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

Abstract

Hirschsprung disease (HSCR) is a complex genetic defect of intestinal innervation mainly ascribed to loss of function mutations of the RET gene. Although RETcoding 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.

Complex pathogenesis of Hirschsprung's disease in a patient with hydrocephalus, vesico-ureteral reflux and a balanced translocation t(3;17)(p12;q11)

“…Our patient showed a marked reduction of RET mRNA (58 vs 100% of the control, P-value ¼ 0.019) ( Figure 5), a finding in agreement with previous data showing an impaired RET expression associated with this haplotype. 18 Our data support the hypothesis of a major role played by the association of L1CAM mutation and the RET predisposing haplotype in the occurrence of the two phenotypes, as previously reported in eight patients. 7 -9 Moreover, based on present results, the hypothesis that the impaired expression of the MYO18A/TIAF1 genes represents an additional risk factor, acting together with L1CAM and RET defects in our patient to the occurrence of the Hirschsprung's phenotype, cannot be excluded.…”

Current concepts in RET-related genetics, signaling and therapeutics

“…Several groups have characterized candidate diseaseassociated polymorphisms [49,[55][56][57]. Two promoter SNPs, rs10900296 and rs10900297 (also named SNP-5G > A and SNP-1A > C, respectively), located just upstream of the RET transcriptional start site, have been shown to reduce RET promoter activity in luciferase reporter assays [49,55], although others have shown that this effect is dependent on the cell lines studied [57].…”

A complex additive model of inheritance for Hirschsprung disease is supported by both RET mutations and predisposing RET haplotypes

“…16 This was the starting point which led to the identification of a predisposing haplotype located at the 5=region of RET, whatever the ethnic background, with some functional data suggesting a putative role of the promoter variants Ϫ200AϾG and Ϫ196CϾA. [17][18][19][20][21][22] In the present report, we report for the first time a mutational screening in the RET proto-oncogene in a series of HSCR patients from Spain, and with the available data derived from previous studies, 19 we have analyzed and compared the RET haplotypic distribution in carriers versus noncarriers of traditional RET germline mutations.…”