Introduction

Coronary artery disease (CAD), clinically manifested with angina pectoris (AP) or myocardial infarction (MI), has become a major health problem in many countries because of its increasing prevalence and high mortality. Although non-genetic factors such as smoking, hypertension, hypercholesterolemia and diabetes mellitus significantly contribute to development of CAD, considerable evidence indicates the involvement of genetic factors in the pathogenesis of CAD (Wang 2005). Recent large scale association studies have accumulated information on the susceptibility genes linked to CAD. Notably, genome-wide association studies repeatedly identified the association of the chromosome 9p21 locus with the susceptibility to CAD in Caucasoid populations (Wellcome Trust Case Control Consortium 2007; McPherson et al. 2007; Helgadottir et al. 2007; Samani et al. 2007). In general, one of the main problems in association studies is the lack of reproducibility, which indicates that contribution of the reported factor is not common or not large enough to be replicated in other studies (Morgan et al. 2007). Therefore, validation studies are indispensable to clarify the genes involved in the pathogenesis, though there might be ethnic differences in the genetic backgrounds or regional differences in the environmental or lifestyle factors even in the same ethnic groups. In this study, we evaluated the association of rs1333049with CAD in Japanese and Korean populations.

Materials and methods

Subjects

The study protocol was approved by the Ethics Review Boards of the Medical Research Institute of Tokyo Medical and Dental University, Kitasato University School of Medicine, Tokyo Metropolitan Geriatric Medical Center, and Samsung Medical Center. Japanese subjects consisted of 604 patients and 1,151 controls, while Korean subjects included 679 patients and 706 controls. The Japanese control group was comprised of healthy volunteers without a history of CAD (n = 633) and consecutive autopsied persons without pathological findings of acute or old MI (n = 518). Korean control subjects were randomly selected from healthy individuals (n = 182) and cancer patients (n = 524) without a history of CAD. The diagnosis of CAD and classical risk factors was based on the standard criteria as described previously (Hohda et al. 2003). Severity of coronary atherosclerosis was classified according to the number of coronary vessels with significant stenosis (angiographic luminal stenosis >50%) as one-, two-, or three-vessel disease (VD).

Genotyping

TaqMan SNP genotyping assay (Applied Biosystems) was used to determine the genotype of rs1333049 in PCR products generated with primer pair rs1333049-F (5′-CCTTCATGCTATTTTGAGGAG) and rs1333049-R (5′-GGAAGATAAGTTGAGAATGTCA).

Statistical analysis

Genotype distributions and allele frequencies were compared between the cases and controls using a chi-square test. When the p value was less than 0.05, the association was considered to be significant. Strength of the association was expressed by odds ratio (OR). Significance of the association with coronary disease severity was examined by Mann–Whitney U-test.

Results and discussion

Five SNPs on the chromosome 9p21 locus, rs1333049, rs10757274, rs2383206, rs2383207, and rs10757278, were associated with CAD, and they were in tight LD in Caucasians. Because the structure of LD based on HapMap database information suggested that they were also in tight LD in Asians, we used rs1333049 in this study. As shown in Table 1, the genotype distribution was in Hardy–Weinberg equilibrium in all tested populations. There was significant association at the allele count model in both Japanese (OR = 1.30, 95% CI; 1.13–1.49, p = 0.00027) and Korean (OR = 1.19, 95% CI; 1.02–1.38, p = 0.025), confirming the association in East Asian populations. A replication study in Korean by using different SNPs was recently reported (Shen et al. 2008). These data strongly suggested that the chromosome 9p21 locus conferred susceptibility to CAD across racial lines.

Table 1 Association of rs1333049 on chromosome 9p21 with CAD in Japanese and Koreans

Because previous studies did not address the correlation between the risk allele and phonotypic background of CAD, we investigated the association between rs1333049 and severity of atherosclerosis. According to the number of significantly affected vessels, CAD patients were classified into three groups, 1VD, 2VD and 3VD. As shown in Table 2, there was no trend of association between the rs1333049 genotype and severity of CAD in both Japanese and Koreans (Table 2). Stratified analyses of rs1333049 with risk factors of CAD such as diabetes mellitus, hyperlipidemia, or hypertension showed again no trend of association between rs1333049 and risk factors (data not shown). Though further studies are required to decipher the mechanism of involvement of this locus to the pathogenesis of CAD, the observations in this study strongly suggested that the chromosome 9p21 locus was a genetic risk factor independent of classical risk factors.

Table 2 Association of rs1333049 on chromosome 9p21 with the severity of coronary atherosclerosis