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Comparison of HapMap and 1000 Genomes Reference Panels in a Large-Scale Genome-Wide Association Study

  • Paul S. de Vries,

    Affiliations Department of Epidemiology, Erasmus MC, Rotterdam, the Netherlands, University of Texas Health Science Center at Houston, Houston, TX, United States of America

  • Maria Sabater-Lleal,

    Affiliation Cardiovascular Medicine Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden

  • Daniel I. Chasman,

    Affiliations Division of Preventive Medicine, Brigham and Women's Hospital, Boston, MA, United States of America, Harvard Medical School, Boston, MA, United States of America

  • Stella Trompet,

    Affiliations Department of Cardiology, Leiden University Medical Center, Leiden, the Netherlands, Department of Gerontology and Geriatrics, Leiden University Medical Center, Leiden, the Netherlands

  • Tarunveer S. Ahluwalia,

    Affiliations Steno Diabetes Center Copenhagen, Gentofte, Denmark, Novo Nordisk Foundation Center For Basic Metabolic Research, Section of Metabolic Genetics, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

  • Alexander Teumer,

    Affiliation Institute for Community Medicine, University Medicine Greifswald, Greifswald, Germany

  • Marcus E. Kleber,

    Affiliation Vth Department of Medicine, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany

  • Ming-Huei Chen,

    Affiliations Department of Neurology, Boston University School of Medicine, Boston, MA, United States of America, Framingham Heart Study, Population Sciences Branch, Division of Intramural Research National Heart Lung and Blood Institute, National Institutes of Health, Framingham, MA, United States of America

  • Jie Jin Wang,

    Affiliation Centre for Vision Research, Department of Ophthalmology, and Westmead Institute for Medical Research, University of Sydney, Sydney, Australia

  • John R. Attia,

    Affiliations Public Health Stream, Hunter Medical Research Institute, University of Newcastle, Newcastle, Australia, School of Medicine and Public Health, University of Newcastle, Newcastle, Australia

  • Riccardo E. Marioni,

    Affiliations Centre for Cognitive Ageing and Cognitive Epidemiology, University of Edinburgh, Edinburgh, United Kingdom, Centre for Genomic and Experimental Medicine, University of Edinburgh, Edinburgh, United Kingdom, Queensland Brain Institute, University of Queensland, Brisbane, Australia

  • Maristella Steri,

    Affiliation Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Monserrato, Cagliari, Italy

  • Lu-Chen Weng,

    Affiliation Division of Epidemiology and Community Health, University of Minnesota, Minneapolis, MN, United States of America

  • Rene Pool,

    Affiliations Department of Biological Psychology, Netherlands Twin Register, VU University, Amsterdam, the Netherlands, EMGO+ institute, VU University & VU medical center, Amsterdam, the Netherlands

  • Vera Grossmann,

    Affiliation Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany

  • Jennifer A. Brody,

    Affiliation Department of Medicine, University of Washington, Seattle WA, United States of America

  • Cristina Venturini,

    Affiliations Division of Infection and Immunology, UCL, London, United Kingdom, Department of Twin Research and Genetic Epidemiology, Kings College London, London, United Kingdom

  • Toshiko Tanaka,

    Affiliation Translational Gerontology Branch, National Institute on Aging, Baltimore, MD, United States of America

  • Lynda M. Rose,

    Affiliation Division of Preventive Medicine, Brigham and Women's Hospital, Boston, MA, United States of America

  • Christopher Oldmeadow,

    Affiliations Public Health Stream, Hunter Medical Research Institute, University of Newcastle, Newcastle, Australia, School of Medicine and Public Health, University of Newcastle, Newcastle, Australia

  • Johanna Mazur,

    Affiliation Institute of Medical Biostatistics, Epidemiology and Informatics, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany

  • Saonli Basu,

    Affiliation Division of Biostatistics, University of Minnesota, Minneapolis, MN, United States of America

  • Mattias Frånberg,

    Affiliations Cardiovascular Medicine Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden, Department of Numerical Analysis and Computer Science, Stockholm University, Stockholm, Sweden

  • Qiong Yang,

    Affiliations Framingham Heart Study, Population Sciences Branch, Division of Intramural Research National Heart Lung and Blood Institute, National Institutes of Health, Framingham, MA, United States of America, Department of Biostatistics, Boston University School of Public Health, Boston, MA, United States of America

  • Symen Ligthart,

    Affiliation Department of Epidemiology, Erasmus MC, Rotterdam, the Netherlands

  • Jouke J. Hottenga,

    Affiliation Department of Biological Psychology, Netherlands Twin Register, VU University, Amsterdam, the Netherlands

  • Ann Rumley,

    Affiliation Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom

  • Antonella Mulas,

    Affiliation Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Monserrato, Cagliari, Italy

  • Anton J. M. de Craen,

    Affiliation Department of Gerontology and Geriatrics, Leiden University Medical Center, Leiden, the Netherlands

  • Anne Grotevendt,

    Affiliation Institute of Clinical Chemistry and Laboratory Medicine, University Medicine Greifswald, Greifswald, Germany

  • Kent D. Taylor,

    Affiliations Institute for Translational Genomics and Population Sciences, Los Angeles Biomedical Research Institute at Harbor/UCLA Medical Center, Torrance, CA, United States of America, Division of Genomic Outcomes, Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, CA, United States of America

  • Graciela E. Delgado,

    Affiliation Vth Department of Medicine, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany

  • Annette Kifley,

    Affiliation Centre for Vision Research, Department of Ophthalmology, and Westmead Institute for Medical Research, University of Sydney, Sydney, Australia

  • Lorna M. Lopez,

    Affiliations Centre for Cognitive Ageing and Cognitive Epidemiology, University of Edinburgh, Edinburgh, United Kingdom, Royal College of Surgeons in Ireland, Department of Psychiatry, Education and Research Centre, Beaumont Hospital, Dublin, Ireland, University College Dublin, UCD Conway Institute, Centre for Proteome Research, UCD, Belfield, Dublin, Ireland

  • Tina L. Berentzen,

    Affiliation Institute of Preventive Medicine, Bispebjerg and Frederiksberg Hospital, The Capital Region, Copenhagen, Denmark

  • Massimo Mangino,

    Affiliations Department of Twin Research and Genetic Epidemiology, Kings College London, London, United Kingdom, NIHR Biomedical Research Centre at Guy’s and St. Thomas’ Foundation Trust, London, United Kingdom

  • Stefania Bandinelli,

    Affiliation Geriatric Unit, Azienda Sanitaria Firenze (ASF), Florence, Italy

  • Alanna C. Morrison,

    Affiliation Department of Epidemiology, Erasmus MC, Rotterdam, the Netherlands

  • Anders Hamsten,

    Affiliation Cardiovascular Medicine Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden

  • Geoffrey Tofler,

    Affiliation Royal North Shore Hospital, Sydney University, Sydney, Australia

  • Moniek P. M. de Maat,

    Affiliation Department of Hematology, Erasmus MC, Rotterdam, the Netherlands

  • Harmen H. M. Draisma,

    Affiliations Department of Biological Psychology, Netherlands Twin Register, VU University, Amsterdam, the Netherlands, Neuroscience Campus Amsterdam, Amsterdam, the Netherlands

  • Gordon D. Lowe,

    Affiliation Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom

  • Magdalena Zoledziewska,

    Affiliation Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Monserrato, Cagliari, Italy

  • Naveed Sattar,

    Affiliation BHF Glasgow Cardiovascular Research Centre, Faculty of Medicine, Glasgow, United Kingdom

  • Karl J. Lackner,

    Affiliation Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center, Johannes Gutenberg University Mainz, Mainz, Germany

  • Uwe Völker,

    Affiliation Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany

  • Barbara McKnight,

    Affiliation Department of Biostatistics, University of Washington, Seattle, WA, United States of America

  • Jie Huang,

    Affiliation Department of Human Genetics, Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom

  • Elizabeth G. Holliday,

    Affiliation Public Health Stream, Hunter Medical Research Institute, and School of Medicine and Public Health, University of Newcastle, Newcastle, Australia

  • Mark A. McEvoy,

    Affiliation School of Medicine and Public Health, University of Newcastle, Newcastle, Australia

  • John M. Starr,

    Affiliations Centre for Cognitive Ageing and Cognitive Epidemiology, University of Edinburgh, Edinburgh, United Kingdom, Alzheimer Scotland Dementia Research Centre, University of Edinburgh, Edinburgh, United Kingdom

  • Pirro G. Hysi,

    Affiliation Department of Twin Research and Genetic Epidemiology, Kings College London, London, United Kingdom

  • Dena G. Hernandez,

    Affiliation Laboratory of Neurogenetics, National Institute on Aging, Bethesda, MD, United States of America

  • Weihua Guan,

    Affiliation Division of Biostatistics, University of Minnesota, Minneapolis, MN, United States of America

  • Fernando Rivadeneira,

    Affiliations Department of Epidemiology, Erasmus MC, Rotterdam, the Netherlands, Department of Internal Medicine, Erasmus MC, Rotterdam, the Netherlands

  • Wendy L. McArdle,

    Affiliation School of Social and Community Medicine, University of Bristol, Bristol, United Kingdom

  • P. Eline Slagboom,

    Affiliation Department of Molecular Epidemiology, Leiden University Medical Center, Leiden, the Netherlands

  • Tanja Zeller,

    Affiliations Department of General and Interventional Cardiology, University Heart Centre, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, German Center for Cardiovascular Research (DZHK), Partner Site Hamburg, Lübeck, Kiel, Hamburg, Germany

  • Bruce M. Psaty,

    Affiliations Department of Medicine, Epidemiology, and Health Services, University of Washington, Seattle WA, United States of America, Group Health Research Institute, Group Health Cooperative, Seattle WA, United States of America

  • André G. Uitterlinden,

    Affiliations Department of Epidemiology, Erasmus MC, Rotterdam, the Netherlands, Department of Internal Medicine, Erasmus MC, Rotterdam, the Netherlands

  • Eco J. C. de Geus,

    Affiliations Department of Biological Psychology, Netherlands Twin Register, VU University, Amsterdam, the Netherlands, EMGO+ institute, VU University & VU medical center, Amsterdam, the Netherlands

  • David J. Stott,

    Affiliation Institute of Cardiovascular and Medical Sciences, Faculty of Medicine, University of Glasgow, Glasgow, United Kingdom

  • Harald Binder,

    Affiliation Institute of Medical Biostatistics, Epidemiology and Informatics, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany

  • Albert Hofman,

    Affiliations Department of Epidemiology, Erasmus MC, Rotterdam, the Netherlands, Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MS, United States of America

  • Oscar H. Franco,

    Affiliation Department of Epidemiology, Erasmus MC, Rotterdam, the Netherlands

  • Jerome I. Rotter,

    Affiliations Los Angeles BioMedical Research Institute at Harbor-UCLA Medical Center, Institute for Translational Genomics and Population Sciences, Torrance, CA, United States of America, Division of Genomic Outcomes, Departments of Pediatrics & Medicine, Harbor-UCLA Medical Center, Torrance, CA, United States of America

  • Luigi Ferrucci,

    Affiliation Translational Gerontology Branch, National Institute on Aging, Baltimore, MD, United States of America

  • Tim D. Spector,

    Affiliation Department of Twin Research and Genetic Epidemiology, Kings College London, London, United Kingdom

  • Ian J. Deary,

    Affiliations Centre for Cognitive Ageing and Cognitive Epidemiology, University of Edinburgh, Edinburgh, United Kingdom, Department of Psychology, University of Edinburgh, Edinburgh, United Kingdom

  • Winfried März,

    Affiliations Vth Department of Medicine, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany, Synlab Academy, Synlab Holding Deutschland GmbH, Mannheim, Germany, Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria

  • Andreas Greinacher,

    Affiliation Institute for Immunology and Transfusion Medicine, University Medicine Greifswald, Greifswald, Germany

  • Philipp S. Wild,

    Affiliations Preventive Cardiology and Preventive Medicine, Center for Cardiology, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany, Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany, German Center for Cardiovascular Research (DZHK), Partner Site RhineMain, Mainz, Germany

  • Francesco Cucca,

    Affiliation Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Monserrato, Cagliari, Italy

  • Dorret I. Boomsma,

    Affiliation Department of Biological Psychology, Netherlands Twin Register, VU University, Amsterdam, the Netherlands

  • Hugh Watkins,

    Affiliation Cardiovascular Medicine Dept/Radcliffe Dept of Medicine, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom

  • Weihong Tang,

    Affiliation Division of Epidemiology and Community Health, University of Minnesota, Minneapolis, MN, United States of America

  • Paul M. Ridker,

    Affiliations Division of Preventive Medicine, Brigham and Women's Hospital, Boston, MA, United States of America, Harvard Medical School, Boston, MA, United States of America

  • Jan W. Jukema,

    Affiliations Department of Cardiology, Leiden University Medical Center, Leiden, the Netherlands, Durrer Center for Cardiogenetic Research, Amsterdam, the Netherlands, Interuniversity Cardiology Institute of the Netherlands, Utrecht, the Netherlands

  • Rodney J. Scott,

    Affiliations Information based Medicine Program, Hunter Medical Research Institute, University of Newcastle, Newcastle, Australia, School of Biomedical Sciences and Pharmacy, University of Newcastle, Newcastle, Australia

  • Paul Mitchell,

    Affiliation Centre for Vision Research, Department of Ophthalmology, and Westmead Institute for Medical Research, University of Sydney, Sydney, Australia

  • Torben Hansen,

    Affiliation Novo Nordisk Foundation Centre for Basic Metabolic Research, Section of Metabolic Genetics, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

  • Christopher J. O'Donnell,

    Affiliations Framingham Heart Study, Population Sciences Branch, Division of Intramural Research National Heart Lung and Blood Institute, National Institutes of Health, Framingham, MA, United States of America, Cardiology Division, Massachusetts General Hospital, Boston, MA, United States of America

  • Nicholas L. Smith,

    Affiliations Group Health Research Institute, Group Health Cooperative, Seattle WA, United States of America, Department of Epidemiology, University of Washington, Seattle WA, United States of America, Seattle Epidemiologic Research and Information Center, Department of Veteran Affairs Office of Research and Development, Seattle, WA, United States of America

  • David P. Strachan,

    Affiliation Population Health Research Institute, St George's, University of London, London, United Kingdom

  •  [ ... ],
  • Abbas Dehghan

    a.dehghan@imperial.ac.uk

    Affiliations Department of Epidemiology, Erasmus MC, Rotterdam, the Netherlands, Department of Epidemiology and Biostatistics, Imperial College London, London, United Kingdom

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Abstract

An increasing number of genome-wide association (GWA) studies are now using the higher resolution 1000 Genomes Project reference panel (1000G) for imputation, with the expectation that 1000G imputation will lead to the discovery of additional associated loci when compared to HapMap imputation. In order to assess the improvement of 1000G over HapMap imputation in identifying associated loci, we compared the results of GWA studies of circulating fibrinogen based on the two reference panels. Using both HapMap and 1000G imputation we performed a meta-analysis of 22 studies comprising the same 91,953 individuals. We identified six additional signals using 1000G imputation, while 29 loci were associated using both HapMap and 1000G imputation. One locus identified using HapMap imputation was not significant using 1000G imputation. The genome-wide significance threshold of 5×10−8 is based on the number of independent statistical tests using HapMap imputation, and 1000G imputation may lead to further independent tests that should be corrected for. When using a stricter Bonferroni correction for the 1000G GWA study (P-value < 2.5×10−8), the number of loci significant only using HapMap imputation increased to 4 while the number of loci significant only using 1000G decreased to 5. In conclusion, 1000G imputation enabled the identification of 20% more loci than HapMap imputation, although the advantage of 1000G imputation became less clear when a stricter Bonferroni correction was used. More generally, our results provide insights that are applicable to the implementation of other dense reference panels that are under development.

Introduction

Most genome-wide association (GWA) studies to date have used their genotyped single nucleotide polymorphisms (SNPs) to impute about 2.5 million SNPs detected in the Phase 2 version of the HapMap Project (HapMap) [113], including mostly common SNPs with a minor allele frequency (MAF) of over 5%. HapMap imputation enabled the interrogation of most common SNPs possible, even while meta-analyzing studies that used different genotyping arrays with low overlap [1]. However, low-frequency and rare variants are not well covered in the HapMap panel [14]. In addition, genetic variants other than SNPs, such as small insertion/deletions (indels) and large structural variants, are not included in HapMap-based imputed projects, and may be possible sources of missing explained heritability.

In contrast, the more recently released Phase 1 version 3 of the 1000 Genomes Project (1000G) is based on a larger set of individuals [15], and comprises nearly 40 million variants, including 1.4 million indels. 1000G allows the interrogation of most common and low-frequency variants (MAF > 1%), and rare variants (MAF < 1%) that were previously not covered [16]. In general, improving reference panels can lead to the identification of additional significant loci both through the addition of new variants and the improved imputation of known variants. 1000G imputation may thus have several advantages, but given that the denser 1000G imputation comes at the cost of an increased computational and analytical burden, it is important to estimate the observed benefits in practice. Furthermore, such empirical data is needed to make informed decisions in the future on the use of newer reference panels such as UK10K, and the Haplotype Reference Consortium [17, 18]. While several GWA studies using 1000G imputation have been published or are in progress, their sample size differs from the previous GWA studies using HapMap imputation, making comparison difficult. Therefore, with the aim of evaluating the benefits of using 1000G imputation in GWA studies compared to HapMap imputation, we carried out meta-analyses of GWA studies of circulating fibrinogen concentration (a quantitative trait), using both HapMap and 1000G imputed data on the same set of 91,953 individuals.

Results

Baseline characteristics of the participants for each of the included studies are shown in S1 Table, and genomic inflation factors are shown separately for the HapMap and 1000G GWA studies in S2 Table. The HapMap GWA study included 2,749,429 SNPs, and the 1000G GWA study included 10,883,314 variants. Summary statistics for all variants in the HapMap and 1000G GWA studies are available via the dbGAP CHARGE Summary Results site [19]. Using a genome-wide significance threshold of 5×10−8, a total of 1,210 SNPs across 30 loci were associated with circulating fibrinogen concentration in the HapMap imputed GWA study compared with 4,096 variants across 35 loci in the 1000G imputed GWA study (S1 Fig and S2 Fig). These loci are described in further detail in S3 Table. Of these loci, six were associated only in the 1000G GWA study and one was associated only in the HapMap GWA study, while 29 were overlapping (Fig 1A). The HapMap and 1000G lead variants of non-overlapping loci are described in Table 1, and leads variants of overlapping loci are described in Table 2. Among significant loci, the correlation coefficient across cohorts of the beta coefficients, P-values, and imputation quality scores of HapMap and 1000G lead variants were 0.925, 0.998, and 0.435 respectively (S3 Fig).

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Fig 1.

Venn diagram of the number of loci significant using HapMap (left circle) and 1000G (right circle) imputation in A) the main analysis, B) the sensitivity analysis applying a significance threshold of 2.5×10−8 to the 1000G GWA analysis, C) the sensitivity analysis without using genomic control corrections, and D) the sensitivity analysis excluding studies that used different imputation software, analysis software, or covariates in the HapMap and 1000G GWA analyses.

https://doi.org/10.1371/journal.pone.0167742.g001

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Table 1. Non-overlapping loci that were significant in either the HapMap or 1000G GWA studies.

https://doi.org/10.1371/journal.pone.0167742.t001

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Table 2. Overlapping loci that were significant in both the HapMap and 1000G GWA studies.

https://doi.org/10.1371/journal.pone.0167742.t002

Non-overlapping loci

The lead variants for the seven non-overlapping loci always differed between the HapMap and 1000G GWA studies, and all P-value differences were greater than one order of magnitude (for example: from 5×10−8 to 5×10−9 or less). Differences between HapMap and 1000G imputation for the seven non-overlapping loci are summarized in Fig 2.

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Fig 2. Summary of the differences between HapMap and 1000G imputation for the seven non-overlapping loci.

https://doi.org/10.1371/journal.pone.0167742.g002

Regional plots of the six loci significant only in the 1000G GWA study are shown in Fig 3. For four of these six loci, the correlation r2 between allelic dosages of the most associated variants imputed using HapMap and 1000G was less than 0.8 (S4 Table). None of the 1000G lead variants among these four loci were included in the HapMap GWA study, and neither were any good proxies (S5 Table).

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Fig 3. Regional plots of non-overlapping loci that were more significantly associated with fibrinogen in the 1000G GWA study, including variants from both the HapMap (red) and 1000G (green) GWA studies.

https://doi.org/10.1371/journal.pone.0167742.g003

A regional plot of the 6p21.3 locus, which was significant only in the HapMap GWA study, is shown in Fig 4. The most significant P-value at the locus was 8.5×10−9 in the HapMap GWA study compared to 7.9×10−6 in the 1000G GWA study. The correlation r2 between imputed dosages of the HapMap and 1000G lead variants was low (0.07). The HapMap lead SNP was included in the 1000G GWA study under a different name, rs114339898, but the imputation quality was only sufficient for inclusion in seven of the studies (S5 Table).

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Fig 4. Regional plot of 6p21.3, a non-overlapping locus that was more significantly associated with fibrinogen in the HapMap GWA study, including variants from both the HapMap (red) and 1000G (green) GWA studies.

https://doi.org/10.1371/journal.pone.0167742.g004

Overlapping loci

Regional plots of the 29 overlapping loci are shown in S4 Table. The lead variants of eight of the 29 overlapping loci were the same for the HapMap and 1000G GWA studies. P-value differences between the HapMap and 1000G GWA studies were often small: they were smaller than or equal to one order of magnitude for 22 loci. P-values differed by more than one order of magnitude for seven loci. Five of these loci were more significant in the 1000G GWA study (2q37.3, 4q31.3, 10q21.3, 12q24.12, and 21q22.2), while two of these loci were more significant in the HapMap GWA study (5q31.1 and 8q24.3).

Among the five overlapping loci with lower P-values in the 1000G GWA study, the correlation r2 between imputed dosages of lead variants from HapMap and 1000G was higher than 0.8 for 4 loci, but was 0.68 for the 12q24.12 locus (S4 Table). There was no good proxy of the 1000G lead variant at the 12q24.12 locus included in the HapMap GWA study.

The 5q31.1 and 8q24.3 loci had lower P-values in the HapMap GWA study. The correlation r2 between imputed dosages from HapMap and 1000G was almost perfect for 5q31.1, but was 0.75 for 8q24.3. The HapMap lead variant of the 8q24.3 locus was also included in the 1000G GWA study. These differences between HapMap and 1000G imputation for the 29 overlapping loci are summarized in Fig 5.

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Fig 5. Summary of the differences between HapMap and 1000G imputation for the 29 overlapping loci.

https://doi.org/10.1371/journal.pone.0167742.g005

Sensitivity analyses

Because more independent variants are included in the 1000G GWA study [20, 21], using the conventional genome-wide significance threshold of 5×10−8 may result in an increased type I error rate. When we used a more stringent genome-wide significance threshold of 2.5×10-8for the 1000G GWA study as suggested by Huang et al. [20], there were 4 loci significant only in the HapMap GWA study, 5 loci significant only in the 1000G GWA study, and 26 overlapping loci (Fig 1B). Three loci that were significant using both HapMap and 1000G imputation thus became non-significant when the stricter significance threshold was applied to the 1000G results.

Genomic inflation factors to correct for genomic control were calculated separately for the HapMap and 1000G analyses of each study. Thus, differences in the genomic inflation factors could explain some of the differences between the HapMap and 1000G results. When we repeated the HapMap and 1000G GWA study without applying genomic control corrections, 2 loci were associated only with circulating fibrinogen concentration in the HapMap GWA study, 6 were only associated in the 1000G GWA study, and 30 were associated in both GWA studies (Fig 1C and S6 Table).

For practical reasons, not all of the studies used the same imputation software, analysis software, or covariates for the HapMap and 1000G analyses. Specifically, fewer studies used principal components in the HapMap GWA study. When we restricted the analysis to those studies that used the same imputation software, analysis software, and covariates in the HapMap and 1000G GWA studies (S7 Table and S8 Table), 3 loci were associated only in the 1000G GWA study, and 6 were associated in both the HapMap and the 1000G GWA studies (Fig 1D and S9 Table). No loci were associated only in the HapMap GWA study.

Discussion

In our fibrinogen GWA study of 91,953 individuals, using 1000G instead of HapMap imputation led to the identification of six additional fibrinogen loci, suggesting an improvement in the detection of associated signals. Nevertheless, there was also one locus that was only identified when using HapMap imputation, and the advantage of 1000G imputation was attenuated when using a more stringent Bonferroni correction for the 1000G GWA study. The inclusion of indels in the 1000G GWA study did not lead to the identification of any new loci. Only one locus in our 1000G GWA study was led by an indel, and it was in strong linkage disequilibrium with a SNP present in HapMap.

While this is the first study of the impact of HapMap and 1000G imputation on genome-wide associations using exactly the same individuals in a large-scale consortium setting, four previous studies have addressed this question on a smaller scale. In the Wellcome Trust Case Control Consortium, consisting of 2000 for seven diseases (bipolar disorder, coronary artery disease, Crohn's disease, hypertension, rheumatoid arthritis, type 1 and 2 diabetes) and 3000 shared controls, Huang et al. re-analyzed GWA studies of these seven diseases with 1000G imputation, and found two novel loci: one for type 1 diabetes and one for type 2 diabetes [20]. A more conservative genome-wide significance threshold of 2.5×10−8 was used in the 1000G GWA studies, while the MAF inclusion threshold was the same at 1%. The second study was a 1000G imputed GWA study of around 2000 cases of venous thrombosis and 2400 controls [22]. Using a conservative P-value threshold of 7.4×10−9, but no MAF threshold, Germain et al. identified an uncommon variant at a novel locus that was not identified in the HapMap GWA study [22]. Third, the National Cancer Institute Breast and Prostate Cancer Cohort Consortium found no new loci by applying 1000G imputation to their existing dataset of 2800 cases and 4500 controls [23, 24]. The conventional genome-wide significance threshold of 5×10−8 was used, but no MAF threshold was used. Fourthly, Wood et al. compared HapMap and 1000G imputation for a total of 93 quantitative traits in 1210 individuals from the InCHIANTI study [25]. Using a significance threshold of 5×10−8 for both the HapMap and 1000G GWA studies, they found 20 overlapping associations, 13 associations that were only significant using 1000G imputation, and one association that was only significant using HapMap imputation. For the association significant only in HapMap, the P-value difference between HapMap and 1000G lead variants was less than one order of magnitude. When the authors lowered their significance threshold to 5×10−11 to reflect the number of tests being done in analyzing multiple traits, 9 associations remained significant based on HapMap imputation and 11 associations remained significant based on 1000G imputation.

All four of these comparison studies used an earlier 1000 genomes reference panel. The present study adds to the literature as it is based on the widely implemented Phase 1 Version 3 of 1000G. Crucially, the large sample size allowed us to examine differences at many non-overlapping and overlapping loci, and improved the generalizability of our results, as ongoing GWA studies are often conducted in large consortia.

Two further studies with different approaches also provide insights. First, Springelkamp et al. found a novel locus using 1000G imputation even though the sample size was smaller than the previous HapMap GWA study [26, 27]. The same genome-wide significance (5×10−8) and MAF (1%) thresholds were used. The lowest P-value at the locus was 1.9×10−8. Because different individuals were included in these GWA studies, the difference between HapMap and 1000G may partially be explained by sampling variability. Second, Shin et al. identified 299 SNP-metabolite associations based on HapMap imputation, and reexamined the associated loci using 1000G imputation in the same individuals [28]. They found that HapMap and 1000G imputation yielded similar P-values and variance explained for all but one loci. For that locus, the 1000G imputation based association was considerably stronger: the explained variance increased from 10% to 16%, and the P-value decreased from 8.8×10−113 to 7.7×10−244. Although Shin et al. did not compare loci identified using HapMap and 1000G, their results do support our finding that large differences in association strengths are possible, albeit not at every locus. All these studies, along with the current study, suggest that additional signals not previously identified in HapMap GWA studies can be found using the 1000G GWA study, with the same sample size.

In the current study we demonstrate that, although 1000G imputation was overall more effective at identifying associated loci, HapMap imputation may outperform 1000G imputation for specific loci. The 6p21.3 locus, corresponding to the major histocompatibility complex (MHC), was significant in the HapMap GWA study but not in the 1000G GWA study. The MHC locus is highly polymorphic and hosts many repetitive sequences, rendering it difficult to genotype and sequence [2931]. The HapMap reference panel was based largely on the genotyping of variants that were known at that time, whereas the 1000G reference panel is based entirely on low-coverage sequencing. This may explain the rather large discrepancy between HapMap and 1000G at this locus.

Differences in associations when GWA studies are based on different participants can be explained by sampling variability, even with the same sample size. Hence, by using exactly the same participants in the HapMap and 1000G comparisons in the present project, we rule out both statistical power and sampling variability as possible explanations for differences between the HapMap and 1000G GWA studies. Several real differences between the HapMap and 1000G reference panels may underlie the net benefit of 1000G imputation. The HapMap reference panel was largely based on genotypes of known variants, whereas the 1000G reference panel was primarily based on low-pass whole genome sequencing, enhancing the inclusion of novel variants. Additionally, most studies used only a small number of European-ancestry participants for HapMap imputation, whereas they used a larger number of participants of all available ancestries for 1000G imputation, introducing further haplotypes into the imputation process.

Nevertheless, some analytical differences between the HapMap and 1000G analyses were not controlled for in our main analysis and therefore remain as potential alternative explanations. First, genomic control corrections were applied to the results of each of the studies before meta-analysis, separately for the HapMap and 1000G GWA studies. As a result, for any given study, there could be differences between the correction applied to the HapMap GWA analysis and to the 1000G GWA analysis. As these differences do not appear to differ systematically between the HapMap and 1000G GWA analyses in our study, the genomic control corrections are unlikely to explain our results. The results from our sensitivity analysis were concordant with this interpretation: when no genomic control corrections were applied there were 6 loci only significant in the 1000G GWA study compared to 2 loci only significant in the HapMap GWA study.

The second difference between the HapMap and 1000G GWA studies that may explain our findings is that in the 1000G GWA study more studies were adjusted for ancestry-informative principal components. This difference reflects common practice, as population stratification is suspected to have a stronger influence on variants with lower MAF, and 1000G includes more of these [32]. However, the adjustments are applied to variants across the spectrum of minor allele frequencies, which may have influenced our results.

Thirdly, some studies used different software for HapMap and 1000G imputation (S1 Table). The imputation quality metrics used by IMPUTE and MACH differ, and this has traditionally been dealt with by applying different imputation quality thresholds: > 0.3 for MACH and > 0.4 for IMPUTE [5, 33]. In studies that used different imputation software for the HapMap and 1000G GWA studies, the filtering of variants can therefore differ. There may, additionally, be real differences in imputation quality. Finally, some studies used different analysis software (S3 Table). When we restricted our analysis to only those studies that used the same covariates, analysis software, and imputation software for the HapMap and 1000G GWA studies, 3 loci were only significant in the 1000G GWA study, while all loci significant in the HapMap GWA study were also significant in the 1000G GWA study. This suggests that differences in imputation software, analysis software, and covariates do not fully explain the observed difference between the HapMap and 1000G GWA studies, and that there are real differences resulting from choice of reference panel.

1000G GWA studies include more independent statistical tests than HapMap GWA studies [20, 21]. Thus, while a P-value threshold of 5×10−8, correcting for 1 million independent tests, maintains the type I error rate at 5% for HapMap GWA studies, this may not be the case for 1000G GWA studies. Using 1000G pilot data, Huang et al. estimated that 2 million independent tests were being done, and thus suggested a P-value threshold of 2.5×10−8 [20]. In our study we used a P-value threshold of 5×10−8 for both the HapMap and 1000G GWA studies, in accordance with the majority of published 1000G GWA studies [26, 3437]. When we used the threshold of 2.5×10−8 in the 1000G imputed GWA study, the difference between the HapMap and 1000G GWA studies became smaller. Thus, while we expect applying 1000G imputation may lead to novel findings using the conventional genome-wide significance threshold, this expectation may not be met when using stricter, and perhaps more appropriate thresholds. In other words, using the traditional significance threshold for 1000G may increase the type 1 error rate, which may account for some additional significant loci detected in 1000G GWA studies.

In this study we only examined variants with a MAF of greater than 1%. This restriction was common practice for HapMap GWA studies, but given the improved coverage of rare variants in 1000G, this may not remain the case for 1000G GWA studies. Different MAF thresholds have been used in published 1000G GWA studies, although many have used 1% [20, 22, 23, 26, 27, 3440]. Therefore, an advantage of 1000G not illustrated by this study may be the identification of rare variants, at new loci or as secondary signals at known loci. The advantage of 1000G imputation will then in part depend on the importance and impact of rare variants in the trait being studied, as well as the distribution of these variants. Rare and uncommon variants are often clustered in genes with previously associated common variants, limiting the new biology revealed through their identification [41, 42]. This appears to be the case for fibrinogen concentration as well [43, 44].

In conclusion, we show that the reference panel used in GWA studies can have an impact on the identification of common variants, although our results do not support the expectation that 1000G imputation always outperforms HapMap imputation, as we found one locus that appeared to be better covered in HapMap. This suggests that GWA studies will continue to be more successful as newer reference panels such as the Haplotype Reference Consortium are adopted. Nevertheless, our results also suggest that the benefits of 1000G are considerably reduced when the additional independent tests introduced by 1000G imputation are corrected for. Given that the bulk of the new information provided by 1000G imputation relates to low-frequency variants, we expect the penalty increased multiple testing burden to become less relevant in future studies as the power to examine these low-frequency variants increases with larger sample sizes and enhanced imputation quality. Imputation using the Haplotype Reference Consortium reference panel improves the imputation quality of low-frequency variants when compared to 1000G, and future reference panels based on the wealth of whole-genome sequencing data currently being generates by efforts such as TOPMed are likely to continue this trend [45].

Methods

Population

The sample for both the HapMap and 1000G GWA studies consists of 22 studies including the same 91,953 European-ancestry participants. The sample is largely a subset of the sample used in our previous work, and when possible the same analyses were used in this project [44, 46]. However, to ensure that only the same individuals were used, one or both of the analyses was rerun using only overlapping individuals when necessary. All studies were approved by appropriate research ethics committees and all respondents signed informed consent prior to participation. The ARIC study was approved by the University of Mississippi Medical Center IRB, Wake Forest University Health Sciences IRB, University of Minnesota IRB, and John Hopkins University IRB. The B58C study was approved by the South East England Multi-Centre Research Ethics Committee and the London & South East Committee of the National Research Ethics Service. The BMES was approved by the University of Sydney and the Western Sydney Area Health Service Human Research Ethics Committees. The CHS was approved by the Wake Forest University Health Sciences IRB, University of California, Davis IRB, John Hopkins University IRB, and University of Pittsburgh IRB, and University of Washington IRB. The FHS was approved by the Bostin University IRB. The GHS was approved by the Ethics Committee of the Landesärztekammer Rheinland-Pfalz (State Chamber of Physicians of Rhineland-Palatinate, Germany). The GOYA-Male study was approved by the regional scientific ethics committee of Copenhagen, Denmark, and the Danish data protection board. The HCS was approved by the University of Newcastle and Hunter New England Human Research Ethics Committee. The InCHIANTI study was approved by the Italian National Institute of Research and Care of Aging Institutional Review and Medstar Research Institute (Baltimore, MD). The LBC1921 study was approved by the Lothian Research Ethics Committee and the Scotland A Research Ethics Committee. The LBC1936 study was approved by the Multi-Centre Research Ethics Committee for Scotland and the Lothian Research Ethics Committee and the Scotland A Research Ethics Committee. The LURIC study was approved by the Ethics Committee at the Ärztekammer Rheinland-Pfalz. The NTR study was approved by the Medical Ethical Committee of the VU University Medical Center Amsterdam, and the Central Committee on Research Involving Human Subjects of the VU University Medical Center Amsterdam. The PROCARDIS study was approved by the Ethics Committee of the Karolinska Institutet. The PROSPER-PHASE study was approved by the Greater Glasgow Community/Primary Care Local Research Ethics Committee, Dumfries and Galloway Health Board Local Research Ethics Committee, Argyll and Clyde Health Board Local Research Ethics Committee, Lanarkshire Research Ethics Committee, Research Ethics Committee of the Cork Teaching Hospitals, and the Medical Ethical Committee of the Leiden University Medical Center. The RS was approved by the Medical Ethics Committee of the Erasmus MC and the Dutch Ministry of Health, Welfare and Sport. The SardiNIA study was approved by the Ethics Committee at Azienda Sanitaria Locale (ASL) n°1 of Sassari, Sardinia, Italy. The SHIP was approved by the Medical Ethics Committee of the University of Greifswald. The TwinsUK study was approved by the NRES Committee London-Westminster (formerly St Thomas' Ethics Committee). The WGHS was approved by Brigham and Women’s Hostpital IRB.

Genotyping and imputation

Genotyping and pre-imputation quality control methods for each study are shown in S7 Table. Studies imputed dosages of genetic variants using reference panels from the 1000 genomes project with MACH [47, 48] or IMPUTE [49]. Studies imputed variant dosages using Phase 2 reference panels from the HapMap project with MACH [47, 48], IMPUTE [49], or BIMBAM [50]. We excluded variants with MACH imputation quality < 0.3, IMPUTE/BIMBAM imputation quality < 0.4, or MAF < 0.01 from each study.

Fibrinogen measurement

Fibrinogen concentration was measured in citrated or EDTA plasma samples using a variety of methods including the Clauss method, immunonephelometric methods, immunoturbidimetric methods, and other functional methods. Fibrinogen concentration was measured in g/L and natural log transformed. Details about the fibrinogen measurement are shown in S10 Table.

Genome-wide association analysis

All analyses were adjusted for age and sex, and study specific covariates such as center or case/control status. In family studies, linear mixed models were used to account for family structure. Some studies adjusted the analysis for principle components to account for population structure and cryptic relatedness. Some studies used a different number of principle components in the HapMap and 1000G analyses. The adjustments and analysis software used by each study are shown in S8 Table. We applied a genomic control correction to the results of each of the studies before meta-analysis to remove any remaining genomic inflation. The genomic inflation factor used in this correction was calculated separately in the HapMap and 1000G analyses for each study. We meta-analyzed the results using an inverse-variance model with fixed effects implemented in METAL [51]. Loci were defined as the 500 Kb area on either side of lead variants (the variant with the smallest P-value). Build 36 positions of HapMap SNPs were converted to build 37 using the UCSC genome browser (http://genome.ucsc.edu/cgi-bin/hgLiftOver). Variants were annotated to genes using ANNOVAR version 2013Mar07. At the meta-analysis level, the imputation quality of each variant was defined as the sample-size weighted mean imputation quality across the studies, not including studies where the variant was filtered out.

Comparison of HapMap and 1000G

When a locus was significant in both the HapMap and 1000G GWA studies we defined it as an overlapping locus. When a locus was significant in only one of the two analyses we defined it as a non-overlapping locus. To compare the strength of association in the HapMap and 1000G GWA studies, we identified loci with P-value differences of 1 order of magnitude or greater (for example: from 5×10−8 compared to 5×10−9 or less).

For each significant locus we used two approaches to assess the relationship between lead variants from HapMap and 1000G. First, we determined whether or not the more significant of the two lead variants or a good proxy (linkage disequilibrium r2 > 0.8) was included in the analysis of the other reference panel. If so, we examined its association in the other reference panel. Thus, if a locus was more significant in the 1000G GWA study, we checked whether the 1000G lead variant or a proxy was included in the HapMap GWA study. Second, we examined the correlation R2 between HapMap and 1000G lead variants in the form of imputed genotype dosages. This was performed for 5966 individuals from the Rotterdam Study (see study description in S1 Text) [52].

Sensitivity analysis

First, we compared the results of the HapMap and 1000G GWA studies when applying a stricter Bonferroni-corrected P-value threshold of 2.5×10−8 to the 1000G GWA study. This threshold was suggested by Huang et al. to keep the type 1 error rate at 5% when using 1000G data [20]. Second, we repeated the analysis without using genomic control corrections. Third, we repeated the analysis in 34,098 participants using only the 10 studies that used the same imputation and analysis software as well as the same covariates for the HapMap and 1000G GWA studies.

Supporting Information

S1 Fig. Quantile-Quantile (QQ) plots comparing the HapMap and 1000G GWA studies.

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S2 Fig. Manhattan plot comparing the HapMap (red) and 1000G (green) GWA studies.

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S3 Fig. Comparison of lead variants of the HapMap and 1000G GWA studies of significant loci.

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S4 Fig. Regional plots of overlapping signals that were significant in both the HapMap (red) and 1000G (green) GWA studies.

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S1 Table. Characteristics of the included studies and their participants.

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S2 Table. Genomic inflation factors by study and imputation panel.

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S3 Table. Annotation of loci significant in the HapMap GWA study, 1000G GWA study, or both.

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S4 Table. Correlation between the lead variants from the HapMap and 1000G GWA studies.

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S5 Table. Differences between HapMap and 1000G for loci with a correlation R2 < 0.8 between imputed dosages of the HapMap and 1000G lead variants.

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S6 Table. Loci that were significant in either the HapMap or 1000G GWA studies with genomic control corrections.

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S7 Table. Genotyping and imputation methods of the included studies.

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S8 Table. Analysis software and covariates used by the included studies.

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S9 Table. Loci that were significant in either the HapMap or 1000G GWAS excluding studies that did not use the same imputation software, analysis software, or covariates.

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S10 Table. Sample and array type used for the fibrinogen measurement in each of the included studies.

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Acknowledgments

The authors acknowledge the essential role of the Cohorts for Heart and Aging Research in Genome Epidemiology (CHARGE) Consortium in development and support of this manuscript. The authors thank the staff and participants of the ARIC study for their important contributions. We would like to thank the University of Minnesota Supercomputing Institute for use of the calhoun supercomputers. A full list of principal CHS investigators and institutions can be found at CHS-NHLBI.org. The analyses reflect intellectual input and resource development from the Framingham Heart Study investigators participating in the SNP Health Association Resource (SHARe) project. The authors would like to thank the men and women participating in the HCS as well as The University of Newcastle, Vincent Fairfax Family Foundation and The Hunter Medical Research Institute. We thank the LBC1936 and LBC1921 participants and research team members. We thank the nurses and staff at the Wellcome Trust Clinical Research Facility, where subjects were tested and the genotyping was performed. We thank the LURIC study team who were either temporarily or permanently involved in patient recruitment as well as sample and data handling, in addition to the laboratory staff at the Ludwigshafen General Hospital and the Universities of Freiburg and Ulm, Germany. This work was performed as part of an ongoing collaboration of the PROSPER study group in the universities of Leiden, Glasgow and Cork. The authors are grateful to the study participants, the staff from the Rotterdam Study and the participating general practitioners and pharmacists. We thank Pascal Arp, Mila Jhamai, Marijn Verkerk, Lizbeth Herrera, Marjolein Peters and Carolina Medina-Gomez for their help in creating the GWAS database, and Karol Estrada and Carolina Medina-Gomez for the creation and analysis of imputed data. We thank the many individuals who generously participated in this study, the Mayors and citizens of the Sardinian towns involved, the head of the Public Health Unit ASL4, and the province of Ogliastra for their volunteerism and cooperation. In addition, we are grateful to the Mayor and the administration in Lanusei for providing and furnishing the clinic site. We are grateful to the physicians Angelo Scuteri, Marco Orrù, Maria Grazia Pilia, Liana Ferreli, Francesco Loi, nurses Paola Loi, Monica Lai and Anna Cau who carried out participant physical exams; the recruitment personnel Susanna Murino; Mariano Dei, Sandra Lai, Andrea Maschio, Fabio Busonero for genotyping; Maria Grazia Piras and Monia Lobina for fibrinogen phenotyping.

Steno Diabetes Center and Synlab Holding Deutschland GmbH provided support in the form of salaries for authors T.S.A. and W.M. respectively, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Infrastructure for the CHARGE Consortium is supported in part by the National Heart, Lung, and Blood Institute grant R01HL105756. ARIC is carried out as a collaborative study supported by National Heart, Lung, and Blood Institute (NHLBI) contracts HHSN268201100005C, HHSN268201100006C, HHSN268201100007C, HHSN268201100008C, HHSN268201100009C, HHSN268201100010C, HHSN268201100011C, and HHSN268201100012C, R01HL087641, R01HL59367 and R01HL086694; National Human Genome Research Institute contract U01HG004402; and National Institutes of Health contract HHSN268200625226C. Infrastructure was partly supported by Grant Number UL1RR025005, a component of the National Institutes of Health and NIH Roadmap for Medical Research. LITE is supported by HL0597367 from the NHLBI. B58C acknowledges use of phenotype and genotype data from the British 1958 Birth Cohort DNA collection, funded by the Medical Research Council grant G0000934 and the Wellcome Trust grant 068545/Z/02. Genotyping for the B58C-WTCCC subset was funded by the Wellcome Trust grant 076113/B/04/Z. The B58C-T1DGC genotyping utilized resources provided by the Type 1 Diabetes Genetics Consortium, a collaborative clinical study sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institute of Allergy and Infectious Diseases (NIAID), National Human Genome Research Institute (NHGRI), National Institute of Child Health and Human Development (NICHD), and Juvenile Diabetes Research Foundation International (JDRF) and supported by U01 DK062418. B58C-T1DGC GWAS data were deposited by the Diabetes and Inflammation Laboratory, Cambridge Institute for Medical Research (CIMR), University of Cambridge, which is funded by Juvenile Diabetes Research Foundation International, the Wellcome Trust and the National Institute for Health Research Cambridge Biomedical Research Centre; the CIMR is in receipt of a Wellcome Trust Strategic Award (079895). The B58C-GABRIEL genotyping was supported by a contract from the European Commission Framework Programme 6 (018996) and grants from the French Ministry of Research. The BMES has been supported by the Australian National Health & Medical Research Council, Canberra Australia (Grant Numbers 974159, 211069, 457349, 512423, 475604, 529912, and the funding for Centre for Clinical Research Excellence in Translational Clinical Research in Eye Diseases, CCRE in TCR-Eye, grant ID 529923); In addition, funding by the Wellcome Trust, UK (to A Viswanathan, P McGuffin, P Mitchell, F Topouzis, P Foster) has supported the genotyping costs of the entire BMES population. This CHS research was supported by NHLBI contracts HHSN268201200036C, HHSN268200800007C, N01HC55222, N01HC85079, N01HC85080, N01HC85081, N01HC85082, N01HC85083, N01HC85086; and NHLBI grants U01HL080295, R01HL087652, R01HL105756, R01HL103612, and R01HL120393 with additional contribution from the National Institute of Neurological Disorders and Stroke (NINDS). Additional support was provided through R01AG023629 from the National Institute on Aging (NIA). The provision of genotyping data was supported in part by the National Center for Advancing Translational Sciences, CTSI grant UL1TR000124, and the National Institute of Diabetes and Digestive and Kidney Disease Diabetes Research Center (DRC) grant DK063491 to the Southern California Diabetes Endocrinology Research Center. The FHS was partially supported by the National Heart, Lung, and Blood Institute’s (NHLBI's) Framingham Heart Study (Contract No. N01-HC-25195) and its contract with Affymetrix, Inc. for genotyping services (Contract No. N02-HL-6-4278). A portion of this research utilized the Linux Cluster for Genetic Analysis (LinGA-II) funded by the Robert Dawson Evans Endowment of the Department of Medicine at Boston University School of Medicine and Boston Medical Center. Partial investigator support was provided by the National Institute of Diabetes and Digestive and Kidney Diseases K24 DK080140 (JB Meigs), the National Institute on Aging and National Institute for Neurological Disorders and Stroke R01 AG033193, NS017950 (S Seshadri). The GOYA Male study was conducted as part of the activities of the Gene-diet Interactions in Obesity project (GENDINOB, www.gendinob.dk) and the MRC centre for Causal Analyses in Translational Epidemiology (MRC CAiTE). We thank the staff of the Copenhagen City Heart Study for their skillful examination of the study subjects in collection of baseline and follow-up data. Tarunveer Singh Ahluwalia received his Postdoctoral Research funding from GENDINOB project and acknowledges the same. The Gutenberg Health Study is funded through the government of Rhineland-Palatinate („Stiftung Rheinland-Pfalz für Innovation“, contract AZ 961-386261/733), the research programs “Wissen schafft Zukunft” and “Center for Translational Vascular Biology (CTVB)” of the Johannes Gutenberg-University of Mainz, and its contract with Boehringer Ingelheim and PHILIPS Medical Systems, including an unrestricted grant for the Gutenberg Health Study. VG, PSW are funded by the Federal Ministry of Education and Research (BMBF 01EO1003). The InCHIANTI study baseline (1998–2000) was supported as a "targeted project" (ICS110.1/RF97.71) by the Italian Ministry of Health and in part by the U.S. National Institute on Aging (Contracts: 263 MD 9164 and 263 MD 821336); This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging. The whole genome association study in LBC1936 and LBC1921 was funded by the Biotechnology and Biological Sciences Research Council (BBSRC; Ref. BB/F019394/1). The LBC1936 research was supported by Age UK. The LBC1921 data collection was funded by the BBSRC. The work was undertaken by The University of Edinburgh Centre for Cognitive Ageing and Cognitive Epidemiology (MR/K026992/1), part of the cross council Lifelong Health and Wellbeing Initiative. Funding from the BBSRC, and MRC is gratefully acknowledged. LURIC has received funding from the 6th Framework Program (integrated project Bloodomics, grant LSHM-CT-2004-503485) and from the 7th Framework Program (Atheroremo, grant agreement number 201668 and RiskyCAD, grant agreement number 305739) of the European Union as well as from the INTERREG IV Oberrhein Program (Project A28, Genetic mechanisms of cardiovascular diseases) with support from the European Regional Development Fund (ERDF) and the Wissenschaftsoffensive TMO. NTR: Funding was obtained from the Netherlands Organization for Scientific Research (NWO) and MagW/ZonMW grants 904-61-090, 985-10-002, 904-61-193,480-04-004, 400-05-717, Addiction-31160008, Middelgroot-911-09-032, Spinozapremie 56-464-14192, Center for Medical Systems Biology (CSMB, NWO Genomics), NBIC/BioAssist/RK(2008.024), Biobanking and Biomolecular Resources Research Infrastructure (BBMRI–NL, 184.021.007). VU University’s Institute for Health and Care Research (EMGO+) and Neuroscience Campus Amsterdam (NCA); the European Science Foundation (ESF, EU/QLRT-2001-01254), the European Community's Seventh Framework Program (FP7/2007-2013), ENGAGE (HEALTH-F4-2007-201413); the European Science Council (ERC Advanced, 230374), Rutgers University Cell and DNA Repository (NIMH U24 MH068457-06), the Avera Institute, Sioux Falls, South Dakota (USA) and the National Institutes of Health (NIH, R01D0042157-01A, MH081802, Grand Opportunity grants 1RC2 MH089951). Part of the genotyping and analyses were funded by the Genetic Association Information Network (GAIN) of the Foundation for the National Institutes of Health. Computing was supported by BiG Grid, the Dutch e-Science Grid, which is financially supported by NWO. PROCARDIS was supported by the European Community Sixth Framework Program (LSHM-CT- 2007–037273), AstraZeneca, the British Heart Foundation, the Wellcome Trust (Contract No. 075491/Z/04), the Swedish Research Council, the Knut and Alice Wallenberg Foundation, the Swedish Heart-Lung Foundation, the Torsten and Ragnar Söderberg Foundation, the Strategic Cardiovascular and Diabetes Programs of Karolinska Institutet and Stockholm County Council, the Foundation for Strategic Research and the Stockholm County Council. Jemma C Hopewell and Robert Clarke acknowledge support from the BHF Centre of Research Excellence, Oxford. M.Sabater-Lleal is supported by the Swedish Heart-Lung Foundation (20130399), and acknowledges funding from Åke Wiberg and Tore Nilssons foundations. B.Sennblad acknowledges funding from the Magnus Bergvall Foundation and the Foundation for Old Servants. PROSPER received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° HEALTH-F2-2009-223004. For a part of the genotyping we received funding from the Netherlands Consortium of Healthy Aging (NGI: 05060810). Measurement of serum fibrinogen was supported by a grant from the Scottish Executive Chief Scientist Office, Health Services Research Committee grant number CZG/4/306. Prof. Dr. J.W. Jukema is an Established Clinical Investigator of the Netherlands Heart Foundation (2001 D 032). The generation and management of GWAS genotype data for the Rotterdam Study is supported by the Netherlands Organisation of Scientific Research NWO Investments (nr. 175.010.2005.011, 911-03-012). This study is funded by the Research Institute for Diseases in the Elderly (014-93-015; RIDE2), the Netherlands Genomics Initiative (NGI)/Netherlands Organisation for Scientific Research (NWO) project nr. 050-060-810. The Rotterdam Study is funded by Erasmus Medical Center and Erasmus University, Rotterdam, Netherlands Organization for the Health Research and Development (ZonMw), the Research Institute for Diseases in the Elderly (RIDE), the Ministry of Education, Culture and Science, the Ministry for Health, Welfare and Sports, the European Commission (DG XII), and the Municipality of Rotterdam. Abbas Dehghan is supported by NWO grant (veni, 916.12.154) and the EUR Fellowship. The SardiNIA (‘‘ProgeNIA”) team was supported by Contract NO1-AG-1–2109 from the NIA. This research was supported by the Intramural Research Program of the NIH, National Institute on Aging, by Sardinian Autonomous Region (L.R. no. 7/2009) grant cRP3-154, and by grant FaReBio2011 “Farmaci e Reti Biotecnologiche di Qualità”. SHIP is part of the Community Medicine Research net of the University of Greifswald, Germany, which is funded by the Federal Ministry of Education and Research (grants no. 01ZZ9603, 01ZZ0103, and 01ZZ0403), the Ministry of Cultural Affairs as well as the Social Ministry of the Federal State of Mecklenburg—West Pomerania. Genome- wide data have been supported by the Federal Ministry of Education and Research (grant no. 03ZIK012) and a joint grant from Siemens Healthcare, Erlangen, Germany and the Federal State of Mecklenburg West Pomerania. Computing resources have been made available by the Leibniz Supercomputing Centre of the Bavarian Academy of Sciences and Humanities (HLRB project h1231). The University of Greifswald is a member of the 'Center of Knowledge Interchange' program of the Siemens AG and the Caché Campus program of the InterSystems GmbH. This work is also part of the research project Greifswald Approach to Individualized Medicine (GANI_MED). The GANI_MED consortium is funded by the Federal Ministry of Education and Research and the Ministry of Cultural Affairs of the Federal State of Mecklenburg–West Pomerania (03IS2061A). TwinsUK. The study was funded by the Wellcome Trust; European Community’s Seventh Framework Programme (FP7/2007-2013). The study also receives support from the National Institute for Health Research (NIHR) Clinical Research Facility at Guy’s & St Thomas’ NHS Foundation Trust and NIHR Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust and King's College London. Tim Spector is an NIHR senior Investigator and is holder of an ERC Advanced Principal Investigator award. SNP Genotyping was performed by The Wellcome Trust Sanger Institute and National Eye Institute via NIH/CIDR. The WGHS is supported by HL043851 and HL080467 from the National Heart, Lung, and Blood Institute and CA047988 from the National Cancer Institute, the Donald W. Reynolds Foundation and the Fondation Leducq, with collaborative scientific support and funding for genotyping provided by Amgen.

Author Contributions

  1. Conceptualization: PSdV CJOD NLS AD.
  2. Formal analysis: PSdV MSL DIC ST TSA AT MEK MHC REM MS LCW RP VG JAB CV TT LMR CO JM S. Basu JJH GED AK LML MM ACM HHMD BM JH PGH WG PES HB WT JWJ DPS AD.
  3. Investigation: DIC JW JRA S. Bandinelli A. Hamsten PSW BMP AGU EJCdG A. Hofman OHF TDS IJD WM A. Greinacher FC DIB HW WT PMR JWJ PM TH CJOD DPS AR AM AJMdC A. Grotevendt TLB MPMdM GDL NS MAM JMS TZ DJS KJL LF NLS ST MEK MF JJH KDT MZ UV JH EGH DGH FR WLM PES RJS.
  4. Supervision: DPS AD.
  5. Visualization: PSdV MSL SL AD.
  6. Writing – original draft: PSdV MSL AD.
  7. Writing – review & editing: PSdV MSL DIC ST TSA AT MEK MHC JJW JRA REM MS LCW RP VG JAB CV TT LMR CO JM S. Basu MF QY SL JJH AR AM AJMdC A. Greinacher KDT GED AK LML TLB MM S. Bandinelli ACM A. Hamsten GT MPMdM HHMD GDL MZ NS KJL UV BM JH EGH MAM JMS PGH DGH WG FR WLM PES TZ BMP AGU EJCdG DJS HB A. Hofman OHF JIR LF TDS IJD WM A. Grotevendt PSW FC DIB HW WT PMR JWJ RJS PM TH CJOD NLS DPS AD.

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