription start site, NT_033985.6:g.975824G > A and NT_033985.6:g.975820C >A, respectively, and silent polymorphism c. 135G > A), which was present in 62% of chromosomes from HSCR patients but only in 22% of control chromosomes. Here we address the question of how this 5' ACA haplotype may functionally act as a predisposing factor in HSCR pathogenesis by performing functional analysis of the same three SNPs. We demonstrate that neither the two promoter variants nor the exon 2 SNP interfere with reporter,gene transcription or RET mRNA splicing, respectively. However, real-time RT-PCR, performed in RNA obtained from lymphoblasts of selected individuals, has shown that homozygosity for the whole ACA haplotype is associated with reduced RET gene expression. We propose that a yet unidentified variant in linkage disequilibrium with the ACA haplotype, rather than the single characterizing SNPs, acts as a HSCR susceptibility allele by affecting the normal amount of RET receptor on the cell surface. (C) 2005 Wiley-Liss, Inc.