Genetic fine mapping and genomic annotation defines causal mechanisms at type 2 diabetes susceptibility loci

Abstract

We performed fine mapping of 39 established type 2 diabetes (T2D) loci in 27,206 cases and 57,574 controls of European ancestry. We identified 49 distinct association signals at these loci, including five mapping in or near KCNQ1. 'Credible sets' of the variants most likely to drive each distinct signal mapped predominantly to noncoding sequence, implying that association with T2D is mediated through gene regulation. Credible set variants were enriched for overlap with FOXA2 chromatin immunoprecipitation binding sites in human islet and liver cells, including at MTNR1B, where fine mapping implicated rs10830963 as driving T2D association. We confirmed that the T2D risk allele for this SNP increases FOXA2-bound enhancer activity in islet- and liver-derived cells. We observed allele-specific differences in NEUROD1 binding in islet-derived cells, consistent with evidence that the T2D risk allele increases islet MTNR1B expression. Our study demonstrates how integration of genetic and genomic information can define molecular mechanisms through which variants underlying association signals exert their effects on disease.

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Figure 1: FOX2A-bound sites are a genomic marker of T2D risk variants.
Figure 2: The lone variant in the 99% credible set at the MTNR1B locus affects FOXA2-bound enhancer activity.

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Acknowledgements

Funding for the research undertaken in this study has been received from the Academy of Finland (including grants 77299, 102318, 10493, 118065, 123885, 124243, 129293, 129680, 136895, 139635, 211119, 213506, 251217 and 263836); Agence National de la Recherche; Association de Langue Française pour l'Etude du Diabète et des Maladies Métaboliques; Association Diabèete Risque Vasculaire; Association Française des Diabétiques; the Association of Danish Pharmacies; the Augustinus Foundation; the Becket Foundation; the British Diabetes Association (BDA) Research; the British Heart Foundation; the Central Norway Health Authority; the Central Finland Hospital District; the Center for Inherited Disease Research (CIDR); the City of Kuopio; the City of Leutkirch; Copenhagen County; the Danish Centre for Evaluation and Health Technology Assessment; the Danish Council for Independent Research; the Danish Heart Foundation; the Danish Research Councils; Deutsche Forschungsgemeinschaft (including project ER 155/6-2); the Diabetes Research Foundation; Diabetes UK; the Doris Duke Charitable Foundation; Erasmus Medical Center; Erasmus University; the Estonian government (SF0180142s08); the European Commission (including ENGAGE HEALTH-F4-2007-201413, FP7-201413, FP7-245536, EXGENESIS LSHM-CT-2004-005272, FP6 LSHM_CT_2006_037197, LSHM-CT-2007-037273, Directorate C-Public Health 2004310, DG XII); the European Regional Development Fund; the Federal Ministry of Education and Research, Germany (including FKZ 01GI1128 and FKZ 01EO1001); the Federal Ministry of Health, Germany; the Finnish Diabetes Association; the Finnish Diabetes Research Foundation; the Finnish Foundation for Cardiovascular Research; the Finnish Medical Society; the Folkhalsan Research Foundation; the Foundation for Life and Health in Finland; the Foundation for Old Servants; the Fredrick och Ingrid Thuring Foundation; the French region of Nord-Pas-de-Calais (Contrat de Projets Etat-Région); the German Center for Diabetes Research; the German Research Council (including grant GRK1041); the German National Genome Research Network; Groupe d'Etude des Maladies Métaboliques et Systémiques; the Health Care Centers in Vasa, Närpes and Korsholm, Finland; the Health Foundation; the Heinz Nixdorf Foundation; Helmholtz Zentrum München; the Helsinki University Central Hospital Research Foundation; the Hospital District of Southwest Finland; the Ib Henriksens Foundation; IngaBritt and Arne Lundberg's Research Foundation (including grant 359); Karolinska Institutet; the Knut and Alice Wallenberg Foundation (including grant KAW 2009.0243); Kuopio University Hospital; the Lundbeck Foundation; the Magnus Bergvall Foundation; the Medical Faculty of University Duisburg-Essen; the Medical Research Council, UK (including grants G0000649 and G0601261); the Ministry for Health, Welfare and Sports, the Netherlands; the Ministry of Education and Culture, Finland (including grants 722 and 627; 2004-2011); the Ministry of Education, Culture and Science, the Netherlands; the Ministry of Health and Prevention, Denmark; the Ministry of Social Affairs and Health, Finland; the Ministry of Innovation, Science, Research and Technology of North Rhine-Westphalia, Germany; the Munich Center of Health Sciences; the Municipal Health Care Center and Hospital in Jakobstad, Finland; the municipality of Rotterdam, the Netherlands; the Närpes Health Care Foundation; the National Health Screening Service of Norway; the National Heart, Lung, and Blood Institute, USA (including grant numbers/contracts HHSN268201100005C, HHSN268201100006C, HHSN268201100007C, HHSN268201100008C, HHSN268201100009C, HHSN268201100010C, HHSN268201100011C, HHSN268201100012C, N01HC25195, N02HL64278, R01HL087641, R01HL59367 and R01HL086694); the National Human Genome Research Institute, USA (including grant numbers/contracts U01HG004402 and N01HG65403); the National Institute for Diabetes and Digestive and Kidney Diseases, USA (including grants R01DK078616, U01DK085526, K24DK080140 and R01DK073490); the National Institute for Health and Welfare, Finland; the National Institutes of Health, USA (including grant numbers/contracts HHSN268200625226C, UL1RR025005, R01DK062370, R01DK072193, 1Z01HG000024, AG028555, AG08724, AG04563, AG10175, AG08861, U01HG004399, DK58845, CA055075, DK085545 and DK098032); the Netherlands Genomics Initiative; the Netherlands Organisation for Health Research and Development; the Netherlands Organisation of Scientific Research NOW Investments (including grants 175.010.2005.011, 911-03-012 and 050-060-810); the Nord-Trondelag County Council; the Nordic Center of Excellence in Disease Genetics; the Norwegian Institute of Public Health; the Norwegian Research Council; the Novo Nordisk Foundation; the Ollquist Foundation; the Oxford National Institute for Health Research (NIHR) Biomedical Research Centre; the Paavo Nurmi Foundation; the Paivikki and Sakari Sohlberg Foundation; the Perklen Foundation; the Pirkanmaa Hospital District, Finland; Programme Hospitalier de Recherche Clinique; Programme National de Recherche sur la Diabète; the Research Institute for Diseases in the Elderly (including grant 014-93-015); the Robert Dawson Evans Endowment, Department of Medicine, Boston University School of Medicine and Boston Medical Center; the Royal Swedish Academy of Sciences; Sarstedt, Germany; the Signe and Ane Gyllenberg Foundation; the Sigrid Juselius Foundation; the Slottery Machine Association, Finland; the Social Insurance Institution of Finland; the South OstroBothnia Hospital District; the state of Baden-Württemberg, Germany; the Stockholm County Council (including grant 560183); the Swedish Cultural Foundation, Finland; the Swedish Diabetes Foundation; the Swedish e-science Research Center; the Swedish Foundation for Strategic Research; the Swedish Heart-Lung Foundation; the Swedish Research Council (including grants SFO EXODIAB 2009-1039, 521-2010-3490, 521-2007-4037, 521-2008-2974, ANDIS 825-2010-5983, LUDC 349-2008-6589 and 8691); the Swedish Society of Medicine; the Tore Nilsson Foundation; the Torsten and Ragnar Soderbergs Stiftelser (including grant MT33/09); University Hospital Essen; University of Tromsø; the University College London NIHR Biomedical Research Centre; the UK NIHR Cambridge Biomedical Research Centre; Uppsala University; Uppsala University Hospital; the Vaasa Hospital District; the Velux Foundation; and the Wellcome Trust (including the Biomedical Collections Grant GR072960 and grants 076113, 083948, 090367, 090532, 083270, 086596, 098017, 095101, 098051 and 098381). We are grateful to R. Scharfmann (INSERM U1016, Cochin Institute Paris) for the gift of EndoC βH1 cells and for providing technical support with their maintenance. We thank P. Johnson and the Oxford NIHR Biomedical Research Centre–funded Islet Isolation facility for providing human islets for this study. Detailed acknowledgments are provided in the Supplementary Note.

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Writing group. K.J.G., T.F., Y. Lee, A.R., R.M., M.E.R., A.L.G., D.A., M. Boehnke, T.M.T., M.I.M., A.P.M. Central meta-analysis group. K.J.G., T.F., Y. Lee, R.M., A. Mahajan, A. Locke, N.W.R., N.R., T.M.T., M.I.M., A.P.M. Annotation and functional analysis group. K.J.G., A.R., M.E.R., S.K.T., J.K.R., N.L.B., M.v.d.B., A.C., I.D., E. Birney, L. Pasquali, J. Ferrer, C.A.O'C., A.L.G., M.I.M. Validation meta-analysis group. R.M., R.A.S., I.P., L.J.S., A.P.M. Metabochip cohort-level primary analysis. Y. Lee, T.G., T.S., D.T., L.Y., H.G., S. Wahl, M.F., R.J.S., H. Kestler, H. Chheda, L.E., S.G., T.M.T., A.P.M. Validation cohort-level primary analysis. V. Steinthorsdottir, G.T., L.Q., L.C.K., E.M.v.L., S.M.W., M. Li, H. Chen, C. Fuchsberger, P. Kwan, C.M., M. Linderman, Y. Lu. Metabochip design. H.M.K., B.F.V. Cohort sample collection, genotyping, phenotyping or additional analysis. B.F.V., G.R.A., P.A., D.B., B.B., R.B., M. Blüher, H.B., L.L.B., E.P.B., N.P.B., J.C., G.C., P.S.C., M.C.C., D.J.C., A.T.C., R.M.v.D., A.S.F.D., M.D., S.E., J.G.E., T.E., E.E., J. Fadista, J. Flannick, P. Fontanillas, C. Fox, P.W.F., K.G., C.G., B.G., O.G., G.B.G., N.G., C.J.G., M.H., C.T.H., C.H., O.L.H., A.B.H., S.E.H., D.J.H., A.U.J., A.J., M.E.J., T.J., W.-H.L.K., N.D.K., L.K., N.K., A.K., P. Kovacs, P. Kraft, J. Kravic, C. Langford, K.L., L. Liang, P.L., C.M.L., E.L., A. Linneberg, C.-T.L., S.L., J.L., V.L., S. Männistö, O. McLeod, J.M., E.M., G.M., T.W.M., M.M.-N., C.N., M.M.N., N.N.O., K.R.O., D.P., S.P., L. Peltonen, J.R.B.P., C.G.P.P., M.R., D. Ruderfer, D. Rybin, Y.T.v.d.S., B.S., G. Sigur∂sson, A.S., G. Steinbach, P.S., K. Strauch, H.M.S., Q.S., B.T., E. Tikkanen, A.T., J. Trakalo, E. Tremoli, T.T., R.W., S. Wiltshire, A.R.W., E.Z. Validation cohort principal investigators. R.J.F.L., J.D., J.C.F., E. Boerwinkle, J.S.P., C.v.D., E.S., J.B.M., F.B.H., U.T., K. Stefansson, P.D., P.J.D., T.M.F., A.T.H., I.B., C. Langenberg, N.J.W., M. Boehnke, M.I.M. Metabochip cohort principal investigators. T.A.L., R.R., M.S., N.L.P., L. Lind, S.M.K.-K., E.K.-H., T.E.S., J.S., J. Kuusisto, M. Laakso, A. Metspalu, R.E., K.-H.J., S. Moebus, S.R., V. Salomaa, E.I., B.O.B., R.N.B., F.S.C., K.L.M., H. Koistinen, J. Tuomilehto, K.H., I.N., P.D., P.J.D., T.M.F., A.T.H., U.d.F., A.H., T.I., A.P., S.C., R.S., P. Froguel, O.P., T.H., A.D.M., C.N.A.P., S.K., O. Melander, P.M.N., L.C.G., I.B., C. Langenberg, N.J.W., D.A., M. Boehnke, M.I.M. Project management. K.J.G., A.L.G., D.A., M. Boehnke, T.M.T., M.I.M., A.P.M. DIAGRAM Consortium management. D.A., M. Boehnke, M.I.M.

Corresponding authors

Correspondence to Kyle J Gaulton or Mark I McCarthy or Andrew P Morris.

Ethics declarations

Competing interests

V. Steinthorsdottir, G.T., A.K., U.T. and K. Stefansson are employed by deCODE Genetics/Amgen, Inc. I.B. and spouse own stock in GlaxoSmithKline and Incyte.

Additional information

A list of members and affiliations appears in the Supplementary Note.

Integrated supplementary information

Supplementary Figure 1 Signal plots for association signals achieving locus-wide significance at the KCNQ1 locus.

Association summary statistics are based on the meta-analysis of Metabochip studies in 27,206 cases and 57,574 controls of European ancestry. Results are presented from exact conditioning after adjusting for all other index variants at the locus. Each point represents a SNP passing quality control in the meta-analysis, plotted with its conditional P value (on a –log10 scale) as a function of genomic position (NCBI Build 37). In each plot, the index variant is represented by the purple symbol. The color coding of all other SNPs indicates LD with the index variant in European-ancestry haplotypes from the 1000 Genomes Project reference panel: red, r2 ≥ 0.8; gold, 0.6 ≤ r2 < 0.8; green, 0.4 ≤ r2 < 0.6; cyan, 0.2 ≤ r2 < 0.4; blue, r2 < 0.2; gray, r2 unknown. The shape of the symbol corresponds to the annotation of the variant: upward triangle, framestop or splice; downward triangle, nonsynonymous; square, synonymous or UTR; and circle, intronic or noncoding. Recombination rates are estimated from HapMap Phase 2, and gene annotations are taken from the UCSC Genome Browser.

Supplementary Figure 2 Signal plots for association signals achieving locus-wide significance at the HNF1A locus.

Association summary statistics are based on the meta-analysis of Metabochip studies in 27,206 cases and 57,574 controls of European ancestry. Results are presented from exact conditioning after adjusting for all other index variants at the locus. Each point represents a SNP passing quality control in the meta-analysis, plotted with its conditional P value (on a –log10 scale) as a function of genomic position (NCBI Build 37). In each plot, the index variant is represented by the purple symbol. The color coding of all other SNPs indicates LD with the index variant in European-ancestry haplotypes from the 1000 Genomes Project reference panel: red, r2 ≥ 0.8; gold, 0.6 ≤ r2 < 0.8; green, 0.4 ≤ r2 < 0.6; cyan, 0.2 ≤ r2 < 0.4; blue, r2 < 0.2; gray, r2 unknown. The shape of the symbol corresponds to the annotation of the variant: upward triangle, framestop or splice; downward triangle, nonsynonymous; square, synonymous or UTR; and circle, intronic or noncoding. Recombination rates are estimated from HapMap Phase 2, and gene annotations are taken from the UCSC Genome Browser.

Supplementary Figure 3 Signal plots for association signals achieving locus-wide significance at the DGKB, CDKN2A-CDKN2B, MC4R and GIPR loci.

Association summary statistics are based on the meta-analysis of Metabochip studies in 27,206 cases and 57,574 controls of European ancestry. Results are presented from exact conditioning after adjusting for all other index variants at the locus. Each point represents a SNP passing quality control in the meta-analysis, plotted with its conditional P value (on a –log10 scale) as a function of genomic position (NCBI Build 37). In each plot, the index variant is represented by the purple symbol. The color coding of all other SNPs indicates LD with the index variant in European-ancestry haplotypes from the 1000 Genomes Project reference panel: red, r2 ≥ 0.8; gold, 0.6 ≤ r2 < 0.8; green, 0.4 ≤ r2 < 0.6; cyan, 0.2 ≤ r2 < 0.4; blue, r2 < 0.2; gray, r2 unknown. The shape of the symbol corresponds to the annotation of the variant: upward triangle, framestop or splice; downward triangle, nonsynonymous; square, synonymous or UTR; and circle, intronic or noncoding. Recombination rates are estimated from HapMap Phase 2, and gene annotations are taken from the UCSC Genome Browser.

Supplementary Figure 4 Signal plots for association signals at the CDKN2A-CDKN2B locus.

Association summary statistics are based on the meta-analysis of Metabochip studies in 27,206 cases and 57,574 controls of European ancestry. (a) Unconditional. (b) After approximate conditioning on the two index SNPs, rs10811660 and rs10757283. Each point represents a SNP passing quality control in the meta-analysis, plotted with its conditional P value (on a –log10 scale) as a function of genomic position (NCBI Build 37). In each plot, the index variant is represented by the purple symbol. The color coding of all other SNPs indicates LD with the index variant in European-ancestry haplotypes from the 1000 Genomes Project reference panel: red, r2 ≥ 0.8; gold, 0.6 ≤ r2 < 0.8; green, 0.4 ≤ r2 < 0.6; cyan, 0.2 ≤ r2 < 0.4; blue, r2 < 0.2; gray, r2 unknown. Recombination rates are estimated from HapMap Phase 2, and gene annotations are taken from the UCSC Genome Browser.

Supplementary Figure 5 Characteristics of index variants for association signals achieving locus-wide significance for T2D susceptibility across fine-mapping regions.

Each association signal was represented by an index variant in the GCTA-COJO joint regression model on the basis of (i) summary statistics from the meta-analysis of Metabochip studies in 27,206 cases and 57,574 controls of European ancestry and (ii) reference genotype data from GoDARTS (3,298 cases and 3,708 controls of European ancestry from the UK) to approximate LD across fine-mapping regions. Each variant is plotted according to risk allele frequency on the x axis and allelic log(OR) on the y axis (with error bars representing the corresponding standard error).

Supplementary Figure 6 Characteristics of the 99% credible sets of variants for each association signal achieving locus-wide significance for T2D susceptibility across fine-mapping regions.

Each point corresponds to an association signal, plotted according to the highest posterior probability of causality of any variant in the credible set on the x axis and the total number of variants in the credible set on the y axis. Distinct association signals at loci are referenced according to their index variant.

Supplementary Figure 7 Enrichment in chromatin state and noncoding RNA elements.

Variants in regulatory enhancer, promoter and insulator elements from 12 cell types and noncoding RNA elements from 25 cell types were tested for enrichment of average posterior causal probability compared to variants in shifted elements. Variants in islet enhancer elements demonstrated significant enrichment (fold = 1.97; P = 0.00022), and variants in islet and HepG2 promoter elements demonstrated nominally significant enrichment (P < 0.01).

Supplementary Figure 8 Null distribution of enriched transcription factors.

Null distribution of mean posterior probabilities of driving association signals for variants in shifted annotations (blue bars) compared to observed mean probability (dashed line) for the most significantly enriched transcription factors.

Supplementary Figure 9 FOXA2 ChIP-seq sites overlap, genome wide, across cell types.

The number of FOXA2 sites identified in ChIP-seq assays from primary pancreatic islets, HepG2 cells and primary liver was obtained, and sites from each cell type were intersected.

Supplementary Figure 10 Electrophoretic mobility shift assay of rs10830963 in HepG2 cellular extracts.

Nuclear extract (NE) from HepG2 cells was treated with labeled probes of 25 bp of sequence containing each allele of rs10830963. We observed protein bands bound to both sequences (black) as well as bands unique to the non-risk (red) and risk (blue) sequences. We then introduced antibodies against HNF3B (FOXA2 alias) and four factors (YY1, TAL1, NEURDO1 and PTF1A) whose known binding motifs match the de novo motif at rs10830963. We observed no shifting of the allele-specific bands with any antibody. Several bands shared by both alleles were removed in the presence of antibody to YY1.

Supplementary Figure 11 Genes at FOXA2-enriched signals are downregulated in Foxa1/Foxa2 null mice.

Pancreatic islet gene expression data were obtained from wild-type and Foxa1/Foxa2 knockout mice (Gao et al.47). Genes within 500 kb of each FOXA2-enriched T2D signal and closest to each FOXA2-enriched signal (orange) were significantly downregulated in the Foxa1/Foxa2 knockout compared to all genes (gray).

Supplementary Figure 12 Comparison of summary statistics for association signals achieving locus-wide significance from the GCTA-COJO joint regression model using genotype data from two reference studies to approximate LD between variants across each fine-mapping region.

Association summary statistics are based on the meta-analysis of Metabochip studies in 27,206 cases and 57,574 controls of European ancestry. Association summary statistics were obtained using (i) 3,298 T2D cases and 3,708 controls of UK ancestry from GoDARTS (represented on the x axis) and (ii) 4,435 T2D cases and 5,757 controls of Scandinavian ancestry from FUSION (represented on the y axis). Left, comparison of P values (PJ) from the GCTA-COJO joint regression model. Right, comparison of allelic log(OR) (blue diamond) and standard error (gray error bars) from the GCTA-COJO joint regression model.

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Gaulton, K., Ferreira, T., Lee, Y. et al. Genetic fine mapping and genomic annotation defines causal mechanisms at type 2 diabetes susceptibility loci. Nat Genet 47, 1415–1425 (2015). https://doi.org/10.1038/ng.3437

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