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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.

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Figure 1: Overview of our study.
Figure 2: Association analysis for the NOD2ADCY7 region on chromosome 16.
Figure 3: Associations between NOD2 variants and Crohn's disease.
Figure 4: Burden of rare, damaging variants in Crohn's disease.
Figure 5: Relative power of this study in comparison to previous GWAS.

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Acknowledgements

We thank all individuals who contributed samples to the study. This work was co-funded by the Wellcome Trust (098051) and the Medical Research Council, UK (MR/J00314X/1). Case collections were supported by Crohn's and Colitis UK. K.M.d.L., L.M., Y.L., C.A.L., C.A.A. and J.C.B. are supported by the Wellcome Trust (098051; 093885/Z/10/Z). K.M.d.L. is supported by a Woolf Fisher Trust scholarship. C.A.L. is a clinical lecturer funded by the NIHR. H.U. is supported by the Crohn's and Colitis Foundation of America (CCFA) and the Leona M. and Harry B. Helmsley Charitable Trust. We acknowledge support from the UK Department of Health via NIHR comprehensive Biomedical Research Centre awards to Guy's and St Thomas' NHS Foundation Trust in partnership with King's College London and to Addenbrooke's Hospital, Cambridge, in partnership with the University of Cambridge, and the BRC to the Oxford IBD cohort study, University of Oxford. This research was also supported by the NIHR Newcastle Biomedical Research Centre. The UK Household Longitudinal Study is led by the Institute for Social and Economic Research at the University of Essex and funded by the Economic and Social Research Council. The survey was conducted by NatCen, and the genome-wide scan data were analyzed and deposited by the Wellcome Trust Sanger Institute. Information on how to access the data can be found on the Understanding Society website. We are grateful for genotyping data from the British Society for Surgery of the Hand Genetics of Dupuytren's Disease consortium and L. Southam for assistance with genotype intensities. This research has been conducted using the UK Biobank Resource.

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Y.L., K.M.d.L., L.J., L.M., J.C.B. and C.A.A. performed statistical analysis. Y.L., K.M.d.L., L.J., L.M., J.C.L., C.A.L., E.G.S., J.R., M. Pollard, S.N. and S.M. processed the data. T.A., C.E., N.A.K., A.H., C.H., J.C.M., J.C.L., C.M., W.G.N., J.S., A.S., M.T., H.U., D.C.W., N.J.P., C.W.L., M. Parkes and C.G.M. contributed samples and/or materials. Y.L., K.M.d.L., L.M., J.C.L., M. Parkes, C.A.L., N.A.K., J.C.B. and C.A.A. wrote the manuscript. All authors read and approved the final version of the manuscript. J.C.M., M. Parkes, C.W.L., T.A., N.J.P., J.C.B. and C.A.A. conceived and designed experiments.

Corresponding authors

Correspondence to Jeffrey C Barrett or Carl A Anderson.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Production pipeline of SNVs and indels.

Supplementary Figure 2 Genotypic accuracy of sequencing data.

Dosage r2 plots for determining sequencing quality when compared against other genotyping data. The x axis is minor allele frequency calculated based on sequencing samples, and the y axis is correlation between dosages for sequencing and genotype data sets. Numbers in parentheses are the number of individuals with both types of data.

Supplementary Figure 3 Biallelic SNV discovery rate compared to the 1000 Genomes Project Phase 3 European panel.

Percentage of biallelic SNVs in all autosomal regions that are shared by the IBD sequencing set and 1000 Genomes Project (1000GP) Phase 3 European panel (503 individuals). Left, percentage of IBD sequencing SNVs that are also found in 1000GP; right, variants identified in the 1000GP set that are also in the IBD sequencing cohort. MAFs on the left were calculated based on SNVs discovered in the IBD sequencing project, and MAFs on the right were calculated based on the 1000GP set. Different lines represent SNVs in different quality control stages of the analysis.

Supplementary Figure 4 Number of copy number variants called per cohort.

Average number of calls per individual per site, across different copy number variant (CNV) lengths. UK10K controls (6×) are shown in yellow, Crohn’s disease cases (4×) are shown in red, and ulcerative colitis cases (2×) are shown in blue.

Supplementary Figure 5 Quantile–quantile plots of genome-wide association studies for variants with MAF ≥ 0.1% in the sequencing data set.

λ1,000 values are reported for ulcerative colitis, Crohn’s disease and inflammatory bowel disease analyses. Gray shapes show the 95% confidence interval.

Supplementary Figure 6 Cluster plots for rs78534766.

(a–c) Cluster plots are shown for rs78534766 for the GWAS3 (a), replication (b) and UK Biobank (c) samples that passed quality control. SNP genotypes have been assigned based on cluster formation in scatterplots of normalized allele intensities X and Y. Each circle represents one individual’s genotype. Blue and red clouds correspond to homozygote genotypes for the SNP (CC/AA), green clouds correspond to the heterozygote genotype (CA) and gray clouds correspond to undetermined genotype.

Supplementary Figure 7 Workflow for heritability estimation.

Supplementary Figure 8 Distribution of INFO scores by cohort, across a range of minor allele frequencies.

(a) INFO scores calculated using genotype probabilities generated directly from the SAMtools Genotype Quality (GQ) field. (b) INFO scores calculated using genotype probabilities after imputation improvement using BEAGLE.

Supplementary Figure 9 Manhattan and quantile–quantile plots showing the results of gene-based burden tests using rare, functional coding variation.

Supplementary Figure 10 Principal-component analysis of the sequencing samples.

IBD samples are plotted with 11 different HapMap 3 populations.

Supplementary Figure 11 Effect of read depth on sensitivity and specificity across the allele frequency spectrum (UC (2×) CD (4×), controls (6×)).

(a–c) Top, full distribution of variant counts per individual at singletons (observed once in the data set) (a), doubletons (observed twice) (b) and variants with a MAF of 5% (c). (d) Plot showing the median of each distribution across a range of MAF values.

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Supplementary Figures 1–11, Supplementary Tables 1–17 and Supplementary Note (PDF 3457 kb)

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Luo, Y., de Lange, K., Jostins, L. et al. Exploring the genetic architecture of inflammatory bowel disease by whole-genome sequencing identifies association at ADCY7. Nat Genet 49, 186–192 (2017). https://doi.org/10.1038/ng.3761

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