Association of Genetic Variants Influencing Lipid Levels with Coronary Artery Disease in Japanese Individuals

Background/Objective In Japanese populations, we performed a replication study of genetic loci previously identified in European-descent populations as being associated with lipid levels and risk of coronary artery disease (CAD). Methods We genotyped 48 single nucleotide polymorphisms (SNPs) from 22 candidate loci that had previously been identified by genome-wide association (GWA) meta-analyses for low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and/or triglycerides in Europeans. We selected 22 loci with 2 parallel tracks from 95 reported loci: 16 significant loci (p<1×10−30 in Europeans) and 6 other loci including those with suggestive evidence of lipid associations in 1292 GWA-scanned Japanese samples. Genotyping was done in 4990 general population samples, and 1347 CAD cases and 1337 controls. For 9 SNPs, we further examined CAD associations in an additional panel of 3052 CAD cases and 6335 controls. Principal Findings Significant lipid associations (one-tailed p<0.05) were replicated for 18 of 22 loci in Japanese samples, with significant inter-ethnic heterogeneity at 4 loci–APOB, APOE-C1, CETP, and APOA5–and allelic heterogeneity. The strongest association was detected at APOE rs7412 for LDL-C (p = 1.3×10−41), CETP rs3764261 for HDL-C (p = 5.2×10−24), and APOA5 rs662799 for triglycerides (p = 5.8×10−54). CAD association was replicated and/or verified for 4 loci: SORT1 rs611917 (p = 1.7×10−8), APOA5 rs662799 (p = 0.0014), LDLR rs1433099 (p = 2.1×10−7), and APOE rs7412 (p = 6.1×10−13). Conclusions Our results confirm that most of the tested lipid loci are associated with lipid traits in the Japanese, further indicating that in genetic susceptibility to lipid levels and CAD, the related metabolic pathways are largely common across the populations, while causal variants at individual loci can be population-specific.


1-1. Japanese general population cohort sample: Amagasaki Study
To investigate lifestyle factors and genetic susceptibility to cardiovascular disease and risk factor traits, we enrolled individuals who sought medical assessment from September 2002 to August 2003 at the Amagasaki Health Medical Foundation, as described elsewhere [26]. The participants were included if they were over 18 years of age and had full clinical examination data, along with a completed questionnaire on their lifestyle. On the basis of these inclusion criteria, 5745 individuals (3435 men and 2310 women) were enrolled in this prospective cohort study, known as the Amagasaki Study. All participants provided written informed consent. Fasting blood samples were collected after ≥6 h fast during a visit to measure glucose and lipid concentrations using standard techniques. People who were receiving current treatment for dyslipidemia and those who did not meet the fasting condition (755 subjects in total) were excluded from the analysis (Table 1).

1-2. Genome-wide association (GWA) scanned sample
Our genetic studies of lipid traits were originally organized as part of the ongoing GWA study for cardiometabolic disorders among the Japanese. After excluding individuals with current treatment for dyslipidemia, 1292 Japanese samples were used for a preliminary screening of lipid trait association. These subjects were enrolled at four separate sites-the Tokyo, Nagoya, Osaka, and Shimane districts. Subjects in the Tokyo district (n=275) were selected from participants in the Hospital-based Cohort Study at the National Center for Global Health and Medicine (NCGM) and outpatients at the Institute for Adult Diseases, Asahi Life Foundation. Subjects in the Nagoya district (n=40) were selected from patients who either visited outpatient clinics of or were admitted to the Nagoya University Hospital and its affiliated medical institutions.

2) Study samples for CAD case-control studies
A total of 2684 Japanese subjects (1347 cases and 1337 controls in the tier-1 panel) were used for testing CAD association of 48 SNPs. The details about the subjects were Subjects with systolic blood pressure ≥140 mm Hg, diastolic blood pressure ≥90 mm Hg and/or the current use of antihypertensive medication were categorized as having hypertension. Dyslipidemia was defined according to the Japan Atherosclerosis Society Guidelines: LDL cholesterol ≥140 mg/dl, HDL cholesterol <40 mg/dl, triglycerides ≥150 mg/dl, or undergoing treatment with lipid-lowering drugs. Type 2 diabetes was defined as fasting plasma glucose levels ≥126 mg/dl, HbA1c ≥6.5%, and/or current treatment for diabetes.
For assessing the overall strength of CAD association in the Japanese, we pooled the genotype counts to combine the genetic effects estimated across the multi-tier scan.

1) SNP genotyping
In the Amagasaki Study panel and CAD case-control study panels, genotyping was performed with the TaqMan assay (Applied Biosystems) unless otherwise indicated. In the GWA study panel, genotyping was performed with Infinium HumanHap550/

2) Quality control of GWA scan data
Quality control of SNPs and samples was performed as previously described [32].
Briefly, data cleaning and analysis were performed using PLINK software [31]. Among the assayed SNPs, we excluded SNPs for which (1) genotype call rate, <0.95; (2) significant (P<10 -6 ) deviation from the Hardy-Weinberg equilibrium; or (3) MAF, <0.01. The remaining 456841 SNPs were analyzed in genome scan. The average call rate for the quality-controlled 456841 SNPs was 99.7% in 1292 samples tested for lipid trait association.