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Ancestral RET haplotype associated with Hirschsprung’s disease shows linkage disequilibrium breakpoint at −1249

BACKGROUND
Hirschsprung disease (HSCR) is a complex disorder with traditional germline mutations in RET in up to 30% of familial cases and in 3% of sporadic cases in a population-based series. We have previously demonstrated that an ancestral haplotype at the 5' end of RET (haplotype 0) was strongly associated with a large subset of isolated HSCR cases and that a putative low penetrance susceptibility locus was encompassed within this ancestral haplotype, anchored by exon 2 SNP A45A.


OBJECTIVE
To determine the 5' extent of the HSCR-associated ancestral haplotype by defining the linkage disequilibrium breakpoint in search for the low penetrance susceptibility locus.


METHODS
Systematic screening of the region upstream of the anchoring A45A SNP, comprising RET intron 1, exon 1, and promoter in 117 population-based HSCR cases and 100 controls. Dual luciferase assay to determine differential activities between SNP combinations near a transcription start site.


RESULTS
New SNP's were found which formed upstream haplotypes, anchored by A45A, in linkage disequilibrium with HSCR (2 = 76.96, p<0.00000001). Linkage disequilibrium appeared to break at the -1249C/T SNP. Further, the HSCR-associated genotype (00) was found in >60% of HSCR but only 2% of controls. Because only 2 variants, -200A>G and -196C>A, lie within the promoter region and are in proximity to the transcriptional start site (at -195), we modelled these combinations into constructs for luciferase reporter assay. The HSCR-associated SNP combination showed the lowest activity and the control-associated combination, the highest.


CONCLUSIONS
Our observations seem to discard the existence of a HSCR-causing mutation as it is conceived in the traditional sense, but strengthen the idea of a specific combination of variants conferring susceptibility to the disease in a low penetrance fashion. The data derived from our functional "in vitro" studies would suggest that the HSCR-associated haplotype 0 may result in a lower level of expression of the RET gene [corrected]