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Although over 30 common genetic susceptibility loci have been identified to be independently associated with coronary artery disease (CAD) risk through genome-wide association studies (GWAS)
genetic risk variants reported to date explain only a small fraction of heritability
To identify novel susceptibility variants for CAD and confirm those previously identified in European population
GWAS and a replication study were performed in the Koreans and Japanese
we genotyped 2123 cases and 3591 controls with 521 786 SNPs using the Affymetrix SNP Array 6.0 chips in Korean
direct genotyping was performed using 3052 cases and 4976 controls from the KItaNagoya Genome study of Japan with 14 selected SNPs
imputation was performed based on 1000 Genome JPT+CHB and 5.1 million SNPs were retained
CAD association was replicated for three GWAS-identified loci (1p13.3/SORT1 (rs599839)
SNP rs3782889 showed a strong association (combined P=3.95 × 10−14)
although the association of SNP rs3782889 doesn’t remain statistically significant after adjusting for SNP rs11066015 (proxy SNP with BRAP (r2=1))
But new possible CAD-associated variant was observed for rs9508025 (FLT1)
even though its statistical significance did marginally reach at the genome-wide a significance level (combined P=6.07 × 10−7)
This study shows that three CAD susceptibility loci
which were previously identified in European can be directly replicated in Koreans and also provides additional evidences implicating suggestive loci as risk variants for CAD in East Asian
we conducted GWAS and a replication study to identify common CAD susceptibility loci and validate the previously reported loci using Affymetrix Genome-Wide Human SNP array 6.0 with 2293 CAD patients from Genomics Research in Cardiovascular Disease (GenRIC) and 4302 healthy controls from a large urban cohort
the Korea Genome Epidemiology Study (KoGES) as Stage I
replication was performed using 3052 cases and 4976 controls from the KItaNagoya Genome (King) study of Japan
we found evidence for genetic variants that may be associated with CAD risk
The study protocol was approved by the institutional review boards at Korea National Institute of Health and at each collaborating institute
Informed consent was obtained from all participants
a GWA scan (GWAS) was conducted with 2293 CAD patients from GenRIC working groups consisting with three teaching hospitals (Samsung Medical Center
Seoul National University Hospital and Yonsei University College of Medicine) in Korea
CAD was confirmed by standard coronary angiography
stable angina (41%) and unstable angina (28%) were classified as CAD subjects
The diagnosis of myocardial infarction was based on typical chest pain with a duration >30 min
on characteristic electrocardiographic patterns of acute myocardial infarction
and on elevated creatine kinase-MB and troponin I levels
Subjects with familial hypercholesterolemia
end-stage renal disease and congenital heart disease were excluded
Genetic variants in samples from 2293 cases and 4302 controls were genotyped using the Affymetrix Genome-Wide Human SNP array 6.0. The BirdSeed (http://www.broadinstitute.org/mpg/birdsuite/birdseed.html) genotyping algorithm was used for genotype calling. The PLINK program (Ver. 1.06) and R statistics (http://www.r-project.org/) (Ver
2.10.1) were used for quality control procedures
Samples with any of the following were removed: (i) gender inconsistencies (n=23 in cases
(iii) outliers in a heterozygosity plot (n=41 in cases
(iv) cryptic first-degree relative>0.8 (n=9 in cases
or (v) outliers in a multidimensional scaling plot (n=3 in cases
Samples with a history of cancer and CAD were also excluded (in controls
duplications and cryptic first-degree relative sibling pairs
genome-wide average IBS values were calculated for each pair of individuals in the present GWA case−control data set using pruned SNPs (51 195 SNPs in 2123 cases and 74 965 SNPs in 3703 controls)
individuals who shared a too high degree of IBS were excluded
To evaluate differences in population structure
an multidimensional scaling calculation was performed using pruned SNPs
SNPs were filtered if: (1) the call rate was <95% (n=92 682 in cases
(2) the minor allele frequency (MAF) was <1% (n=83 902 in cases
(3) the difference between case and control was missing (P<5 × 10−5) (41 142 SNPs in cases and controls)
(4) differential genotype calling rate between the cases and controls (case missing rate >1% or control missing rate >1%
and (5) significant deviation from Hardy-Weinberg equilibrium (HWE P⩽1 × 10−6) were also filtered
521 786 autosomal SNPs in 2123 cases and in 3591 controls remained for association analysis
For replication, fourteen significant candidate loci in GWAS scan were examined for replication using 3052 cases and 4976 controls using TaqMan SNP genotyping assay (Supplementary Figure S1)
The minimum genotyping success rate in replication study was 0.993 and Hardy–Weinberg equilibrium test P-value showed >0.05 in control group
suggesting no fault in genotyping procedure
We selected the SNPs if they were implicated in previous GWAS studies and reported from the National Human Genome Research Institute catalog (http://www.genome.gov/gwastudies)
We included SNPs only if they have assigned reference allele
and estimated OR or β-coefficient and 95% confidence interval (CI)
All SNPs successfully imputed were used (imputation QC R2<0.3)
Regional plots of the –log P-values for three replicated loci
(a–c) Results (–log P) are shown for the association of directly genotyped and imputed SNPs for a 1-Mb region centered on SNP reported in previous genome-wide association studies (GWAS) (diamond)
Genotyped SNPs are represented as square and imputed SNPs are marked by circle
SNPs for replication were selected on the basis of the following criteria among the 521,786 SNPs directly genotyped that had passed QC procedure: SNPs (a) with an MAF of >5% (b) with very clear genotyping clusters (c) with P-value of <5 × 10−4
and (d) not in strong LD (R2<0.5) with any of the GWAS-identified risk variants
when multiple SNPs showed LD within 100kb (R2>0.2)
the SNP with the lowest P-value was selected for replication
SNPs with MAF of between 1% and 5% is ∼50 000 and
fifty SNPs (0.1%) have the P-value of <5 × 10−4
these SNPs didn’t meet other replication criteria
we did performed logistic regression by adjusting age and gender
The combined analysis was performed by fixed effects and weighted numbers of samples
and combined P-values and ORs were calculated using the Mantel-Haenszel test in RMETA in the R package
as we found CAD association of variant previously identified on 12q24 in the Japanese
we examined independent test at chromosomal position 12q24.11 using SNP (rs11066011) as a proxy of BRAP
Manhattan plots for genome-wide association studies (GWAS) of coronary artery disease (CAD) in the Koreans
The horizontal solid red line indicates the preset threshold of P=5 × 10−8
After SNP quality control and mapping of genomic positions to build37, a total of 5 125 961 SNPs for 5714 samples were retained as input genotype data for imputation. Among previously identified CAD loci, imputed SNPs for 9p21.3 and 12q24 loci were satisfied the P-value of GWAS significance (5 × 10−8) (Supplementary Table S7)
the associations of other SNPs were not significant in the study possibly owing to the limited power of current study
For the established risk locus within TCF21 (rs12190287)
we could not evaluate the same SNPs as reported because they were not genotyped or successfully imputed (imputation QC R2<0.3)
capturing TCF21 for rs12190287 (R2=0.24 in CHB+JTP; R2=0.35 in CEU) and rs9457925 in replacement for LPA rs3798220 (R2=0.31 in CHB+JPT; R2=none)
and rs2246833 in replacement for LIPA rs1412444 (R2=0.86 in CHB+JPT; R2=1.00 in CEU) on the basis of 1000 genomes pilot1 data
Even though the association of rs3782889 (in MYL2) was identified with Korean GWAS and directly replicated in the Japanese
it couldn’t escape the possibility that the potential presence of LD block around MYL2 is not independent of the one around BRAP previously identified in Japanese
The multi-ethnic studies will be required to contribute a better understanding of genetic architecture of CAD and pathogenesis
we have confirmed four SNPs in three loci of CAD that were initially identified in Europeans
provided additional evidence suggesting association in the risk of CAD susceptibility variants
Future studies including fine mapping study
functional assay and replication study with large sample sizes from diverse ethnic populations need to validate our results
Global and regional burden of disease and risk factors
2001: systematic analysis of population health data
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Genetic susceptibility to death from coronary heart disease in a study of twins
Thirty-five common variants for coronary artery disease: the fruits of much collaborative labour
Large-scale association analysis identifies 13 new susceptibility loci for coronary artery disease
Design of the Coronary ARtery DIsease Genome-Wide Replication And Meta-Analysis (CARDIoGRAM) Study: a genome-wide association meta-analysis involving more than 22 000 cases and 60 000 controls
Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls
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Coronary Artery Disease (C4D) Genetics Consortium
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SNPs in BRAP associated with risk of myocardial infarction in Asian populations
Genome-wide association identifies a susceptibility locus for coronary artery disease in the Chinese Han population
Genome-wide association study of coronary artery disease in the Japanese
Genetics of myocardial infarction: a progress report
Association of the 9p21.3 locus with risk of first-ever myocardial infarction in Pakistanis: case-control study in South Asia and updated meta-analysis of Europeans
Plasma resistin concentration determined by common variants in the resistin gene and associated with metabolic traits in an aged Japanese population
A large-scale genome-wide association study of Asian populations uncovers genetic factors influencing eight quantitative traits
Genome-wide association of early-onset myocardial infarction with single nucleotide polymorphisms and copy number variants
A genome-wide meta-analysis identifies 22 loci associated with eight hematological parameters in the HaemGen consortium
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clinical and population relevance of 95 loci for blood lipids
Genome-wide association study in Han Chinese identifies four new susceptibility loci for coronary artery disease
Association of SNP rs17465637 on chromosome 1q41 and rs599839 on 1p13.3 with myocardial infarction in an American caucasian population
Factors of risk in the development of coronary heart disease--six year follow-up experience
Correcting "winner's curse" in odds ratios from genomewide association findings for major complex human diseases
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We thank all participants and investigators of the GenRIC study and the Korea Genome Epidemiology Study (KoGES)
This work was supported by grants from the Korea Centers for Disease Control and Prevention (4845-301)
an intramural grant from the Korea National Institute of Health (2011-N73007-00
Dong-Jik Shin and Kyung Woo Park: These authors contributed equally to this work
Jeong Euy Park and Jong-Young Lee: These authors jointly directed this work
Sungkyunkwan University School of Medicine
Interdisciplinary Program in Bioinformatics
Division of Pulmonary and Critical Care Medicine
Osong Health Technology Administration complex
The authors declare no conflict of interest
Supplementary Information accompanies the paper on Journal of Human Genetics website
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