Genome-wide association study identifies NRG1 as a susceptibility locus for Hirschsprung's disease

Abstract

Hirschsprung's disease (HSCR), or aganglionic megacolon, is a congenital disorder characterized by the absence of enteric ganglia in variable portions of the distal intestine. RET is a well-established susceptibility locus, although existing evidence strongly suggests additional loci contributing to sporadic HSCR. To identify these additional genetic loci, we carried out a genome-wide association study using the Affymetrix 500K marker set. We successfully genotyped 293,836 SNPs in 181 Chinese subjects with sporadic HSCR and 346 ethnically matched control subjects. The SNPs most associated with HSCR were genotyped in an independent set of 190 HSCR and 510 control subjects. Aside from SNPs in RET, the strongest overall associations in plausible candidate genes were found for 2 SNPs located in intron 1 of the neuregulin1 gene (NRG1) on 8p12, with rs16879552 and rs7835688 yielding odds ratios of 1.68 [CI(95%):(1.40, 2.00), P = 1.80 x 10(-8)] and 1.98 [CI(95%):(1.59, 2.47), P = 1.12 x 10(-9)], respectively, for the heterozygous risk genotypes under an additive model. There was also a significant interaction between RET and NRG1 (P = 0.0095), increasing the odds ratio 2.3-fold to 19.53 for the RET rs2435357 risk genotype (TT) in the presence of the NRG1 rs7835688 heterozygote, indicating that NRG1 is a modifier of HSRC penetrance. Our highly significant association findings are backed-up by the important role of NRG1 as regulator of the development of the enteric ganglia precursors. The identification of NRG1 as an additional HSCR susceptibility locus not only opens unique fields of investigation into the mechanisms underlying the HSCR pathology, but also the mechanisms by which a discrete number of loci interact with each other to cause disease.

Fig. 1.

Results of the Genomewide Association Study. (A) Cochran-Armitage significance test results after EIGENSTRAT correction for population stratification. (B) Q-Q-plot revealing deviation of association from expected, starting from P = 5 × 10⁻⁴ (purple before and blue after EIGENSTRAT correction).

Fig. 2.

(A) A −log(p) value for GWA NRG1 SNPs (dark green) and imputed SNPs (light green) under the additive model of association. (B) The cumulative recombination rate (genetic distance) measured away from the most highly associated genotyped SNP. (C) The fine-scale recombination rate across the region. The vertical dashed lines on the plot delineate the main region of association. (D) Schematic representation of the NRG1 isoforms overlapping the associated region. Boxes represent exons and fine lines introns. Notice that exon 1 is in the associated region.

Fig. 3.

Expression of NRG1 in the intestinal mucosa of (A) a non-HSCR infant, and in the (B) normoganglionic (C) and aganglionic mucosa of a HSCR-affected infant. NRG1 expression in the (D) enteric plexuses of a non-HSCR infant, (E) HSCR infant, and (F) in the hypertrophic nerve trunk. The abnormal nerve bundles characteristic of the aganglionic bowel are extrinsic cholinergic fibers of sacral origin. cm, circular muscle; hn, hypertrophic nerve; lm, longitudinal muscle; mm, muscularis mucosa; mp, myenteric plexuses; mu, intestinal mucosa.