Extended haplotype association study in Crohn's disease identifies a novel, Ashkenazi Jewish-specific missense mutation in the NF-κB pathway gene, HEATR3

Abstract

The Ashkenazi Jewish population has a several-fold higher prevalence of Crohn's disease (CD) compared with non-Jewish European ancestry populations and has a unique genetic history. Haplotype association is critical to CD etiology in this population, most notably at NOD2, in which three causal, uncommon and conditionally independent NOD2 variants reside on a shared background haplotype. We present an analysis of extended haplotypes that showed significantly greater association to CD in the Ashkenazi Jewish population compared with a non-Jewish population (145 haplotypes and no haplotypes with P-value <10(-3), respectively). Two haplotype regions, one each on chromosomes 16 and 21, conferred increased disease risk within established CD loci. We performed exome sequencing of 55 Ashkenazi Jewish individuals and follow-up genotyping focused on variants in these two regions. We observed Ashkenazi Jewish-specific nominal association at R755C in TRPM2 on chromosome 21. Within the chromosome 16 region, R642S of HEATR3 and rs9922362 of BRD7 showed genome-wide significance. Expression studies of HEATR3 demonstrated a positive role in NOD2-mediated NF-κB signaling. The BRD7 signal showed conditional dependence with only the downstream rare CD-causal variants in NOD2, but not with the background haplotype; this elaborates NOD2 as a key illustration of synthetic association.

Figure 1

Manhattan plot of haplotype associations in non-Ashkenazi Jewish (A) and Ashkenazi Jewish (B) European Ancestry cohorts. In the non-Jewish analysis, no regions demonstrated evidence for association. In contrast, in the Ashkenazim, we observed 145 haplotypes demonstrating nominal evidence for association with P-values < 10⁻³, including three distinct regions with P-values < 10⁻⁴. Associated haplotypes that overlapped with established CD loci, and which are detailed in Table 1, are labeled.

Figure 2

Single-marker associations in the chromosome 16 haplotype region. We observed genome-wide significant associations at rs2076756 (P-value = 2.32 × 10⁻²⁰) in NOD2 and rs9922362 (P-value = 3.26 × 10⁻¹⁶) in BRD7. Positions of two nominally associated Ashkenazi Jewish haplotypes are shown by black bars at top.

Figure 3

HEATR3 is a positive component of the NOD2 signaling pathway. siRNA mediated knock down of HEATR3 diminished NOD2-dependent NF-κB induction (A) and IL-8 secretion (B) relative to non-targeting siRNA controls. Furthermore, over-expression of HEATR3 increased NF-κB signaling (C) and IL-8 production (D). Cells were left unstimulated (black bars) or stimulated overnight with 20 ng/mL of MDP (grey bars). Together these results support a positive role for HEATR3 in NOD2 signaling. * P<0.05, ** P<0.01, *** P<0.001.

Figure 4

Venn diagram of carriers for chromosome 16 haplotype variants among healthy controls. Numbers indicate the percentage of haploid chromosomes (n = 2440) that carry each combination of mutations. Colors indicate causal mutations within NOD2: peach = R702W; green = N852S; pink = G908R; blue = L1007insC. Missing genotypes and phasing of haploid carriers were inferred using BEAGLE with default parameters. Regions representing less than 0.5% of haploid carriers are not shown. These results indicate that both the common BRD7 and NOD2 SNPs serve as backgrounds for the NOD2 uncommon causal variants, which in turn serve as the basis for association at the these two common background variants.