Genome-Wide Association Study Identifies a Novel Locus Contributing to Type 2 Diabetes Susceptibility in Sikhs of Punjabi Origin From India

We performed a genome-wide association study (GWAS) and a multistage meta-analysis of type 2 diabetes (T2D) in Punjabi Sikhs from India. Our discovery GWAS in 1,616 individuals (842 case subjects) was followed by in silico replication of the top 513 independent single nucleotide polymorphisms (SNPs) (P < 10−3) in Punjabi Sikhs (n = 2,819; 801 case subjects). We further replicated 66 SNPs (P < 10−4) through genotyping in a Punjabi Sikh sample (n = 2,894; 1,711 case subjects). On combined meta-analysis in Sikh populations (n = 7,329; 3,354 case subjects), we identified a novel locus in association with T2D at 13q12 represented by a directly genotyped intronic SNP (rs9552911, P = 1.82 × 10−8) in the SGCG gene. Next, we undertook in silico replication (stage 2b) of the top 513 signals (P < 10−3) in 29,157 non-Sikh South Asians (10,971 case subjects) and de novo genotyping of up to 31 top signals (P < 10−4) in 10,817 South Asians (5,157 case subjects) (stage 3b). In combined South Asian meta-analysis, we observed six suggestive associations (P < 10−5 to < 10−7), including SNPs at HMG1L1/CTCFL, PLXNA4, SCAP, and chr5p11. Further evaluation of 31 top SNPs in 33,707 East Asians (16,746 case subjects) (stage 3c) and 47,117 Europeans (8,130 case subjects) (stage 3d), and joint meta-analysis of 128,127 individuals (44,358 case subjects) from 27 multiethnic studies, did not reveal any additional loci nor was there any evidence of replication for the new variant. Our findings provide new evidence on the presence of a population-specific signal in relation to T2D, which may provide additional insights into T2D pathogenesis.

and 40 and were ≤45 years when they first joined the study. On the basis of clinical history (independence from insulin for at least 6 months after diagnosis), biochemistry and immunological testing (absence of anti-GAD antibodies) and exclusion for mitochondrial diabetes (mt3243 A>G) [11], 879 were considered to have clinical T2D and available for inclusion in this study. The mean (SD) age at diagnosis for this group was 37.3 (5.2) years and the mean BMI was 24.9 (3.9) kgm -2 . Genotyping was performed using Applied Biosystems TaqMan® SNP genotyping assays on an Applied Biosystems 7900HT system.

East Asian Replication Studies Bio Bank Japan
The BioBank Japan Project aims to gather basic information for personalized medicine. GWAS data are available on 4,878 individuals with T2D and 3,345 controls using Illumina HumanHap610-Quad and 550K BeadChip, respectively. We then selected 7,541 subjects belonging to the Hondo cluster (4,470 cases and 3,071 controls) for an association study with T2D. We performed genome-wide imputation by using IMPUTE (https://mathgen.stats.ox.ac.uk/impute/impute.html) with data from 89 HapMap samples (44 JPT and 45 CHB in HapMap phase 2) [12]. T2D cases were selected from individuals registered as having T2D. Control groups were individuals registered as not having T2D but affected by other illnesses, or healthy volunteers.

RIKEN T2D study
We selected another 5,339 T2D cases from the BioBank Japan or from subjects with T2D who visited outpatient clinics at one of 4 different institutions: St. Marianna University School of Medicine, Kawasaki Medical School, Toyama University Hospital; or the Shiga University of Medical Science. We also examined 2,141 controls enrolled during an annual health check-up at Keio University, St. Marianna University School of Medicine; or Toyama University Hospital. Diabetes was diagnosed according to World Health Organization (WHO) criteria. We excluded individuals who were positive for antibody to glutamic acid decarboxylase (GAD) or those with diabetes due to (1) liver dysfunction, (2) steroids and other drugs that might raise glucose levels, (3) malignancy, or (4) a monogenic disorder known to cause diabetes [13].

AGEN Consortium Korea Association Resource Study (KARE)
The KARE study was initiated in 2007 to undertake a large scale GWA analysis for T2D and numerous complex quantitative traits amongst the 10,038 participants (aged between 40 and 69) of the Ansung (N=5,018) and Ansan (N=5,020) population-based cohorts. About 10,000 subjects from KARE study cohorts were genotyped with Affymetrix Genome-Wide Human SNP array 5.0. Two KARE study cohorts were established as part of the Korean Genome Epidemiology Study (KoGES) in 2001. The sampling base for both cohorts is in KyungGi-Do province, close to Seoul, the capital of the Republic of Korea. Both cohorts were designed to allow longitudinal prospective study and adopted the same investigational strategy. Participants have been examined every two years since baseline (2001). More than 260 traits have been extensively examined through epidemiological surveys, physical examinations, and laboratory tests applied to Ansung and Ansan cohort members. Among a total of 10,038 KARE study participants, 1,042 subjects were included as T2D cases according to the following criteria: (1) treatment of T2D, (2) fasting plasma glucose ≥7 mmol/L or plasma glucose 2-h after ingestion of 75g oral glucose load ≥11.1 mmol/L and (3) age of disease onset ≥40 years. About 10,000 (1,042 T2D cases and 8,958 controls) participants from KARE study cohorts were genotyped with Affymetrix Genome-Wide Human SNP array 5.0.

Singapore Diabetes Cohort Study (SDCS)/Singapore Prospective Study Program (SP2)
T2D cases were selected from The Singapore Diabetes Cohort Study (SDCS). SDCS is a research initiative to identify genetic and environmental risk factors for diabetic complications. Since 2004, all T2D patients seen at participating polyclinics and hospitals were invited to be part of the cohort. Questionnaire data as well as clinical data of consenting patients were obtained together with bio-specimens such as blood and urine archived at -80°C. For the purpose of this study, 2,202 Chinese subjects were available for genome-wide analysis [14]. T2D controls were selected from The Singapore Prospective Study Program (SP2). From 2003-2007, participants were invited to participate by linking their unique national identification numbers with national registries, where 7,742 attended the interview and of these 7,742 participants, 5,163 attended the clinical examination. A total of 5,499 Chinese, 1,405 Malays and 1,138 Asian-Indians were available at the time of the study and only the Chinese were used for this study. Detailed population selection and methodology have been previously reported [14,15]. Controls were individuals with no prior history of diabetes and had a fasting glucose level of not more than 6.0 mmol/L.

Singapore Malay Eye Study (SiMES)
The Singapore Malay Eye Study (SiMES) is a population-based, cross-sectional study of Malay adults, aged 40 -80 years living in Singapore (N=3,280). Of the 4,168 eligible participants invited, 3,280 participated in the study with a 78.7% response rate. Briefly, age-stratified random sampling of all Malay adults aged from 40-80 years residing in 15 residential districts in the southwestern part of Singapore was performed. Details of the study participants and methods have been published previously [14,16]. For the SiMES study, 3,072 samples from the population based study were genotyped on the Illumina HumanHap 610Quad. Diabetic cases were defined as having either a history of diabetes or had HbA1c level ≥6.5%. Controls had no history of diabetes and HbA1c level <6%. This yielded 794 Malay diabetic cases with 1,240 controls.

Cardiometabolic Genome Epidemiology (CAGE) Network
In the case-control panel of the CAGE Network, T2D cases (N=1,110) were enrolled according to the 1999 WHO criteria, while unaffected controls (N=1,014) were enrolled according to the following criteria: (1) no past history of urinary glucose or glucose intolerance; (2) HbA1c, <5.6% or a normal glucose (75g) tolerance test; and (3) age at examination, ≥55 years. In addition, population-based subjects (740 cases and 4,889 controls from the CAGE-Fukuoka Study) and the Biobank Japan (http://biobankjp.org/) cases (N=3,403, BBJ samples independent of those used for the stage-2 analysis) were included in this case-control study. In the CAGE-Fukuoka Study (12,569 participants in total), diabetes was defined as HbA1c ≥7.0% or under treatment of diabetes; the controls were chosen as nondiabetic subjects who met the following conditions: age, ≥55 years; HbA1c, ≤5.0%; no previous and/or current treatment for diabetes; and absence of renal failure (serum creatinine, <3.0 mg/dL), as previously described [17].

Shanghai Diabetes Genetic Study (SDGS)
The SDGS includes genome wide scan data of 1,019 diabetes case and 1,710 controls. Details of the study design have been described elsewhere [18]. Briefly, diabetes cases in the SDGS included 886 incident T2D cases identified in the Shanghai Women's Health Study (SWHS) [19], a population-based cohort study of 75,000 women, and 133 prevalent T2D cases identified from female controls of the Shanghai Breast Cancer Study (SBCS), a population-based case control study [20]. The 886 diabetes cases identified from the SWHS all met the following criteria: (1) age ≤65 with a self-reported diabetes diagnosed after study enrollment; (2) used diabetes medication; (3) had fasting glucose level >7 mmol/L at least twice, and (4) donated a blood sample. The 133 diabetes cases identified from the controls of SBCS were women who were diagnosed with T2D and were on diabetes medication or had a blood glucose level >7 mmol/L (measured by study). The 1,710 controls used in this GWAS were shared with a GWAS of breast cancer that was recently completed and was based primarily on the Shanghai Breast Cancer Study (SBCS). Excluded from the control group are women who (1) had a self-reported history of diabetes; or (2) had a blood glucose level between 5.5 and 7mmol/L and had HbA1c >6.1% or had no HbA1C data.

Taiwan T2D study (TDS)
A total of 2,798 unrelated individuals with T2D, age >20 years, were recruited from China Medical University Hospital (CMUH), Taichung, Taiwan; Chia-Yi Christian Hospital (CYCH), Chia-Yi, Taiwan; and National Taiwan University Hospital (NTUH), Taipei, Taiwan. All of the T2D cases were diagnosed according to medical records and fasting plasma glucose levels using American Diabetic Association criteria. Subjects with type 1 diabetes, gestational diabetes, and maturity-onset diabetes of the young (MODY) were excluded from this study. The controls were randomly selected from the Taiwan Han Chinese Cell and Genome Bank. The criteria for controls in the association study were (1) no past diagnostic history of T2D, (2) HbA1c ranging from 3.4% to 6%, and (3) BMI ≤32 kg/m 2 . The two control groups were comparable with respect to BMI, gender, age at study, and level of HbA1c. All of the participating T2D cases and controls were of Han Chinese origin, which is the origin of 98% of the Taiwan population [21].

Cebu Longitudinal Health and Nutrition Survey (CLHNS)
The CLHNS is a community-based birth cohort study that originally enrolled 3,327 pregnant women from the Metropolitan Cebu, Philippines area in 1983-4 (3,080 singleton live births), and has since followed them and their offspring. For this study of CLHNS mothers, glucose levels were collected in the year 2005, and the study sample consists of 159 T2D cases and 1624 controls. T2D was defined as overnight fasting plasma glucose level ≥7.0 mmol/L or current use of anti-diabetic medication. Controls were defined as overnight fasting plasma glucose level <7.0 mmol/L and treatment naive. Whole blood glucose levels were converted to plasma glucose level by subtracting 0.97 mmol/L [22].

European Replication Studies DIAbetes Genetics Replication And Meta-analysis (DIAGRAM)
The DIAGRAM consortium is focused on performing large-scale studies to characterize the genetic basis of T2D, with a principal focus on samples of European descent. An incremental meta-analysis (DIAGRAM)+ is comprised of a total of 8,130 cases and 38,987 controls available with GWAS genotyping. T2D case-control status was defined using study-specific criteria. SNP associations were tested using an additive genetic model, and combined across studies by inverse variance meta-analysis using a fixed effects model [23].

Diabetes Genetics in Pakistan study
We would like to thank all the diabetic patients and control subjects for agreeing to participate in this study. We are grateful to Dr. Asher Fawwad, Dr. Waheed Iqbal and Dr. Abdul Razzaq for recruiting the study subjects and coordinating data collection. We are particularly grateful to Dr. Muhammad Zafar Iqbal Hydrie for overseeing the DGP collection and for the assistance of Prof. A. Samad Shera in setting up collaborative links between the UK and Pakistan. Funding for the collection of DNA and clinical data was provided by Diabetes UK (project number 07/0003512).

RIKEN/ Bio Bank Japan
For RIKEN T2D study, we thank Dr. Minoru Iwata at First Department of Internal Medicine, University of Toyama for providing DNA samples and clinical information. We also thank the technical staff of the Laboratory for Endocrinology and Metabolism at the RIKEN Center for Genomic Medicine for providing technical assistance. Japanese studies were supported by a grant from Ministry of Education, Culture, Sports, Science, and Technology, Japan.