Fine mapping of the NRG1 Hirschsprung's disease locus

Abstract

The primary pathology of Hirschsprung's disease (HSCR, colon aganglionosis) is the absence of ganglia in variable lengths of the hindgut, resulting in functional obstruction. HSCR is attributed to a failure of migration of the enteric ganglion precursors along the developing gut. RET is a key regulator of the development of the enteric nervous system (ENS) and the major HSCR-causing gene. Yet the reduced penetrance of RET DNA HSCR-associated variants together with the phenotypic variability suggest the involvement of additional genes in the disease. Through a genome-wide association study, we uncovered a ∼350 kb HSCR-associated region encompassing part of the neuregulin-1 gene (NRG1). To identify the causal NRG1 variants contributing to HSCR, we genotyped 243 SNPs variants on 343 ethnic Chinese HSCR patients and 359 controls. Genotype analysis coupled with imputation narrowed down the HSCR-associated region to 21 kb, with four of the most associated SNPs (rs10088313, rs10094655, rs4624987, and rs3884552) mapping to the NRG1 promoter. We investigated whether there was correlation between the genotype at the rs10088313 locus and the amount of NRG1 expressed in human gut tissues (40 patients and 21 controls) and found differences in expression as a function of genotype. We also found significant differences in NRG1 expression levels between diseased and control individuals bearing the same rs10088313 risk genotype. This indicates that the effects of NRG1 common variants are likely to depend on other alleles or epigenetic factors present in the patients and would account for the variability in the genetic predisposition to HSCR.

Figure 1. Fine mapping association results of NRG1 SNPs.

Association results shown before (upper panel) and after imputation (lower panel). Diamonds and circles indicate genotyped and imputed SNPs respectively. Colour gradient (red r² = 1 to white r² = 0) marks the LD of the SNPs with rs10088313, except green for rs7835688, dark green for rs16879552 and blue for itself; grey indicates no information on LD. The 21 kb region we narrowed down to is highlighted in pink. The fine-scale recombination rate across the region is represented by the light-blue line. Green lines symbolize a schematic representation of the NRG1 isoforms overlapping the associated region. The bottom green line represents the GGF2 isoform associated with schizophrenia that expands 900 kb upstream the transcription start site for the rest of NRG1 isoforms.

Figure 2. NRG1 expression in human gut tissues.

Quantitative RT-PCR analysis of log-transformed expression ratio of NRG1 to 18S in the colon tissues from the normal portions of HSCR patients and non-HSCR patients stratified according to the rs10088313 (G/T; G risk allele). Bars represent the standard error (SE). * p<0.05; ** p<0.005.