Multi-ancestry study of blood lipid levels identifies four loci interacting with physical activity

Many genetic loci affect circulating lipid levels, but it remains unknown whether lifestyle factors, such as physical activity, modify these genetic effects. To identify lipid loci interacting with physical activity, we performed genome-wide analyses of circulating HDL cholesterol, LDL cholesterol, and triglyceride levels in up to 120,979 individuals of European, African, Asian, Hispanic, and Brazilian ancestry, with follow-up of suggestive associations in an additional 131,012 individuals. We find four loci, in/near CLASP1, LHX1, SNTA1, and CNTNAP2, that are associated with circulating lipid levels through interaction with physical activity; higher levels of physical activity enhance the HDL cholesterol-increasing effects of the CLASP1, LHX1, and SNTA1 loci and attenuate the LDL cholesterol-increasing effect of the CNTNAP2 locus. The CLASP1, LHX1, and SNTA1 regions harbor genes linked to muscle function and lipid metabolism. Our results elucidate the role of physical activity interactions in the genetic contribution to blood lipid levels.

details of the study design for the CARDIA study have been previously published 3 . Nine examinations have been completed since initiation of the study, respectively in the years 0, 2, 5, 7, 10, 15, 20, 25 and 30. Written informed consent was obtained from participants at each examination and all study protocols were approved by the institutional review boards of the participating institutions. All participants were asked to fast for 12 hours before each clinic visit. Serum and plasma blood samples were drawn from the antecubital vein and stored at −70°C until analyzed. Plasma total cholesterol, HDL-c, and triglyceride levels were measured using enzymatic methods; HDL-c levels were measured after dextran-sulfate-magnesium precipitation of other lipoproteins. LDL-c levels were estimated with the Friedewald equation for individuals with fasting triglyceride values less than 400 mg/dL. Baseline measures were used in this analyses.

CHS (Cardiovascular Health Study):
CHS is a population-based cohort study of risk factors for cardiovascular disease in adults 65 years of age or older conducted across four field centers 4 . The original predominantly European ancestry cohort of 5,201 persons was recruited in 1989-1990 from random samples of the Medicare eligibility lists and an additional predominately African-American cohort of 687 persons was enrolled in 1992-93 for a total sample of 5,888. Blood samples were drawn from all participants at their baseline examination and DNA was subsequently extracted from available samples. European ancestry participants were excluded from the GWAS study sample due to prevalent coronary heart disease, congestive heart failure, peripheral vascular disease, valvular heart disease, stroke, or transient ischemic attack at baseline. After QC, genotyping was successful for 3271 European ancestry and 823 African-American participants. CHS was approved by institutional review committees at each site and individuals in the present analysis gave informed consent including consent to use of genetic information for the study of cardiovascular disease.

GenSalt (Genetic Epidemiology Network of Salt Sensitivity):
GenSalt is a multi-center, family based study designed to identify, through dietary sodium and potassium intervention, salt-sensitivitivity susceptibility genes which may underlie essential hypertension in rural Han Chinese families.
Approximately 629 families with at least one "proband" with high blood pressure were recruited and tested for a wide variety of physiological, metabolic and biochemical measures at baseline and at multiple times during the 3-week intervention. The intervention consisted of one week on a low sodium diet, followed by one week on a high sodium diet, and finally one week on a high sodium diet with a potassium supplement.

GS:SFHS (Generation Scotland: Scottish Family Health Study):
The GS:SFHS (www.generationscotland.org) is a family-based genetic epidemiology cohort with DNA, other biological samples (serum, urine and cryopreserved whole blood) and socio-demographic and clinical data from approximately 24,000 volunteers, aged 18-98 years, in ~7,000 family groups. An important feature of GS:SFHS is the breadth of phenotype information, including detailed data on cognitive function, personality traits and mental health. Although data collection was cross-sectional, GS:SFHS becomes a longitudinal cohort as a result of the ability to link to routine NHS data, using the community health index (CHI) number.

HANDLS (Healthy Aging in Neighborhoods of Diversity across the Life Span):
HANDLS is a community-based, longitudinal epidemiologic study examining the influences of race and socioeconomic status (SES) on the development of age-related health disparities among a sample of socioeconomically diverse African Americans and whites. This unique study will assess over a 20-year period physical parameters and also evaluate genetic, biologic, demographic, and psychosocial, parameters of African American and white participants in higher and lower SES to understand the driving factors behind persistent black-white health disparities in overall longevity, cardiovascular disease, and cognitive decline. The study recruited 3,722 participants from Baltimore, MD with a mean age of 47.7 years, 2,200 African Americans and 1,522 whites, with 41% reporting household incomes below the 125% poverty delimiter.
Genotyping was done on a subset of self-reporting African American participants by the Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health (NIH). A larger genotyping effort included a small subset of self-reporting European ancestry samples. This research was supported by the Intramural Research Program of the NIH, NIA and the National Center on Minority Health and Health Disparities.
Health ABC (Health, Aging, and Body Composition): Cohort description: The Health ABC study is a prospective cohort study investigating the associations between body composition, weight-related health conditions, and incident functional limitation in older adults. Health ABC enrolled wellfunctioning, community-dwelling black (n=1281) and white (n=1794) men and women aged 70-79 years between April 1997 and June 1998. Participants were recruited from a random sample of white and all black Medicare eligible residents in the Pittsburgh, PA, and Memphis, TN, metropolitan areas. Participants have undergone annual exams and semi-annual phone interviews. The current study sample consists of 1559 white participants who attended the second exam in 1998-1999 with available genotyping data. Genotyping was performed by the Center for Inherited Disease Research (CIDR) using the Illumina Human1M-Duo BeadChip system.

HUFS (Howard University Family Study):
HUFS followed a population-based selection strategy designed to be representative of African American families living in the Washington, DC metropolitan area. The major objectives of the HUFS were to study the genetic and environmental basis of common complex diseases including hypertension, obesity and associated phenotypes. Participants were sought through door-to-door canvassing, advertisements in local print media and at health fairs and other community gatherings. In order to maximize the utility of this cohort for the study of multiple common traits, families were not ascertained based on any phenotype. During a clinical examination, demographic information was collected by interview.

HyperGEN (Hypertension Genetic Epidemiology Network):
HyperGEN is a family-based study that looks at the genetic causes of hypertension and related conditions in EA and AA subjects. HyperGEN recruited hypertensive sibships, along with their normotensive adult offspring, and an agematched random sample. HyperGEN has collected data on 2,471 Caucasian-American subjects and 2,300 African-American subjects, from five field centers in Alabama, Massachusetts, Minnesota, North Carolina, and Utah.

JHS (Jackson Heart Study):
The Jackson Heart Study is a longitudinal, community-based observational cohort study investigating the role of environmental and genetic factors in the development of cardiovascular disease in African Americans [8][9][10] . Between 2000 and 2004, a total of 5306 participants were recruited from a tri-county area (Hinds, Madison, and Rankin Counties) that encompasses Jackson, MS. Details of the design and recruitment for the Jackson Heart Study cohort has been previously published.1-3 Briefly, approximately 30% of participants were former members of the Atherosclerosis Risk in Communities (ARIC) study. The remainder were recruited by either 1) random selection from the Accudata list, 2) commercial listing, 3) a constrained volunteer sample, in which recruitment was distributed among defined demographic cells in proportions designed to mirror those in the overall population, or through the Jackson Heart Study Family Study.

MESA (Multi-Ethnic Study of Atherosclerosis):
The Multi-Ethnic Study of Atherosclerosis (MESA) is a study of the characteristics of subclinical cardiovascular disease and the risk factors that predict progression to clinically overt cardiovascular disease or progression of the subclinical disease 11 . MESA consisted of a diverse, population-based sample of an initial 6,814 asymptomatic men and women aged 45-84. 38 percent of the recruited participants were white, 28 percent African American, 22 percent Hispanic, and 12 percent Asian, predominantly of Chinese descent. Participants were recruited from six field centers across the United States: Wake Forest University, Columbia University, Johns Hopkins University, University of Minnesota, Northwestern University and University of California -Los Angeles. Participants are being followed for identification and characterization of cardiovascular disease events, including acute myocardial infarction and other forms of coronary heart disease (CHD), stroke, and congestive heart failure; for cardiovascular disease interventions; and for mortality. The first examination took place over two years, from July 2000 -July 2002. It was followed by four examination periods that were 17-20 months in length. Participants have been contacted every 9 to 12 months throughout the study to assess clinical morbidity and mortality.

NEO (The Netherlands Epidemiology of Obesity study):
The NEO was designed for extensive phenotyping to investigate pathways that lead to obesity-related diseases. The NEO study is a population-based, prospective cohort study that includes 6,671 individuals aged 45-65 years, with an oversampling of individuals with overweight or obesity. At baseline, information on demography, lifestyle, and medical history have been collected by questionnaires. In addition, samples of 24-h urine, fasting and postprandial blood plasma and serum, and DNA were collected. Genotyping was performed using the Illumina HumanCoreExome BeadChip, which was subsequently imputed to the 1000 genome reference panel. Participants underwent an extensive physical examination, including anthropometry, electrocardiography, spirometry, and measurement of the carotid artery intima-media thickness by ultrasonography. In random subsamples of participants, magnetic resonance imaging of abdominal fat, pulse wave velocity of the aorta, heart, and brain, magnetic resonance spectroscopy of the liver, indirect calorimetry, dual energy X-ray absorptiometry, or accelerometry measurements were performed. The collection of data started in September 2008 and completed at the end of September 2012. Participants are currently being followed for the incidence of obesity-related diseases and mortality.

Pelotas Birth Cohort Study (The 1982 Pelotas Birth Cohort Study, Brazil):
The maternity hospitals in Pelotas, a southern Brazilian city (current population ~330,000), were visited daily in the year of 1982. The 5,914 liveborns whose families lived in the urban area were examined and their mothers interviewed. Information was obtained for more than 99% of the livebirths. These subjects have been followed-up at the following mean ages: 11.3 months (all children born from January to Abril 1982; n=1457), 19.4 months (entire cohort; n=4934), 43.1 months (entire cohort; n=4742), 13.1 years (random subsample; n=715), 14.7 years (systematic subsample; n=1076); 18.2 (male cohorts attending to compulsory Army recruitment examination; n=2250), 18.9 (systematic subsample; n=1031), 22.8 years (entire cohort; n=4297) and 30.2 years (entire cohort; n=3701). Details about follow-up visits and available data can be found in the two Cohort Profile papers [12][13] . DNA samples (collected at the mean age of 22.8 years) were genotyped for ~2.5 million of SNPs using the Illumina HumanOmni2.5-8v1 array (which includes autosomal, X and Y chromosomes, and mitochondrial variants). After quality control, the data were prephased using SHAPEIT and imputed using IMPUTE2 based on 1000 Genomes haplotypes.

RS (Rotterdam Study):
The Rotterdam Study is a prospective, population-based cohort study among individuals living in the well-defined Ommoord district in the city of Rotterdam in The Netherlands. The aim of the study is to determine the occurrence of cardiovascular, neurological, ophthalmic, endocrine, hepatic, respiratory, and psychiatric diseases in elderly people. The cohort was initially defined in 1990 among approximately 7,900 persons, aged 55 years and older, who underwent a home interview and extensive physical examination at the baseline and during follow-up rounds every 3-4 years (RS-I

SCHS-CHD (Singapore Chinese Health Study -Coronary Heart Disease):
SCHS-CHD is a casecontrol study of coronary heart disease that was nested within the Singapore Chinese Health Study (SCHS), a prospective cohort study of 63,257 Singaporean Chinese men and women aged 45-74 years living in Singapore. We selected cases and controls from participants that provided blood samples and were free of coronary heart disease and stroke at the time of blood collection (N=24,454). Cases (N=760) had acute myocardial infarction (AMI) or died of coronary heart disease. AMI was identified through the Singapore Myocardial Infarction Registry or through the nationwide hospital discharge database followed by confirmation of AMI by cardiologists" review of medical records using the Multi-Ethnic Study of Atherosclerosis criteria (available at: http://www.mesa-nhlbi.org/manuals.aspx). Coronary heart disease deaths were identified through the Singapore Registry of Births and Deaths (ICD9 410-414 as first stated cause of death). Matched controls (N=1,491) were selected using a riskset sampling strategy. Controls were participants who were alive and free of coronary heart disease at the time of the diagnosis or death of the index cases and were matched for age, sex, dialect group, year of recruitment and date of blood collection. In-person interviews and phlebotomy were conducted before the onset of disease and non-fasting venous blood was stored at -80 0 C for extraction of DNA and blood biochemistry. . LDL-C was calculated from Friedewald formula. Participants completed both the physical activity questionnaire in SP2 (SP2PAQ) and IPAQ long form 14 . Data from this re-visit were utilized for this study 15,16 .

WGHS (Women's Genome Health Study):
WGHS is a prospective cohort of female North American health care professionals representing participants in the Women"s Health Study (WHS) trial who provided a blood sample at baseline and consent for blood-based analyses. Participants in the WHS were 45 years or older at enrollment and free of cardiovascular disease, cancer or other major chronic illness. The current data are derived from 23,294 WGHS participants for whom whole genome genotype information was available at the time of analysis and for whom self-reported European ancestry could be confirmed by multidimensional scaling analysis of 1,443 ancestry informative markers in PLINK v. 1.06. At baseline, lifestyle habits related to smoking, consumption of alcohol, and physical activity as well as other general clinical information were ascertained by a self-reported questionnaire, an approach which has been validated in the WGHS demographic, namely female health care professionals.

WHI (Women's Health Initiative):
WHI is a long-term national health study that focuses on strategies for preventing common diseases such as heart disease, cancer and fracture in postmenopausal women. A total of 161,838 women aged 50-79 years old were recruited from 40 clinical centers in the US between 1993 and 1998. WHI consists of an observational study, two clinical trials of postmenopausal hormone therapy (HT, estrogen alone or estrogen plus progestin), a calcium and vitamin D supplement trial, and a dietary modification trial 17 . Study recruitment and exclusion criteria have been described previously 17 . Recruitment was done through mass mailing to age-eligible women obtained from voter registration, driver"s license and Health Care Financing Administration or other insurance list, with emphasis on recruitment of minorities and older women 18 . Exclusions included participation in other randomized trials, predicted survival < 3 years, alcoholism, drug dependency, mental illness and dementia. For the CT, women were ineligible if they had a systolic BP > 200 mm Hg or diastolic BP > 105 mm Hg, a history of hypertriglyceridemia or breast cancer. Study protocols and consent forms were approved by the IRB at all participating institutions. Socio-demographic characteristics, lifestyle, medical history and self-reported medications were collected using standardized questionnaires at the screening visit. Physical measures of height, weight and blood pressure were measured at a baseline clinical visit 18 . The genome wide association study (GWAS) non-overlapping samples are composed of a case-control study (WHI Genomics and Randomized Trials Network -GARNET, which included all coronary heart disease, stroke, venous thromboembolic events and selected diabetes cases that happened during the active intervention phase in the WHI HT clinical trials and aged matched controls), women selected to be "representative" of the HT trial (mostly younger white HT subjects that were also enrolled in the WHI memory study -WHIMS) and the WHI SNP Health Association Resource (WHI SHARe), a randomly selected sample of 8,515 African American and 3,642 Hispanic women from WHI. GWAS was performed using

DESIR (Data from an Epidemiological Study on the Insulin Resistance):
The DESIR cohort study aims to: describe and understand the relations between the abnormalities of the syndrome, their evolution, according to age and sex; search for risk factors of insulin resistance, in particular factors associated with the environment, lifestyle and genetic markers; quantify the links between the syndrome and both cardiovascular disease and diabetes; evaluate the frequency of the syndrome in terms of its consequences on public health.

DFTJ (Dongfeng-Tongji Cohort Study):
The DFTJ-cohort study includes 27,009 retired employees from a state-owned automobile enterprise in China. This study was launched in 2008 and will be followed up every 5 years. In 2013 we conducted the first follow-up. By using semi-structural questionnaire and health examination, those having cancer or severe diseases were excluded. Fasting blood samples and detailed epidemiology data were collected. The main goal of the cohort was to identify the environmental and genetic risk factors and the gene-environment interactions on chronic diseases, and to find novel biomarkers for chronic disease and mortality prediction. Finally, 1,461 included in the present study with GWAS data. All of the participants wrote informed consent and the ethical committees in the Tongji Medical College approved this research project. Detailed information has been described in elsewhere 21 .

QC criteria and imputation methods:
We did the GWAS scan on the DFTJ-cohort with Affymetrix Genome-Wide Human SNP Array 6.0 chips. In total, we genotyped 906,703 SNPs among 1,461 subjects. After stringent QC filtering, SNPs with MAF < 0.01, Hardy-Weinberg Equilibrium (HWE) < 0.0001, and SNP call rate < 95% were excluded. Individuals with call rates < 95% were also not included for further analysis. In total, we retained 1,452 subjects with 658,288 autosomal SNPs for statistical analyses, with an overall call rate of 99.68%. We used MACH 1.0 software to impute untyped SNPs using the LD information from the HapMap phase II database (CHB+JPT as a reference set (2007-08_rel22, released 2007-03-02). Imputed SNPs with high genotype information content (Rsq > 0.3 for MACH) were kept for the further association analysis.

DHS (Diabetes Heart Study):
The Diabetes Heart Study (DHS) is an ongoing family-based cohort study investigating the epidemiology and genetics of cardiovascular disease (CVD) in a populationbased sample. The DHS recruited T2D-affected siblings without advanced renal insufficiency from 1998 through 2005 in western North Carolina. DHS has collected genetic data on 1,220 self-described European American (EA) individuals from 475 families. Genotyping was completed using an Affymetrix Genome-Wide Human SNP Array 5.0 with imputation of 1,000 Genomes project SNPs from this array using IMPUTE2 and the Phase I v2, cosmopolitan (integrated) reference panel, build 37.

DR's EXTRA (Dose-Responses to Exercise Training):
The Dose-Responses to Exercise Training (DR"s EXTRA) Study is a 4-year RCT on the effects of regular physical exercise and healthy diet on endothelial function, atherosclerosis and cognition in a randomly selected population sample (n=3,000) of Eastern Finnish men and women, identified from the national population register, aged 55-74 years. Of the eligible sample, 1,410 individuals were randomized into one of the 6 groups: aerobic exercise, resistance exercise, diet, combined aerobic exercise and diet, combined resistance exercise and diet, or reference group following baseline assessments. During the four year intervention the drop-out rate was 15%.

EGCUT (Estonian Genome Center -University of Tartu (Estonian Biobank)): The Estonian
Biobank is the population-based biobank of the Estonian Genome Center at the University of Tartu (www.biobank.ee; EGCUT). The entire project is conducted according to the Estonian Gene Research Act and all of the participants have signed the broad informed consent. The cohort size is up to 51,535 individuals from 18 years of age and up, which closely reflects the age, sex and geographical distribution of the Estonian population. All of the subjects are recruited randomly by general practitioners and physicians in hospitals. A Computer Assisted Personal interview is filled within 1-2 hours at a doctor"s office, which includes personal, genealogical, educational, occupational history and lifestyle data. Anthropometric measurements, blood pressure and resting heart rate are measured and venous blood taken during the visit. Medical history and current health status is recorded according to ICD-10 codes.

EPIC (European Prospective Investigation into Cancer and Nutrition)-Norfolk: The European
Prospective Investigation of Cancer (EPIC) began as a large multi-centre cohort study primarily looking at the connection between diet, lifestyle factors and cancer, although the study was broadened from the outset to include other conditions. The EPIC-Norfolk participants are men and women who were aged between 40 and 79 when they joined the study and who lived in Norwich and the surrounding towns and rural areas. They have been contributing information about their diet, lifestyle and health through questionnaires and health checks over two decades. The Norwich Local Research Ethics Committee granted ethical approval for the study. All participants gave written informed consent.

FUSION (Finland-United States Investigation of NIDDM Genetics): The Finland-United States
Investigation of NIDDM Genetics (FUSION) study is a long-term effort to identify genetic variants that predispose to type 2 diabetes (T2D) or that impact the variability of T2D-related quantitative traits.

GeneSTAR (Genetic Studies of Atherosclerosis Risk):
GeneSTAR is a family-based prospective study of more than 4000 participants begun in 1983 to determine phenotypic and genetic causes of premature cardiovascular disease. Families were identified from 1983-2006 from probands with a premature coronary disease event prior to 60 years of age who were identified at the time of hospitalization in any of 10 hospitals in the Baltimore, Maryland area. Their apparently healthy 30-59 year old siblings without known coronary disease were recruited and screened between 1983 and 2006. From 2003-2006, adult offspring over 21 years of age of all participating siblings and probands, as well as the coparents of the offspring were recruited and screened. Genotyping was performed in 3,232 participants on the Illumina 1Mv1_c platform.

GLACIER (Gene x Lifestyle Interactions and Complex Traits Involved in Elevated Disease Risk):
The Gene-Lifestyle interactions And Complex traits Involved in Elevated disease Risk (GLACIER) Study 23 is nested within the Västerbotten Intervention Programme, which is part of the Northern Sweden Health and Disease Study, a population-based prospective cohort study from northern Sweden. Participants were genotyped with Illumina CardioMetaboChip array. This array contains ~200,000 variants, the majority being common variants. Analysis of serum lipids (HDL-C, triglycerides and total cholesterol) were undertaken at the Department of Clinical Chemistry at Umeå University Hospital using routine methods. LDL-C was determined using the Friedewald formula. All participants completed a detailed, optically readable, health and lifestyle questionnaire including questions about smoking status and alcohol intake (FFQ).

GRAPHIC (Genetic Regulation of Arterial Pressure of Humans in the Community):
The GRAPHIC Study comprises 2024 individuals from 520 nuclear families recruited from the general population in Leicestershire, UK between 2003-2005 for the purpose of investigating the genetic determinants of blood pressure and related cardiovascular traits. A detailed medical history was obtained from study subjects by standardized questionnaires and clinical examination was performed by research nurses following standard procedures. Measurements obtained included height, weight, waist-hip ratio, clinic and ambulatory blood pressure and a 12-lead ECG. Respondents were removed if they had missing genotype or phenotype data.

HyperGEN-AXIOM (Hypertension Genetic Epidemiology Network):
HyperGEN is a family-based study that investigates the genetic causes of hypertension and related conditions in EA and AA subjects. HyperGEN recruited hypertensive sibships, along with their normotensive adult offspring, and an age-matched random sample. HyperGEN has collected data on 2,471 Caucasian-American subjects and 2,300 African-American subjects, from five field centers in Alabama, Massachusetts, Minnesota, North Carolina, and Utah. HyperGEN participates as a discovery study using GWAS available in a large subset of the samples. The remaining AA subjects without GWAS data were genotyped on the Affymetrix Axiom chip as part of a HyperGEN admixture mapping ancillary study. After excluding subjects already included in the original HyperGEN (or with family members included), this subset of approximately 450 AA subjects are included in the HyperGEN-AXIOM study which participates in replications.

INGI-CARL and INGI-FVG (Italian Network Genetic Isolates): INGI-FVG and INGI-CARL studies include samples coming from isolated populations and belong to the ITALIAN NETWORK OF GENETIC ISOLATES (INGI). INGI-CARL examined about 1000 subjects between 1998 and 2005
coming from a small village of the South of Italy situated in the extreme northern part of Puglia Region, while INGI FVG involved about 1700 subjects between 2008 and 2011 coming from six different villages located in the North-East of Italy in Friuli Venezia Giulia region. A questionnaire was administered to each participant to obtain socio-demographic information, as well as data on professional activity, family history, eating habits and lifestyle, such as smoking, coffee and alcohol consumption, physical activity. Furthermore, a medical screening, including anamnesis, blood pressure, drugs and clinical chemistry evaluation (blood count and different biochemical parameters, such as lipids) were made. All participants gave their written informed consent.

IRAS Family Study (Insulin Resistance Atherosclerosis Study):
The IRASFS was a family study designed to examine the genetic and epidemiologic basis of glucose homeostasis traits and abdominal adiposity. Briefly, self-reported Mexican pedigrees were recruited in San Antonio, TX and San Luis Valley, CO. Probands with large families were recruited from the initial non-family-based IRAS, which was modestly enriched for impaired glucose tolerance and T2D. Inclusion of IRASFS data is limited to 1040 normoglycemic individuals in 88 pedigrees with genotype data from the Illumina OmniExpress and Omni 1S arrays and imputation to the 1000 Genome Integrated Reference Panel (phase I).

JUPITER (Justification for the Use of Statins in Primary Prevention: An Intervention Trial
Evaluating Rosuvastatin): Genetic analysis was performed in a sub-population from JUPITER (Justification for the Use of statins in Prevention: an Intervention Trial Evaluating Rosuvastatin), an international, randomized, placebo-controlled trial of rosuvastatin (20mg/day) in the primary prevention of cardiovascular disease conducted among apparently healthy men and women with LDL-C < 130 mg/dL and hsCRP ≥ 2 mg/L 26,27 . Individuals with diabetes or triglyceride concentration >500mg/dL were excluded. The present analysis includes only individuals who provided consent for genetic analysis, had successfully collected genotype information, and who had either verified European or verified South African black ancestry. Lifelines (Netherlands Biobank): Lifelines (https://lifelines.nl/) is a multi-disciplinary prospective population-based cohort study using a unique three-generation design to examine the health and healthrelated behaviors of 165,000 persons living in the North East region of The Netherlands 29 . It employs a broad range of investigative procedures in assessing the biomedical, socio-demographic, behavioral, physical and psychological factors which contribute to the health and disease of the general population, with a special focus on multimorbidity. In addition, the Lifelines project comprises a number of crosssectional sub-studies which investigate specific age-related conditions. These include investigations into metabolic and hormonal diseases, including obesity, cardiovascular and renal diseases, pulmonary diseases and allergy, cognitive function and depression, and musculoskeletal conditions. All survey participants are between 18 and 90 years old at the time of enrollment. Recruitment has been going on since the end of 2006, and over 130,000 participants had been included by April 2013. At the baseline examination, the participants in the study were asked to fill in a questionnaire (on paper or online) before the first visit. During the first and second visit, the first or second part of the questionnaire, respectively, are checked for completeness, a number of investigations are conducted, and blood and urine samples are taken. Lifelines is a facility that is open for all researchers. Information on application and data access procedure is summarized on www.lifelines.nl.

METSIM (Metabolic Syndrome In Men):
The METSIM Study includes 10,197 men, aged from 45 to 73 years at recruitment, randomly selected from the population register of the Kuopio town, Eastern Finland, and examined in 2005-2010 30 . The aim of the study is to investigate genetic and non-genetic factors associated with type 2 diabetes and cardiovascular disease and its risk factors.

NESDA (Netherlands Study of Depression and Anxiety):
NESDA is a multi-center study designed to examine the long-term course and consequences of depressive and anxiety disorders (http://www.nesda.nl) 31 . NESDA included both individuals with depressive and/or anxiety disorders and controls without psychiatric conditions. Inclusion criteria were age 18-65 years and self-reported western European ancestry while exclusion criteria were not being fluent in Dutch and having a primary diagnosis of another psychiatric condition (psychotic disorder, obsessive compulsive disorder, bipolar disorder, or severe substance use disorder).

PREVEND(The Prevention of REnal and Vascular ENd stage Disease study):
The PREVEND study is an ongoing prospective study investigating the natural course of increased levels of urinary albumin excretion and its relation to renal and cardiovascular disease. Inhabitants 28 to 75 years of age (n=85,421) in the city of Groningen, The Netherlands, were asked to complete a short questionnaire, 47% responded, and individuals were then selected with a urinary albumin concentration of at least 10 mg/L (n = 7,768) and a randomly selected control group with a urinary albumin concentration less than 10 mg/L (n = 3,395). Details of the protocol have been described elsewhere 32 .

RHS (Ragama Health Study):
The Ragama Health Study (RHS) is a population-based study of South Asian men and women aged 35-64yrs living in the Ragama Medical Officer of Health (MOH) area, near Colombo, Sri Lanka. 33 Consenting adults attended a clinic after a 12-h fast with available health records, and were interviewed by trained personnel to obtain information on medical, sociodemographic, and lifestyle variables. A 10-mL sample of venous blood was obtained from each subject. The concurrent study was performed in two tea plantation estates in the Lindula MOH area, near Nuwara Eliya (180 km from Colombo), to investigate the gene-environment interaction in a community with differing lifestyles (e.g., physical activity and diet). The RHS is a collaborative effort between the Faculty of Medicine, University of Kelaniya and the National Center for Global Health and Medicine, Japan.

SHEEP (Stockholm Heart Epidemiology Project):
The SHEEP is a population based case-control study of risk factors for first episode of acute myocardial infarction. The study base comprised all Swedish citizens resident in the Stockholm county 1992-1994 who were 45-70 years of age and were free of previous clinically diagnosed myocardial infarction.
Cases were identified using three different sources: 1) coronary units and internal medicine wards for acute care in all Stockholm hospitals; 2) the National Patient Register; and 3) death certificates. For the present study, only cases who survived at least 28 days were considered (n=1213).
First time incident myocardial infarction cases (n=1213) were identified during a 2-year period (1992)(1993) for men and during a 3-year period (1992-1994) for women. Controls (n=1561) were randomly recruited from the study population continuously over time within 2 days of the case occurrence and matched to cases on age (5-years interval), sex and hospital catchment area using computerized registers of the population of Stockholm. Five control candidates were sampled simultaneously to be able to replace potential non-respondent controls. Occasionally, because of late response of the initial control, both the first and alternative controls were considered resulting in the inclusion of more controls than cases. Postal questionnaires covering a wide range of exposure areas including occupational exposures, life style factors, social factors and health related factors were distributed to the participants. Clinical investigations were performed at least three months after myocardial infarction of cases and their matched controls. The investigations included blood samplings under fasting conditions with collection of whole blood for DNA extraction, serum and plasma. A biobank was established containing DNA, serum and plasma.
Exposure information based on both the questionnaire and biological data from the health examination was available for 78% of the male and 67% of the female non-fatal cases; the corresponding figures for their controls were 68% and 64%.

SWHS/SMHS (Shanghai Women's Health Study/ Shanghai Men's Health Study): The Shanghai
Women's Health Study (SWHS) is an ongoing population-based cohort study of approximately 75,000 women who were aged 40-70 years at study enrollment and resided in in urban Shanghai, China; 56,832 (75.8%) provided a blood samples. Recruitment for the SWHS was initiated in 1997 and completed in 2000. The self-administered questionnaire includes information on demographic characteristics, disease and surgery histories, personal habits (such as cigarette smoking, alcohol consumption, tea drinking, and ginseng use), menstrual history, residential history, occupational history, and family history of cancer. Included in the current project were all women who had GWAS data and lipid measurements at the baseline interview.
The Shanghai Men"s Health Study (SMHS) is an ongoing population-based cohort study of 61,480 Chinese men who were aged between 40 and 74 years, were free of cancer at enrollment, and lived in urban Shanghai, China; 45,766 (74.4%) provided a blood samples. Recruitment for the SMHS was initiated in 2002 and completed in 2006. The self-administered questionnaire includes information on demographic characteristics, disease and surgery histories, personal habits (such as cigarette smoking, alcohol consumption, tea drinking, and ginseng use), residential history, occupational history, and family history of cancer. Included in the current project were 298 men who had GWAS data and lipid measurements at the baseline interview.
Genotyping and imputation: Genomic DNA was extracted from buffy coats by using a Qiagen DNA purification kit (Valencia, CA) or Puregene DNA purification kit (Minneapolis, MN) according to the manufacturers" instructions and then used for genotyping assays. The GWAS genotyping was performed using the Affymetrix Genome-Wide Human SNP Array 6.0 (Affy6.0) platform or Illumina 660, following manufacturers" protocols. After sample quality control, we exclude SNPs with 1) MAF <0.01; 2) call rate <95%; 2) bad genotyping cluster; and 3) concordance rate <95% among duplicated QC samples. Genotypes were imputed using the program MACH (http://www.sph.umich.edu/csg/abecasis/MACH/download/), which determines the probable distribution of missing genotypes conditional on a set of known haplotypes, while simultaneously estimating the fine-scale recombination map. Phased autosome SNP data from HapMap Phase II Asians (release 22) were used as the reference. To test for associations between the imputed SNP data with BMI, linear regression (additive model) was used, in which SNPs were represented by the expected allele count, an approach that takes into account the degree of uncertainty of genotype imputation (http://www.sph.umich.edu/csg/abecasis/MACH/download/).
The lipid profiles were measured at Vanderbilt Lipid Laboratory. Total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides (TG) were measured using an ACE Clinical Chemistry System (Alfa Wassermann, Inc, West Caldwell, NJ). Low-density lipoprotein (LDL) cholesterol levels were calculated by using the Friedewald equation. The levels of LDL cholesterol were directly measured using an ACE Clinical Chemistry System for subjects with TG levels ≥ 400 mg/dL. Fasting status was defined as an interval between the last meal and blood draw of 8 hours or longer.

TRAILS (Tracking Adolescents' Individual Lives Survey):
TRAILS is a prospective cohort study of Dutch adolescents and young adults, with bi-or triennial measurements from age 11 onwards, which started in 2001. TRAILS consists of a general population and a clinical cohort (https://www.trails.nl/en/home). In the population cohort, six assessment waves have been completed to date, at mean ages 11.1 (SD = 0.6), 13.6 (SD = 0.5), 16 Invitations to the study contained information of the study and its purpose. Along with the invitations consent forms and health questionnaire were sent to the subjects. When the signed consent forms where returned, the subjects were sent blood sampling equipment and asked to contact a local health facility for blood sampling. The study population was recruited among twins participating in the Screening Across the Lifespan Twin Study (SALT) which was a telephone interview study conducted in 1998-2002. Other inclusion criteria were that both twins in the pair had to be alive and living in Sweden. Subjects were excluded from the study if they preciously declined participation in future studies or if they had been enrolled in other STR DNA sampling projects. The subjects were asked to make an appointment for a health check-up at their local health-care facility on the morning Monday to Thursday and not the day before a national holiday, this to ensure that the sample would reach the KI biobank the following morning by overnight mail. The subjects were instructed to fast from 20.00 the previous night. By venipuncture a total of 50 ml of blood was drawn from each subject. Tubes with serum and blood for biobanking as well as for clinical chemistry tests were sent to KI by overnight mail. One 7ml EDTA tube of whole blood is stored in -80°C while a second 7ml EDTA tube of blood is used for DNA extraction using Puregene extraction kit (Gentra systems, Minneapolis, USA). After excluding subjects in which the DNA concentration in the stock-solution was below 20ng/µl as well as subset of 302 female monozygous twin pairs participating in a previous genome wide effort DNA from 9896 individual subjects was sent to SNP&SEQ Technology Platform Uppsala, Sweden for genome wide genotyping with Illumina OmniExpress bead chip (all available dizygous twins + one twin from each available MZ twin pair).

YFS (The Cardiovascular Risk in Young Finns Study):
The YFS is a population-based follow upstudy started in 1980. The main aim of the YFS is to determine the contribution made by childhood lifestyle, biological and psychological measures to the risk of cardiovascular diseases in adulthood. In 1980, over 3,500 children and adolescents all around Finland participated in the baseline study. The follow-up studies have been conducted mainly with 3-year intervals. The latest 30-year follow-up study was conducted in 2010-11 (ages 33-49 years) with 2,063 participants. The study was approved by the local ethics committees (University Hospitals of Helsinki, Turku, Tampere, Kuopio and Oulu) and was conducted following the guidelines of the Declaration of Helsinki. All participants gave their written informed consent.

STAGE 1 STUDY ACKNOWLEDGMENTS:
Infrastructure for the CHARGE Consortium is supported in part by the National Heart, Lung, and Blood Institute grant R01HL105756. Infrastructure for the Gene-Lifestyle Working Group is supported by the National Heart, Lung, and Blood Institute grant R01HL118305. Tuomas O. Kilpeläinen was supported in part by the Danish Council for Independent Research (DFF-1333-00124 and DFF-1331-00730B) and the Novo Nordisk Foundation (NNF18CC0034900, NNF17OC0026848 and NNF15CC0018486).

AGES (Age Gene/Environment Susceptibility Reykjavik Study):
This study has been funded by NIH contract N01-AG012100, the NIA Intramural Research Program, an Intramural Research Program Award (ZIAEY000401) from the National Eye Institute, an award from the National Institute on Deafness and Other Communication Disorders (NIDCD) Division of Scientific Programs (IAA Y2-DC_1004-02), Hjartavernd (the Icelandic Heart Association), and the Althingi (the Icelandic Parliament). The study is approved by the Icelandic National Bioethics Committee, VSN: 00-063. The researchers are indebted to the participants for their willingness to participate in the study.

ARIC (Atherosclerosis Risk in Communities) Study:
The ARIC study is carried out as a collaborative study supported by National Heart, Lung, and Blood Institute 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. The authors thank the staff and participants of the ARIC study for their important contributions. Research (NWO-RFBR 047.017.043). ERF was further supported by the ZonMw grant (project 91111025). We are grateful to all study participants and their relatives, general practitioners and neurologists for their contributions and to P. Veraart for her help in genealogy, J. Vergeer for the supervision of the laboratory work, P. Snijders for his help in data collection and E.M. van Leeuwen for genetic imputation.

FamHS (Family Heart Study):
The FamHS is funded by R01HL118305 and R01HL117078 NHLBI grants, and 5R01DK07568102 and 5R01DK089256 NIDDK grant.

FHS (Framingham Heart Study):
This research was conducted in part using data and resources from the Framingham Heart Study of the National Heart Lung and Blood Institute of the National Institutes of Health and Boston University School of Medicine. The analyses reflect intellectual input and resource development from the Framingham Heart Study investigators participating in the SNP Health Association Resource (SHARe) project. This work was partially supported by the National Heart, Lung and Blood Institute's Framingham Heart Study (Contract Nos. N01-HC-25195 and HHSN268201500001I) and its contract with Affymetrix, Inc for genotyping services (Contract No. Examination Services (Alençon, Angers, Blois, Caen, Chartres, Chateauroux, Cholet, LeMans, Orléans and Tours), Research Institute for General Medicine (J. Cogneau), the general practitioners of the region and the Cross-Regional Institute for Health (C. Born, E. Caces, M. Cailleau, N. Copin, J.G. Moreau, F. Rakotozafy, J. Tichet, S. Vol).

IRAS Family Study (Insulin Resistance Atherosclerosis Study):
The IRASFS is supported by the National Heart Lung and Blood Institute (HL060944, HL061019, and HL060919). Genotyping for this study was supported by the GUARDIAN Consortium with grant support from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK; DK085175) and in part by UL1TR000124 (CTSI) and DK063491 (DRC). The authors thank study investigators, staff, and participants for their valuable contributions.

JUPITER (Justification for the Use of Statins in Primary Prevention: An Intervention Trial
Evaluating Rosuvastatin): Support for genotype data collection and collaborative genetic analysis in JUPITER was provided by Astra-Zeneca.

KORA (Cooperative Health Research in the Augsburg Region):
The KORA study was initiated and financed by the Helmholtz Zentrum München -German Research Center for Environmental Health, which is funded by the German Federal Ministry of Education and Research (BMBF) and by the State of Bavaria. Furthermore, KORA research was supported within the Munich Center of Health Sciences (MC-Health), Ludwig-Maximilians-Universität, as part of LMUinnovativ.