Fine mapping of the NRG1 Hirschsprung's-associated gene

Abstract

Hirschsprung's disease (HSCR, aganglionic megacolon), is a congenital disorder characterized by the absence of enteric ganglia in variable portions of the distal intestine. HSCR has a complex pattern of inheritance and presents mainly sporadically. Besides the major HSCR gene, RET, there is evidence that other loci contribute to HSCR. Through a genome-wide association study (GWAS) on Chinese individuals we identified the association of a 350kb genomic region encompassing the NRG1 gene with HSCR. Within the region, the strongest associations were found for two physically close SNPs, rs16879552 and rs7835688 with association values of p=1.80x10-8 and p=1.12x10-9, respectively. These NRG1 HSCR-associated SNPs are not predicted to functionally affect the gene. Thus, we hypothesized that a common causative/functional variant must lie within the 350kb region and was not revealed by the 500K Affymetrix chips used in the GWAS. To identify the functional variant/s, we resorted to increase the marker density within the region by genotyping 325 SNPs in 380 HSCR Chinese HSCR patients and 380 Chinese controls and test for association. Genotype imputation was also used to increase the SNPs density. We identified a SNPs (rs10088313T/G) highly associated with HSCR, with an EIGENSTRAT corrected p-association value of 6.71x10-5, which is lower than those obtained for the NRG1 intron 1 SNPs identified through the GWAS after correction. Importantly, rs10088313 maps ≈20kb upstream the NRG1 transcription start site. Since rs10088313 could be either functional or/and be in perfect LD with other functionally relevant markers, we focused on the region encompassed by Chinese Han from Beijing (CHB) HapMap markers whose r2 with rs10088313 equals 1 (rs7830563; rs10113578; rs10107065; rs10113593; rs10094655). This delineated a 10.2 kb region. As a) further genotyping of these 5 SNPs in an expanded sample would not help discern among these SNPs in perfect LD and, b) the finding of association across an intron implies involvement in regulation, we used comparative genomics to investigate the 10.2kb region. rs7830563, rs10088313 and rs10113593 create/disrupt binding sites of transcription factors with a prominent in the development of the enteric nervous system. The predicted changes caused by the SNP HSCR-associated alleles are bound to alter the regulation of NRG1 transcription and could possibly affect the NRG1 signalling during ENS development.