Low-frequency and rare exome chip variants associate with fasting glucose and type 2 diabetes susceptibility

Fasting glucose and insulin are intermediate traits for type 2 diabetes. Here we explore the role of coding variation on these traits by analysis of variants on the HumanExome BeadChip in 60,564 non-diabetic individuals and in 16,491 T2D cases and 81,877 controls. We identify a novel association of a low-frequency nonsynonymous SNV in GLP1R (A316T; rs10305492; MAF=1.4%) with lower FG (β=−0.09±0.01 mmol l−1, P=3.4 × 10−12), T2D risk (OR[95%CI]=0.86[0.76–0.96], P=0.010), early insulin secretion (β=−0.07±0.035 pmolinsulin mmolglucose−1, P=0.048), but higher 2-h glucose (β=0.16±0.05 mmol l−1, P=4.3 × 10−4). We identify a gene-based association with FG at G6PC2 (pSKAT=6.8 × 10−6) driven by four rare protein-coding SNVs (H177Y, Y207S, R283X and S324P). We identify rs651007 (MAF=20%) in the first intron of ABO at the putative promoter of an antisense lncRNA, associating with higher FG (β=0.02±0.004 mmol l−1, P=1.3 × 10−8). Our approach identifies novel coding variant associations and extends the allelic spectrum of variation underlying diabetes-related quantitative traits and T2D susceptibility.

(a) Regional associa�on results (--log 10 p) for fas�ng glucose of the ABO locus and within 500KB around the lead SNV (rs651007, purple dot); rs579459, rs653634 and rs507666 are also shown and are in strong linkage disequilibrium (LD) with rs651007 (r 2 =0.95--1). r 2 indicated by color scale legend. Triangle symbols indicate variants with MAF>5%, square symbols indicate variants with MAF1--5%, and circle symbols indicate variants with MAF <1%. (b) Inset of ABO gene with lead SNVs from FG analysis (labeled "FG associated") depic�ng low LD (r 2 ) with the major blood group variants (labeled "Blood Type") in European (CEU, top) and African (YRI, bo�om) individuals. Major blood group variants were not genotyped on the exome chip; therefore r 2 was calculated from the 1000 Genomes project (Phase 1, version 3). (c) An expressed sequence tag (EST)--supported an�sense transcript from islets overlaps ABO exon 1. RED arrow: Genomic span of the ABO gene. The panel highlighted (light green, below) is the UCSC Expressed Sequence Tags (EST) Track. BLUE arrow: Genomic span of the EST--supported an�sense transcript. BLUE Ellipse: ESTs suppor�ng an�sense transcrip�on. (d) Inset of the ABO upstream region, promoter,5' untranslated region (5'UTR) and part of intron 1. The intronic SNV rs507666 is near the transcrip�on start site of the expressed sequence tag (EST) CK821046 from a human islet cDNA library. Two other ESTs, also from human islets, support this an�sense non--coding transcript. This EST is an�sense to exon 1 of ABO, sugges�ng that rs507666 may func�on as a promoter SNV of a previously uncharacterized ABO an�sense non--coding RNA transcript in islet cells. The intergenic SNVs rs651007 and rs579459 reside in a DNAsel hypersensi�ve site cluster, overlapping an H3K27Ac peak and par�ally overlapping a transcrip�on factor binding site (TFBS) ChIP--seq peak upstream of the ABO promoter. The sequences encompassing these SNVs may, therefore, represent puta�ve ac�ve chroma�n regulatory elements whose func�on may be altered by these SNVs. The intergenic SNV rs635634 had less evidence for transcrip�onal or regulatory ac�vity. Purple Boxes: Two variants (rs507666, rs635634) were annotated as eQTLs for SLC2A6 (gene to the right of ABO in panel A) from GTEx analysis. Blue Box: The ENCODE H3K4Me1 (enhancer), H3K27Ac (ac�ve regulatory element), DNAse I hypersensi�ve sites, and TFBS ChIP--Seq tracks, all with signals overlapping the SNVs, are shown. The intronic SNV rs560887 was assessed as significant in 11 independent NHGRI--catalogued GWAS studies (green, top, A) of serum metabolites, pregnancy--associated glycemia, fas�ng glucose levels, atherosclerosis, and body mass index (h�p://genome.ucsc.edu/cgi--bin/hgc? hgsid=369635347_bV0VuuwNQelM7MRwqH5tEyMLIvjx&c=chr2&o=169763147&t=169763148& g=gwasCatalog&i=rs560887). The SNV resides at the 5'end of an ENCODE H3K4Me1 (puta�ve enhancer) signature, sugges�ng that it may have a regulatory role (Layered H3K4Me1, middle, A). There are several ENCODE transcrip�on factor binding sites evident in ChIP--seq data in the last exon of G6PC2 (bo�om, a), further sugges�ng that the region may have regulatory func�ons impac�ng the expression of G6PC2 or other genes. The intronic SNV rs560887 is also exonic with respect to the EST DB031634, a posi�ve--strand (same as G6PC2) transcript that may represent a cryp�c minor isoform of G6PC2 ini�a�ng from an internal promoter in the intron where this SNV resides (b). This SNV is 25 bases upstream of the intron's splice acceptor (b), sugges�ng that it may also func�on as a regulator of G6PC2 splicing. c.

Supplementary Table 17. Top ten pathways with lowest p-values in MAGENTA analysis of FG, analyzing all genes (A) and excluding those with known associations with FG (B)
A.

Database
Pathway

Age, Gene/Environment, Susceptibility-Reykjavik (AGES) Study
The AGES study has been described previously. The study was initiated in 2002 to examine genetic susceptibility and gene/environment interactions related to disease and disability in old age. The AGES study is comprised of 5,764 individuals drawn from the Reykjavik Study, a population-based cohort comprised of individuals born between 1907 and 1935 and followed since 1967 by the Icelandic Heart Association. 2983 individuals have ExomeChip genotypes 1 .

Atherosclerosis Risk in Communities (ARIC) Study
The ARIC Study is a prospective cohort study of cardiovascular disease risk in four US communities 2 . Between 1987 and 1989, 7,082 men and 8,710 women aged 45-64 years were recruited from Forsyth County, North Carolina; Jackson, Mississippi (African Americans only); suburban Minneapolis, Minnesota; and Washington County, Maryland. The ARIC Study protocol was approved by the institutional review board of each participating university. After written informed consent was obtained, including that for genetic studies, participants underwent a baseline clinical examination (Visit 1) and four subsequent follow-up exams (Visits 2 -5).

Coronary Artery Risk Development in young Adults (CARDIA)
The CARDIA study is a prospective, multi-center investigation of the natural history and etiology of cardiovascular disease in African Americans and whites 18-30 years of age at the time of initial examination. The initial examination included 5,115 participants selectively recruited to represent proportionate racial, gender, age, and education groups from four communities: Birmingham, AL; Chicago, IL; Minneapolis, MN; and Oakland, CA. Participants from the Birmingham, Chicago, and Minneapolis centers were recruited from the total community or from selected census tracts. Participants from the Oakland center were randomly recruited from the Kaiser-Permanente health plan membership. From the time of initiation of the study in 1985-1986, seven follow-up examinations have been conducted at years 2, 5, 7, 10, 15, 20, and 25.

The Chinese American Eye Study (CHES)
is a population-based study designed to assess the prevalence and risk factors of visual impairment, diabetic retinopathy, agerelated macular degeneration, lens opacities, glaucoma and myopia, and to determine potential unique genetic associations with ocular disease in 4,582 (79% participation) non-institutionalized Chinese Americans aged 50 years and older living in Monterey Park, in Los Angeles County. Household residence were determined eligible if they lived in Monterey Park, were 50 years or older, and self-reported Chinese Americans. Eligible participants were asked to complete an in-home questionnaire which included demographic and ocular history information. Participants were then invited to complete a comprehensive eye examination, including presenting and best-corrected visual acuity measurements, visual field, intraocular pressure, random glucose, Hba1c, lens grading, fundus photography and diabetes status.

Cardiovascular Health Study (CHS)
CHS is an NHLBI-funded observational study of risk factors for cardiovascular disease in adults 65 years or older 3 . Starting in 1989, and continuing through 1999, participants underwent annual extensive clinical examinations. Measurements included traditional risk factors such as blood pressure and lipids as well as measures of subclinical disease, including echocardiography of the heart, carotid ultrasound, and cranial magnetic-resonance imaging (MRI). At six month intervals between clinic visits, and once clinic visits ended, participants were contacted by phone to ascertain hospitalizations and health status. The main outcomes are coronary heart disease (CHD), angina, heart failure (HF), stroke, transient ischemic attack (TIA), claudication, and mortality. Participants continue to be followed for these events. Participants of either European or African-American Ancestry were included in this study and signed informed test for genetic testing.

CoLaus Study
The CoLaus study is a community-based study of 6188 European white subjects aged 35 ~75 years 4 . Participants were drawn from the CHUV University Hospital in Lausanne Switzerland and studied for cardiovascular and metabolic phenotypes.

CROATIA-Korcula (Korcula) Study
The CROATIA-Korcula study includes 969 Croatians between the ages of 18 and 98. The field work was performed in 2007 and 2008 in the eastern part of the island, targeting healthy volunteers from the town of Korcula and the villages of Lumbarda, Zrnovo and Racisce. Ethical approval was obtained from appropriate regulatory bodies in both Scotland and Croatia and participants gave informed consent prior to joining the study. After all quality control measures, 855 European individuals were successfully genotyped using the Illumina HumanExome BeadChip array 5 .

European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam Study
The European Prospective Investigation into Cancer and Nutrition (EPIC) -Potsdam recruited men aged 40 to 64 years and women aged 35 to 64 years from the general population in Potsdam and surrounding municipalities from 1994 -1998 6 and is part of the multi-center prospective cohort study EPIC 7 . The baseline assessment included a blood collection, self-administered questionnaires for assessment of lifestyle, sociodemographic factors and dietary habits, a personal interview on lifestyle habits and medical history as well as a physical examination to measure among others anthropometric characteristics 8 . A total number of 27,548 individuals participated in the study. From all participants that provided blood samples (n=26,444), a subcohort sample of 2,500 individuals was drawn randomly for measurement of genetic and biomarkers. Genotyping was additionally performed in 795 incident type 2 diabetes cases. Incident type 2 diabetes cases (N=849, mean follow-up time =7 years) were identified by an active follow up procedure 9 . Self-reports of diabetes diagnosis, diabetes-relevant medication, or dietary treatment due to diabetes were verified by questionnaires mailed to the diagnosing physician. Only subjects with a verified type 2 diabetes diagnosis were included in the analysis. The EPIC-Potsdam study was approved by the Ethics Committee of the Medical Association of the State of Brandenburg (Germany). All participants provided written, informed consent at study entry.

The Exeter Family Study of Childhood Health (EFSOCH)
The EFSOCH is a prospective study, set up to test the fetal insulin hypothesis, and to identify genetic polymorphisms that play a role in determining birthweight and early postnatal growth. We recruited 1,017 families from a postcode-defined area in central Exeter. Specific inclusion criteria were established to obtain a homogeneous, nondiabetic, UK Caucasian cohort. Detailed anthropometric measurements were taken from both parents at 28 weeks' gestation, and from their children at birth, 12 weeks, 1 year and 2 years of age. Insulin and other biochemical analysis were measured in fasting parental samples and an umbilical cord blood sample taken at delivery. Parental and offspring DNA were extracted to allow molecular genetic analysis of candidate genes implicated in fetal growth 10 .

The Erasmus Rucphen Family (ERF) Study
The ERF Study is a family-based cohort study in a genetically isolated population in the southwest of the Netherlands. Approximately 3,200 individuals, spanning an age range from 18 to 86 years old, participated in the study. The cross-sectional examinations took place between 2002 and 2005. The rationale and study design have been described elsewhere 11,12 .

Family Heart Study (FamHS)
The FamHS began in 1992 with the ascertainment of 1,200 European American families, half randomly sampled, and half selected because of an excess of coronary heart disease or risk factor abnormalities as compared with age-and sex-specific population rates 13 . The families, with approximately 6,000 individuals, were sampled on the basis of information on probands from four population-based parent studies: the Framingham Heart Study (Boston, MA), the Utah Family Tree Study (Salt Lake City, UT), and two Atherosclerosis Risk in Communities (ARIC) study centers (Minneapolis, MN, and Forsyth County, NC). Eight years later, study participants belonging to the largest pedigrees were invited for a second clinical exam (2002-04). Approximately 82% of the recruited subjects returned for the second visit and 275 newly eligible family members were also recruited. In addition, a sample of 633 family members including 153 T2D subjects from 215 African American families was recruited at an additional ARIC field center (Birmingham, AL).

FENLAND Study
The Fenland Study is an ongoing, population-based cohort study (started in 2005) designed to investigate the association between genetic and lifestyle environmental factors and the risk of obesity, insulin sensitivity, hyperglycemia and related metabolic traits in men and women aged 30 to 55 years. Potential volunteers were recruited from General Practice sampling frames in the Fenland, Ely and Cambridge areas of the Cambridgeshire Primary Care Trust in the UK. Exclusion criteria for the study were: prevalent diabetes, pregnant and lactating women, inability to participate due to terminal illness, psychotic illness, or inability to walk unaided. All participants had measurements done at the MRC Epidemiology Unit Clinical Research Facilities in Ely, Wisbech and Cambridge. Participants attended after an overnight fast for a detailed clinical examination, and blood samples were collected. The Local Research Ethics Committee granted ethical approval for the study and all participants gave written informed consent.

Framingham Heart Study (FHS)
The FHS is a three generational prospective cohort that has been described in detail previously 14

Gene-Lifestyle interactions And Complex traits Involved in Elevated disease Risk (GLACIER) Study
The Gene-Lifestyle interactions And Complex traits Involved in Elevated disease Risk (GLACIER) Study is nested within the Västerbotten Health Survey, which is part of the Northern Sweden Health and Disease Study, a population-based prospective cohort study from northern Sweden. A total of 1000 non-diabetic participants from the GLACIER Study were genotyped with Illumina HumanExome Beadchip 12 v1.1 15 .

Generation Scotland: Scottish Family Health Study (GS:SFHS)
The Generation Scotland: Scottish Family Health Study is a collaboration between the Scottish Universities and the NHS, funded by the Chief Scientist Office of the Scottish Government. GS:SFHS is a family-based genetic epidemiology cohort with DNA, other biological samples (serum, urine and cryopreserved whole blood) and sociodemographic and clinical data from ~24,000 volunteers, aged 18-98 years, in ~7,000 family groups. Participants were recruited across Scotland, with some family members from further afield, from 2006 -2011. Most (87%) participants were born in Scotland and 96% in the UK or Ireland. The cohort profile has been published. GS:SFHS operates under appropriate ethical approvals, and all participants gave written informed consent. After all quality control procedures had been carried out, 9955 participants of European ancestry were genotyped using the Illumina HumanExome BeadChip array 16 .

The Johns Hopkins Genetic Study of Atherosclerosis Risk (GeneSTAR)
GeneSTAR is a longitudinal family-based study examining determinants of incident coronary artery disease, stroke, and vascular disease among apparently healthy first degree adult relatives of probands who were hospitalized in Baltimore, Maryland, with documented coronary disease prior to 60 years of age. African American and white family members who were 21-80 years of age were enrolled between 1983 and 2007, and have been followed at regular five-year intervals.

GoMAP (Genetic Overlap between Metabolic and Psychiatric traits)
The GOMAP study includes 2871 unrelated Greek nationals of European ancestry 18-98 years of age recruited from five different hospitals in Athens Greece between 2012 and 2014. The study aims to explore shared inherited risk factors for metabolic and psychiatric disease. Genome-wide association scan (GWAS) was applied to biological samples from 422 individuals with type 2 diabetes (T2D). Clinical and haematological/biochemical measurements, interview-based lifestyle and sociodemographic information, detailed anthropometric measurements as well as information on treatment of T2D volunteers were collected. The cohort included in the present work comprises candidates with T2D regularly followed up in the outpatient diabetes centers (more than three outpatient visits on separate days) and receiving a prescriptions for insulin or oral antidiabetic agents. For newly diagnosed T2D patients ascertainment was conducted according to the WHO criteria. Ethical permission was obtained from the appropriate regulatory bodies and all volunteers gave written informed consent.

Health, Aging, and Body Composition (Health ABC) Study
Health ABC is a longitudinal, prospective study that tracks functional limitation onset and progression in a cohort of high-functioning older men and women recruited from Memphis, TN and Pittsburgh, PA using Medicare records. Recruitment began in 1997-1998 when participants were between 70 and 79 years of age and entry into study was dependent on participants' ability to walk one-quarter mile and climb 10 steps without difficulty. At baseline, the Health ABC cohort included 3,075 men and women, including 552 African-American men and 729 African-American women. The goal of the study is to understand how change in body composition and weight-related health conditions contributed to and affected incident functional limitation. Clinic examinations were conducted at baseline and at years 2, 3, 4, 5, 6, 8, 10, 11, and 16. Follow-up interviews by telephone have been conducted every six months since study initiation.

Health2008
Health2008 is a population-based epidemiological study of general health, diabetes and cardiovascular disease comprising 771 participants. An oral glucose tolerance test was performed with measurement of plasma glucose and serum insulin at fasting and 30 and 120 min after glucose intake. Health2008 was conducted at the Research Centre for Prevention and Health in Glostrup, Denmark. Informed written consent was obtained from all study participants. The studies were conducted in accordance with the Declaration of Helsinki II and were approved by the local Ethical Committee.

Hellenic Isolated Cohorts (HELIC) Study
The HELIC (Hellenic Isolated Cohorts) MANOLIS (Minoan Isolates) and Pomak collections focus on the Cretan Mylopotamos villages and the Pomak villages in Greece, respectively. Recruitment of these population-based samples was primarily carried out at the village medical centres. The study includes biological sample collection for DNA extraction and lab-based blood measurements, and interview-based questionnaire filling. The phenotypes collected include anthropometric and biometric measurements, clinical evaluation data, biochemical and haematological profiles, selfreported medical history, demographic, socioeconomic and lifestyle information. Biochemical measurements were obtained using enzymatic colorimetric assays and included glucose (hexokinase method, insulin and ferritin were measured via chemiluminescence and C-reaction protein (CRP) through an immunoturbidimetric method. The study was approved by the appropriate institutional review board (IRB) and appropriate informed consent was obtained from human subjects.

INCIPE Study
The INCIPE study (Initiative on Nephropathy, of relevance to public health, which is Chronic, possibly in its Initial stages, and carries a Potential risk of major clinical Endpoints) aims to explore the prevalence of CKD and related cardiometabolic traits in Italy 17 . Six thousand and two hundred (6,200) individuals, all Caucasians, ≥40-years old by January 1, 2006, were randomly chosen from the lists of patients of 62 randomly selected general practitioners (GPs) based in four geographical areas in the Veneto region, NE Italy. After exclusion of pregnant or lactating women, a written informed consent was obtained from a total of 3,870 subjects (62%). Each participant filled a selfadministered questionnaire on family and personal medical history, pharmacologic treatments, smoking habits etc. Participants attended after an overnight fast for a detailed clinical examination performed locally in four units by trained medical doctors and blood and urine samples were collected with a standardized protocol. All determinations were centralized (Hospital Trust of Verona, Central Laboratory, Verona, Italy). Patients were asked to refrain from smoking beginning from the night before. BP, waist circumference, body weight, and height were measured as in the NHANES study 18 . About 2,500 study participants underwent Illumina ExomeChip v.1.0 genotyping, 1,933 of them (1,749 non-T2D, 184 affected by T2D) had complete information to contribute to the present study.

Inter99
The Inter99 cohort is a randomized, non-pharmacological intervention study for the prevention of ischaemic heart disease, conducted on 6,784 randomly ascertained participants aged 30 to 60 years at the Research Centre for Prevention and Health in Glostrup, Denmark (ClinicalTrials.gov: NCT00289237). An oral glucose tolerance test was performed with measurement of plasma glucose and serum insulin at fasting and 30 and 120 min after glucose intake. Subsequently, 6,094 participants of Danish nationality and with available DNA were classified as having normal glucose tolerance (n=4,525), impaired fasting glycaemia (n=504), impaired glucose tolerance (n=693), screen-detected type 2 diabetes (n=253), or previously diagnosed type 2 diabetes (n=119) according to World Health Organization (WHO) 1999 criteria. Informed written consent was obtained from all study participants. The studies were conducted in accordance with the Declaration of Helsinki II and were approved by the local Ethical Committee.

InterAct Consortium
The InterAct study 19

Insulin Resistance Atherosclerosis Study (IRAS)
IRAS was an epidemiologic cohort study designed to examine the relationship between insulin resistance and carotid atherosclerosis across a range of glucose tolerance. Individuals of self-reported African-American ethnicity were recruited in Oakland, CA and Los Angeles, CA. Recruitment was balanced across age and glucose tolerance status. The overall cohort consisted of 464 African-American individuals.

The Insulin Resistance Atherosclerosis Study Family Study (IRASFS)
IRASFS was a family study designed to examine the genetic and epidemiologic basis of glucose homeostasis traits and abdominal adiposity. Self-reported Mexican-American 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 Study. The overall cohort consisted of 1,414 Mexican-American individuals from 90 families and 596 African-American individuals from 42 families.

Jackson Heart Study (JHS) Study Description
The Jackson Heart Study is a community-based observational study of African Americans in the Jackson, MS, metropolitan area. Beginning in 2000 5,301 participants (mean age 54.9 ± 12.9) were enrolled to explore the causes of the large burden of common complex diseases in this population. Participants have been evaluated during three clinic examinations (2000-2004, 2004-2008, and 2009-2012). ExomeChip genotyping has been performed for 2,790 participants 20 .

LOLIPOP Study
A population-based study of 21,915 subjects, primarily of Indian Asians and Northwestern Europeans aged 35-75 years, identified from the lists of 58 general practitioners in West London 21 . 549 European whites with exome sequencing data available from the GSK discovery sequence project were included in the current study.

Metabolic Syndrome GEMS Study
The GEMS study is a large multinational study designed to explore the genetic basis of the metabolic syndrome control 22 . Subjects were recruited from two centers in Europe (Oulu, Finland and Lausanne, Switzerland), one in the United States (Dallas, TX), one in Canada (Ottawa, Ontario), and one in Australia (Adelaide, South Australia). Dyslipidemic subjects were required to have the combination of an elevated plasma triglyceride (greater than 75th percentile) and a low serum HDL-cholesterol (less than 25th percentile) for their age, sex and country threshold (age 18-75 years) and were non-diabetic. Unrelated normolipidemic controls were required to have plasma triglyceride lower than 50th percentile, serum HDL-cholesterol greater than 50th percentile for their age, sex and country threshold, body mass index (BMI) greater than 25 kg/m2, and be greater than 40 years of age. Dyslipidemic subjects (n=787 subjects) and normolipidemic controls (n=792 subjects), matched by sex, age and collection center were sequenced in the GSK discovery sequence project and included in the current study.

METSIM (METabolic Syndrome In Men) Study
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 23 . Study protocol included interview on cardiovascular risk factors, measurement of height, weight, waist, hip, blood pressure, and bioimpedance for the evaluation of fat percentage. Laboratory studies include an oral glucose tolerance test to evaluate glucose tolerance (samples for glucose and insulin at 0, 30, and 120 minutes), as well as fasting laboratory measurements including lipids, lipoproteins, inflammatory markers, etc.

MRC Ely
The MRC Ely Study is a population-based cohort randomly selected from people living in Ely and surrounding villages (East Anglia, UK), an ethnically homogenous European ancestry population. The study design, methods and measurements of the three phases have been described in detail elsewhere. The current analyses included individuals aged 35-79 years, from phase 3. Ethical permission was granted by the Cambridgeshire Research Ethics Committee, and study participants provided written informed consent. Data from 1394 individuals were included in the current analyses.

Precocious Coronary Artery Disease (PROCARDIS) Study
The PROCARDIS (Precocious Coronary Artery Disease) Study recruited subjects with coronary artery disease at/before 65 years. Control subjects free of CAD before 65 years, were recruited from the same centers as cases. Ethics approval was granted by the each recruitment center (Sweden, UK, Germany, Italy). All participants completed a questionnaire including baseline characteristics, lifestyle, cardiovascular risk factors and medication. Standard biochemical phenotyping was performed 25 .

Relationship between Insulin Sensitivity and Cardiovascular disease (RISC) Study
The RISC (Relationship between Insulin Sensitivity and Cardiovascular disease) Study is being carried out in 19 European recruiting centers to examine whether insulin sensitivity (directly measured with the euglycemic clamp technique) predicts cardiovascular disease (CVD) independently of other factors. The study makes use of ultrasound scans of the carotid artery and takes the thickness of the intima-media layer in the artery wall (IMT) as an early marker of atherosclerosis 26 .

Rotterdam Study
The Rotterdam Study is an ongoing prospective population-based cohort study, focused on chronic disabling conditions of the elderly. The study comprises an outbred ethnically homogenous population of Dutch Caucasian origin. The rationale of the study has been described in detail elsewhere 27 . In summary, 7,983 men and women aged 55 years or older, living in Ommoord, a suburb of Rotterdam, the Netherlands, were invited to participate. A total of 2986 individuals from the initial study were included in the current study 27,28 .

Stockholm Coronary Artery Risk Factor (SCARF) Study
Subjects <60 years admitted to 3 coronary care units in Stockholm (Danderyd Hospital, Karolinska Hospital and Norrtälje Hospital) for first MI were included in this study (n=387). Healthy age, county and sex-matched control were also recruited for each case. Blood sampling was carried out 3 months after the cardiac event at a medical examination under fasting conditions. In addition, an interview of social-economic factors, lifestyle and medical history was completed. Ethics approval was granted by the ethics committee at Karolinska 29 .

Singapore Chinese Eye (SCES) Study
SCES is a population-based cross-sectional study of eye diseases in Chinese adults 40 years of age or older residing in the south-western part of Singapore. The study was designed to ascertain the prevalence and impact of major eye disease in Chinese persons in Singapore. An age-stratified (by 10-year age group) random sampling strategy was used to select participants from a computer-generated list provided by the Ministry of Home Affairs, Singapore. Between 2009 and 2011, 3,353 (72.8%) of 4,605 eligible individuals underwent a comprehensive physical and ophthalmologic examinations. Of these 2492 had ExomeChip data. The study adhered to the tenets of the Declaration of Helsinki, and ethical approval was obtained from the Institutional Review Board of the Singapore Eye Research Institute. All participants provided written informed consent.

Taiwan US Diabetic Retinopathy (TUDR) Study
Taiwan US Diabetic Retinopathy (TUDR) is a cohort that enrolled subjects with Type 2 diabetes receiving care at Taichung Veteran General Hospital (Taichung VGH), and a small number of subjects from Taipei Tri-Service General Hospital. All TUDR subjects underwent a complete ophthalmic and fundus examination to carefully document the presence and extent of retinopathy.

TEENAGE (TEENs of Attica: Genes and Environment)
The TEENAGE study comprises 857 adolescents of Greek origin randomly recruited from public secondary schools located in the wider Athens area of Attica in Greece 30 .

ULSAM
All men born between 1920 and 1924 in Uppsala, Sweden were invited to participate at age 50 in this longitudinal cohort study that was started in 1970. Participants were reinvestigated at the ages of 60, 70, 77, 82 and 88 years.1 Blood samples for DNA extraction and main cardiovascular risk factors were available from the investigation at age 70 (n=1,146 with DNA and data on CVD risk factors). The participants have undergone extensive phenotyping at repeated time points, including euglycemic clamps, oral glucose tolerance tests, DXA, echocardiography, 24-h ambulatory blood pressure measurement, and a range of biomarkers.

Val Borbera (INGI-VB) Study
The INGI--Val Borbera population is a collection of 1803 samples recruited in the Val Borbera Valley, a geographically isolated valley located within the Appennine Mountains