title: A common haplotype at the 5' end of the RET proto-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.
authors.affiliation: Laboratorio di Genetica Molecolare, Istituto G. Gaslini, Genova, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Griseri
authors.forename: Tiziana
authors.initials: T
authors.lastname: Bachetti
authors.forename: Francesca
authors.initials: F
authors.lastname: Puppo
authors.forename: Francesca
authors.initials: F
authors.lastname: Lantieri
authors.forename: Roberto
authors.initials: R
authors.lastname: Ravazzolo
authors.forename: Marcella
authors.initials: M
authors.lastname: Devoto
authors.forename: Isabella
authors.initials: I
authors.lastname: Ceccherini
mesh_terms: D024506:5' Flanking Region; D002478:Cells, Cultured; D005091:Exons; D005260:Female; D015870:Gene Expression; D020022:Genetic Predisposition to Disease; D006239:Haplotypes; D006627:Hirschsprung Disease / Q000235:genetics* / Q000378:metabolism; D006720:Homozygote; D006801:Humans; D008297:Male; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D000090063:Proto-Oncogene Mas; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human