Identifying Candidate Hirschsprung Disease–Associated RET Variants

Abstract

Patients with sporadic Hirschsprung disease (HSCR) show increased allele sharing at markers in the 5' region of the RET locus, indicating the presence of a common ancestral RET mutation. In a previous study, we found a haplotype of six SNPs that was transmitted to 55.6% of our patients, whereas it was present in only 16.2% of the controls we used. Among the patients with that haplotype, 90.8% had it on both chromosomes, which led to a much higher risk of developing HSCR than when the haplotype occurred heterozygously. To more precisely define the HSCR-associated region and to identify candidate disease-associated variant(s), we sequenced the shared common haplotype region from 10 kb upstream of the RET gene through intron 1 and exon 2 (in total, 33 kb) in a patient homozygous for the common risk haplotype and in a control individual homozygous for the most common nonrisk haplotype. A comparison of these sequences revealed 86 sequence differences. Of these 86 variations, 8 proved to be in regions highly conserved among different vertebrates and within putative transcription factor binding sites. We therefore considered these as candidate disease-associated variants. Subsequent genotyping of these eight variants revealed a strong disease association for six of the eight markers. These six markers also showed the largest distortions in allele transmission. Interspecies comparison showed that only one of the six variations was located in a region also conserved in a nonmammalian species, making it the most likely candidate HSCR-associated variant.

RET polymorphisms and the risk of Hirschsprung’s disease in a Chinese population

“…Recent studies have suggested that the HSCR-associated RET mutations may be enhancer mutations, located approximately in the middle of intron 1 (Burzynski et al 2005;Emison et al 2005). Our previous study was consistent with this hypothesis.…”

“…This high risk may be related to features of their structures: both are located in conserved regions that contain binding sites for relevant transcription factors. Interestingly, rs1864410 is recognized by ubiquitous transcription factors, such as SP1-erk1(1) and AP2/SP1 (Burzynski et al 2005). These data suggest that rs1864410 and rs752975 may function as candidate disease-associated variants.…”

“…They found that only one region, located in intron 1, was conserved among all species (including avians) and that only one SNP, rs2506004, was present in this region (Burzynski et al 2005). However, they neither tested larger samples nor performed a functional analysis.…”

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].…”

“…Two groups have described two closely located SNPs, rs2435357 and rs2506004, in intron 1 as disease-causing candidates on the basis of association studies, functional assays and comparative genomics [52,56]. In particular, the last approach seems to be useful for judging the functional relevance of noncoding mutations, because it shows that the two disease-causing candidates are located in regulatory binding sites, which in turn are in regions that are conserved among different vertebrate species.…”