Exploring the genetic architecture of inflammatory bowel disease by whole-genome sequencing identifies association at ADCY7

Abstract

To further resolve the genetic architecture of the inflammatory bowel diseases ulcerative colitis and Crohn's disease, we sequenced the whole genomes of 4,280 patients at low coverage and compared them to 3,652 previously sequenced population controls across 73.5 million variants. We then imputed from these sequences into new and existing genome-wide association study cohorts and tested for association at ∼12 million variants in a total of 16,432 cases and 18,843 controls. We discovered a 0.6% frequency missense variant in ADCY7 that doubles the risk of ulcerative colitis. Despite good statistical power, we did not identify any other new low-frequency risk variants and found that such variants explained little heritability. We detected a burden of very rare, damaging missense variants in known Crohn's disease risk genes, suggesting that more comprehensive sequencing studies will continue to improve understanding of the biology of complex diseases.

Figure 1. Overview of our study.

Variants were called from raw sequence reads in three groups of samples, and jointly filtered using support vector machines. The resulting genotypes were refined using BEAGLE and incorporated into the reference panel for a GWAS-imputation based meta-analysis, which discovered a low frequency association in ADCY7. A separate gene-based analysis identified a burden of rare damaging variants in certain known Crohn’s disease genes.

Figure 2. Association analysis for the NOD2/ADCY7 region in chromosome 16.

Results from the single variant association analysis are presented in gray, and results after conditioning on seven known NOD2 risk variants in blue. Results for Crohn’s disease (CD) are shown in the top half, and ulcerative colitis (UC) in the bottom half. The dashed red lines indicate genome-wide significance, at α = 5x10^-8.

Figure 3. Associations between NOD2 and Crohn’s disease.

Each point represents the contribution of an individual variant to our NOD2 burden test. Three common variants (rs2066844, rs2066845, rs2066847) are shown for scale, and the six rare variants identified by targeted sequencing are starred. Exonic regions (not to scale) are marked in blue, with their corresponding protein domains highlighted.

Figure 4. Burden of rare damaging variants in Crohn’s disease.

Each point represents a gene in our confidently implicated (green) or generically implicated (blue) gene sets. Genes are ranked on the x-axis from most enriched in cases to most enriched in controls, and position on the y-axis represents significance. The purple shaded region indicates where 75% of all genes tested lie. Our burden signal is driven by a mixture of genes where rare variants are risk increasing (e.g. NOD2) and risk decreasing (IL23R).

Figure 5. Relative power of this study compared to previous GWAS.

The black line shows the path through frequency-odds ratio space where the latest IIBDGC meta-analysis had 80% power. The purple line (imputed GWAS) and green line (sequencing) shows the same for this study. The earlier study had more samples but restricted their analysis to MAF > 1%. Purple density and points show known GWAS loci, with our novel ADCY7 association (p.Asp439Glu) highlighted as a star. Green points show a subset of our sequenced NOD2 rare variants, and the green star shows their equivalent position when tested by gene burden, rather than individually.