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Deep resequencing of GWAS loci identifies independent rare variants associated with inflammatory bowel disease

Abstract

More than 1,000 susceptibility loci have been identified through genome-wide association studies (GWAS) of common variants; however, the specific genes and full allelic spectrum of causal variants underlying these findings have not yet been defined. Here we used pooled next-generation sequencing to study 56 genes from regions associated with Crohn's disease in 350 cases and 350 controls. Through follow-up genotyping of 70 rare and low-frequency protein-altering variants in nine independent case-control series (16,054 Crohn's disease cases, 12,153 ulcerative colitis cases and 17,575 healthy controls), we identified four additional independent risk factors in NOD2, two additional protective variants in IL23R, a highly significant association with a protective splice variant in CARD9 (P < 1 × 10−16, odds ratio ≈ 0.29) and additional associations with coding variants in IL18RAP, CUL2, C1orf106, PTPN22 and MUC19. We extend the results of successful GWAS by identifying new, rare and probably functional variants that could aid functional experiments and predictive models.

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Figure 1: Overview.
Figure 2: CARD9 protective splice-site variant and predicted transcript.
Figure 3: Identification of additional rare variants in NOD2 associated with Crohn's disease.
Figure 4: Functional analyses of NOD2 variants.

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References

  1. Loftus, E.V. Jr. Clinical epidemiology of inflammatory bowel disease: Incidence, prevalence, and environmental influences. Gastroenterology 126, 1504–1517 (2004).

    Article  Google Scholar 

  2. Bengtson, M.B. et al. Familial aggregation in Crohn's disease and ulcerative colitis in a Norwegian population-based cohort followed for ten years. J. Crohns Colitis 3, 92–99 (2009).

    Article  Google Scholar 

  3. Brant, S.R. Update on the heritability of inflammatory bowel disease: the importance of twin studies. Inflamm. Bowel Dis. 17, 1–5 (2011).

    Article  Google Scholar 

  4. Rioux, J.D. & Abbas, A.K. Paths to understanding the genetic basis of autoimmune disease. Nature 435, 584–589 (2005).

    Article  CAS  Google Scholar 

  5. Nadeau, J.H. Single nucleotide polymorphisms: tackling complexity. Nature 420, 517–518 (2002).

    Article  CAS  Google Scholar 

  6. Plenge, R. & Rioux, J.D. Identifying susceptibility genes for immunological disorders: patterns, power, and proof. Immunol. Rev. 210, 40–51 (2006).

    Article  CAS  Google Scholar 

  7. Barrett, J.C. et al. Genome-wide association defines more than 30 distinct susceptibility loci for Crohn's disease. Nat. Genet. 40, 955–962 (2008).

    Article  CAS  Google Scholar 

  8. Franke, A. et al. Genome-wide meta-analysis increases to 71 the number of confirmed Crohn's disease susceptibility loci. Nat. Genet. 42, 1118–1125 (2010).

    Article  CAS  Google Scholar 

  9. McGovern, D.P. et al. Genome-wide association identifies multiple ulcerative colitis susceptibility loci. Nat. Genet. 42, 332–337 (2010).

    Article  CAS  Google Scholar 

  10. Anderson, C.A. et al. Meta-analysis identifies 29 additional ulcerative colitis risk loci, increasing the number of confirmed associations to 47. Nat. Genet. 43, 246–252 (2011).

    Article  CAS  Google Scholar 

  11. Altshuler, D. & Daly, M. Guilt beyond a reasonable doubt. Nat. Genet. 39, 813–815 (2007).

    Article  CAS  Google Scholar 

  12. Altshuler, D., Daly, M.J. & Lander, E.S. Genetic mapping in human disease. Science 322, 881–888 (2008).

    Article  CAS  Google Scholar 

  13. Hugot, J.P. et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature 411, 599–603 (2001).

    Article  CAS  Google Scholar 

  14. Duerr, R.H. et al. A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science 314, 1461–1463 (2006).

    Article  CAS  Google Scholar 

  15. The 1000 Genomes Project Consortium. A map of human genome variation from population-scale sequencing. Nature 467, 1061–1073 (2010).

  16. Nejentsev, S. et al. Rare variants of IFIH1, a gene implicated in antiviral responses, protect against type 1 diabetes. Science 324, 387–389 (2009).

    Article  CAS  Google Scholar 

  17. Calvo, S.E. et al. High-throughput, pooled sequencing identifies mutations in NUBPL and FOXRED1 in human complex I deficiency. Nat. Genet. 42, 851–858 (2010).

    Article  CAS  Google Scholar 

  18. Zaghloul, N.A. et al. Functional analyses of variants reveal a significant role for dominant negative and common alleles in oligogenic Bardet-Biedl syndrome. Proc. Natl. Acad. Sci. USA 107, 10602–10607 (2010).

    Article  CAS  Google Scholar 

  19. Emison, E.S. et al. Differential contributions of rare and common coding and noncoding Ret mutations to multifactorial Hirschsprung disease liability. Am. J. Hum. Genet. 87, 60–74 (2010).

    Article  CAS  Google Scholar 

  20. Cohen, J.C., Boerwinkle, E., Mosley, T.H.J. & Hobbs, H.H. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N. Engl. J. Med. 354, 1264–1272 (2006).

    Article  CAS  Google Scholar 

  21. Cohen, J.C. et al. Multiple rare variants in NPC1L1 associated with reduced sterol absorption and plasma low-density lipoprotein levels. Proc. Natl. Acad. Sci. USA 103, 1810–1815 (2006).

    Article  CAS  Google Scholar 

  22. Rioux, J.D. et al. Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis. Nat. Genet. 39, 596–604 (2007).

    Article  CAS  Google Scholar 

  23. Marth, G.T. et al. The functional spectrum of low-frequency coding variation. Genome Biol. 12, R84 (2011).

    Article  Google Scholar 

  24. Chamaillard, M. et al. Gene-environment interaction modulated by allelic heterogeneity in inflammatory diseases. Proc. Natl. Acad. Sci. USA 100, 3455–3460 (2003).

    Article  CAS  Google Scholar 

  25. Ogura, Y. et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature 411, 603–606 (2001).

    Article  CAS  Google Scholar 

  26. Miceli-Richard, C. et al. CARD15 mutations in Blau syndrome. Nat. Genet. 29, 19–20 (2001).

    Article  CAS  Google Scholar 

  27. King, K. et al. Mutation, selection, and evolution of the Crohn disease susceptibility gene CARD15. Hum. Mutat. 27, 44–54 (2006).

    Article  CAS  Google Scholar 

  28. Barnich, N., Aguirre, J.E., Reinecker, H.C., Xavier, R.J. & Podolsky, D.K. Membrane recruitment of NOD2 in intenstinal epithelial cells is essential for nuclear factor-κB activation in muramyl dipeptide recognition. J. Cell Biol. 170, 21–26 (2005).

    Article  CAS  Google Scholar 

  29. Tanabe, T. et al. Regulatory regions and critical residues of NOD2 involved in muramyl dipeptide recognition. EMBO J. 23, 1587–1597 (2004).

    Article  CAS  Google Scholar 

  30. Roth, M.P. et al. Geographic origins of Jewish patients with inflammatory bowel disease. Gastroenterology 97, 900–904 (1989).

    Article  CAS  Google Scholar 

  31. Tukel, T. et al. Crohn disease: frequency and nature of CARD15 mutations in Ashkenazi and Sephardi/Oriental Jewish families. Am. J. Hum. Genet. 74, 623–636 (2004).

    Article  CAS  Google Scholar 

  32. Momozawa, Y. et al. Resequencing of positional candidates identifies low frequency IL23R coding variants protecting against inflammatory bowel disease. Nat. Genet. 43, 43–47 (2011).

    Article  CAS  Google Scholar 

  33. Di Meglio, P. et al. The IL23R R381Q gene variant protects against immune-mediated diseases by impairing IL-23-induced TH17 effector response in humans. PLoS ONE 6, e17160 (2011).

    Article  CAS  Google Scholar 

  34. Ghoreschi, K. et al. Generation of pathogenic T(H)17 cells in the absence of TGF-β signalling. Nature 467, 967–971 (2010).

    Article  CAS  Google Scholar 

  35. Festen, E.A. et al. A meta-analysis of genome-wide association scans identifies IL18RAP, PTPN2, TAGAP, and PUS10 as shared risk loci for Crohn's disease and celiac disease. PLoS Genet. 7, e1001283 (2011).

    Article  CAS  Google Scholar 

  36. Behrends, C., Sowa, M.E., Gygi, S.P. & Harper, J.W. Network organize of the human autophagy system. Nature 466, 68–76 (2010).

    Article  CAS  Google Scholar 

  37. Barrett, J.C. et al. Genome-wide association study and meta-analysis find that over 40 loci affect risk of type 1 diabetes. Nat. Genet. 41, 703–707 (2009).

    Article  CAS  Google Scholar 

  38. Stahl, E.A. et al. Genome-wide association study meta-analysis identifies seven new rheumatoid arthritis risk loci. Nat. Genet. 42, 508–514 (2010).

    Article  CAS  Google Scholar 

  39. Jin, Y. et al. Variant of TYR and autoimmunity susceptibility loci in generalized vitiligo. N. Engl. J. Med. 362, 1686–1697 (2010).

    Article  CAS  Google Scholar 

  40. Pearson, K. Mathematical contributions to the theory of evolution VIII: On the inheritance of characters not capable of exact quantitative measurement. Phil. Trans. R. Soc. Lond. A 195, 79–150 (1900).

    Article  Google Scholar 

  41. Fisher, R.A. The correlation between relatives on the supposition of Mendelian inheritance. Trans. R. Soc. Edinb. 52, 399–433 (1918).

    Article  Google Scholar 

  42. Ahmad, T., Satsangi, J., McGovern, D., Bunce, M. & Jewell, D.P. The genetics of inflammatory bowel disease. Aliment. Pharmacol. Ther. 15, 731–748 (2001).

    Article  CAS  Google Scholar 

  43. Gnirke, A. et al. Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat. Biotechnol. 27, 182–189 (2009).

    Article  CAS  Google Scholar 

  44. Li, H., Ruan, J. & Durbin, R. Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res. 18, 1851–1858 (2008).

    Article  CAS  Google Scholar 

  45. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  46. Krawczak, M. et al. PopGen: population-based recruitment of patients and controls for the analysis of complex genotype-phenotype relationships. Community Genet. 9, 55–61 (2006).

    PubMed  Google Scholar 

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Acknowledgements

We thank L. Solomon for designing figures, R. Onofrio for designing PCR primers, K. Ardlie and S. Mahan for assistance in DNA sample preparation, J. Wilkinson and L. Ambrogio for Illumina sequence project management, B. Thomson, G. Crenshaw and A. Taylor for genotyping assistance, E.S. Lander for helpful comments, the Broad Institute Sequencing Platform for sequence data production, J. Flannick and J. Maguire for assistance with pooled sequence analysis, and M. dePristo and the Broad Institute Genome Sequence Analysis Group for sequencing analysis help and useful discussions. The work was supported by US National Human Genome Research Institute sequencing grant DK83756; Funds from the Helmsley Trust to M.J.D. and R.J.X.; and US National Institutes of Health grants AI062773, DK060049, DK086502, HG005923 and DK043351 (to R.J.X.). A.G. was supported by fellowships from La Fondation pour la Recherche Medicale and the CCFA. J.D.R. holds a Canada Research Chair and is funded by grants from the US National Institutes of Allergy and Infectious Diseases (AI065687; AI067152) from the NIDDK (DK064869; DK062432), the CCFA (SRA512), La Fondation de l'Institut de Cardiologie de Montréal, the Crohn's and Colitis Foundation of Canada (CCFC), the Fonds de Recherche en Santé du Québec (17199) and the Canadian Institutes of Health Research (01038). Genotyping of the Italian samples was supported by the Italian Ministry for Health GR-2008-1144485 and unrestricted research grant of Giuliani, with case collections provided by the Italian Group for IBD. The NIDDK IBDGC is funded by the following grants: DK062431 (S.R.B.), DK062422 (J.H.C.), DK062420 (R.H.D.), DK062432 (J.D.R.), DK062423 (M.S.), DK062413 (D.P.B.M.) and DK062429 (J.H.C.). D.P.B.M. is supported by NCRR grant M01-RR00425 to the Cedars-Sinai General Research Center Genotyping Core, U01-DK062413 (IBD Genetics Research Center), P01-DK046763 (IBD Program Project Grant) and Southern California Diabetes Endocrinology Research Center grants DK063491, R21-DK84554-01 and U01 DK062413. Genotyping of the German samples was supported by the German Ministry of Education and Research through the National Genome Research Network with infrastructure support through the Deutsche Forschungsgemeinschaft cluster of excellence Inflammation at Interfaces. Genotyping of the Swedish samples was supported by the Swedish Research Council, the Bengt Ihre Foundation and the Örebro University Hospital Research Foundation.

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M.A.R. and M.J.D. conceived and designed the study. Functional characterization of NOD2 mutants was coordinated and designed by A.G. and R.J.X. Study subject recruitment and phenotyping was supervised by R.H.D., M.C.D., D.P.B.M., M.D., R.J.X., J.H.C., J.D.R., M.C.D., M.D., A.F., D.E., M.S.S., and A.L. Sequenom assay designs were developed by P.G., T.H., J.H., L.T., and A.K. NIDDK IBDGC BeadXpress typing was coordinated and supervised by Y.S. and J.H.C. The pooled sequencing protocol was designed and established at the Broad Institute by N.B., M.A.R., C.S., D.A., M.J.D. and S.G. NIDDK IBDGC, UK IBDGC and IIBDGC contributed sample collection and Immunochip genotype data for replication. The project was managed by M.A.R., G.L., M.S., J.D.R., J.H.C., R.J.X., D.P.B.M., R.H.D., S.R.B. and M.J.D. C.S. and M.B. carried out pooling. C.S., Y.S., P.G., C.L., D.E. and M.B. carried out genotyping. M.A.R. and M.J.D. designed and carried out the statistical and computational analyses, with assistance from K.S.L., G.B., B.N., J.M.K., T.G., S.R., F.K., T.F., P.S. and C.K.Z. S.R. assisted with quality control, principal-component analysis and analysis of Immunochip data. Syzygy was developed by M.A.R. and M.J.D. M.J.D. supervised all aspects of the study. The manuscript was written by M.A.R., J.D.R., R.J.X. and M.J.D.

Corresponding authors

Correspondence to Manuel A Rivas or Mark J Daly.

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The authors declare no competing financial interests.

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A full list of consortium members is provided in the Supplementary Note.

A full list of consortium members is provided in the Supplementary Note.

A full list of consortium members is provided in the Supplementary Note.

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Supplementary Note, Supplementary Tables 1–7 and Supplementary Figures 1–7 (PDF 5855 kb)

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Rivas, M., Beaudoin, M., Gardet, A. et al. Deep resequencing of GWAS loci identifies independent rare variants associated with inflammatory bowel disease. Nat Genet 43, 1066–1073 (2011). https://doi.org/10.1038/ng.952

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