Associations Between Single Nucleotide Polymorphisms on Chromosome 9p21 and Risk of Coronary Heart Disease in Chinese Han Population
Objectives— We aimed to determine whether the single nucleotide polymorphisms (SNPs) on chromosome 9p21 were associated with coronary heart disease (CHD) in a Chinese Han population.
Methods and Results— We determined the genotypes of rs2383206 and rs2383207 on chromosome 9p21 in 1360 CHD patients and 1360 age- and sex-frequency–matched controls from an unrelated Chinese Han population. GG genotypes in rs2383207 occurred more frequently in CHD patients compared to controls, and the odds ratio (OR) was 1.52 (95% CI 1.13 to 2.04), after adjusting for conventional risk factors. In stratified analysis, the risk associated with the GG genotype of the two SNPs was stronger in subjects who were males, less than 60 years old, overweight, and smokers. The SNP rs2383207 had significant interactions with gender and smoking (P=0.018 and 0.037, respectively). The risk allele G of rs2383207 plus family history of CHD had a cumulative association with CHD (P for trend, 1.0×10−6); the OR for CHD was 4.59 (95% CI 2.52 to 8.37) for those with all the risk factors as compared with subjects without any of the factors.
Conclusions— The SNP rs2383207 on chromosome 9p21 is significantly associated with CHD in Chinese. This SNP combined with family history has a cumulative association with CHD.
Coronary heart disease (CHD) is the leading cause of death worldwide.1,2 The development of CHD is a complex process. It is caused by multiple genetic and environmental factors, and interactions among these factors.3 Many risk factors have been identified for CHD, including smoking, advanced age, male gender, diabetes mellitus, and high blood pressure.4 However, few genetic factors for CHD have been identified. Recently, genome-wide association studies (GWAS) using hundreds of thousands of markers and targeted gene-based resequencing have facilitated the gene discovery for CHD.5,6 In genome-wide association studies, a susceptibility locus for CHD has been mapped to chromosome 9p21, adjacent to the tumor suppressor genes CDKN2A and CDKN2B.7,8 Helgadottir et al7 found that the variant rs2383207 was associated with myocardial infarction (MI). Meanwhile, in another GWAS, McPherson et al8 found that the homozygotes of the risk alleles of rs2383206 were associated with an increased risk of CHD. The same genetic locus was identified by a genome-wide association study from a British population by the Wellcome Trust Case Control Consortium and replicated in a German population.9
However, these associations need to be confirmed by further replication studies, particularly in other ethnic groups. Therefore, we carried out a large case-control association study including 1360 CHD patients and 1360 age- and sex-frequency–matched controls in an unrelated Chinese Han population. We selected 2 SNPs (rs2383206 and rs2383207) in the locus of interest and aimed to determine whether the SNPs on chromosome 9p21 were associated with CHD in a Chinese Han population.
The study population was composed of 1360 case patients and 1360 age- and sex-frequency–matched controls. Patients were consecutively recruited from 3 hospitals (Tongji Hospital, Union Hospital, and Wugang Hospital) in Wuhan (Hubei, China) between May 2004 and October 2006. The diagnostic criteria for CHD cases included 1 of the following: (1) The presence of a stenosis ≥50% in at least 1 of the major segments of coronary arteries (the right coronary artery, left circumflex, or left anterior descending arteries) on coronary angiography; (2) Based on World Health Organization criteria in terms of elevations of cardiac enzymes, electrocardiographic changes and clinical symptoms10; (3) A documented history of coronary artery bypass graft or percutaneous coronary intervention. Patients with congenital heart disease, cardiomyopathy, and valvular disease were excluded. A total of 1440 patients diagnosed as having CHD were recruited; 1360 of them (94.4%) consented to participate in the study and provided questionnaire information and blood samples. After cases were diagnosed with CHD, they were interviewed in person by a trained interviewer within 3 days. The control subjects, residing in the same communities as the cases, were determined to be free of CHD and peripheral atherosclerotic arterial disease by medical history, clinical examinations, and electrocardiography. The response rate for the controls was 90.7% (1360 of 1500). Subjects with severe liver or kidney disease were excluded. Medical history, family history of CHD among first degree relatives, medication use, home environment, and lifestyle factors were obtained through questionnaire interview.
Subjects were classified as smokers and nonsmokers. Those who had smoked less than 100 cigarettes in the lifetime were defined as nonsmokers; otherwise, they were defined as smokers. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters. Subjects were considered to be hypertensive if their systolic blood pressure was ≥140 mm Hg or diastolic pressure ≥90 mm Hg or they were already being treated with antihypertensive drugs. Diabetes was defined either by 1999 World Health Organization criteria11 or self-report of being previously diagnosed as diabetic. Family history was positive if first-degree relatives (parents, siblings) had CHD. All subjects gave written consent after receiving a full explanation of the study. The Ethics Committee of Tongji Medical College approved this study.
Fasting venous blood was collected in 5-mL EDTA tubes, and genomic DNA was isolated with a Puregene kit (Gentra Systems Inc). Genotyping was performed with TaqMan SNP allelic discrimination by means of an ABI 7900HT (Applied Biosystems), in 384-well format. The TaqMan Assay kit was purchased from Applied Biosystems. It included the forward target-specific polymerase chain reaction (PCR) primer, the reverse primer, and the TaqMan MGB probes labeled with 2 special dyes: FAM and VIC. PCR reactions were carried out in reaction volume of 5 μL containing 5 ng DNA, 2.5 μL 2×Taqman universal PCR Master MixNo AmpErase UNG (Applied Biosystems), 0.125 μL 40×Assay MIX. PCR conditions included 95°C for 10 minutes followed by 40 cycles of 15s at 92°C and 1 minute at 60°C. Two blank controls (DNA hydration) and 2 replicate quality control samples were included in each 384-well format, and 2 replicate samples were genotyped with 100% concordance. Automatic allele calling, with the default settings (the quality value of auto caller ≥95.0), was carried out by ABI 7900HT data collection and analysis software version 2.2.1 (SDS 2.2.1).
Continuous variables were reported as the mean value±SD. Normal distribution of data was checked using the Kolmogorov-Smirnov normality test. Data with a normal distribution were compared by Student t test, and those with unequal variance or without a normal distribution were analyzed by a Mann-Whitney rank sum test. Categorical values were compared by the χ2 test, which was also used to test for deviation of genotype distribution from Hardy-Weinberg equilibrium. The association between variants in the two SNPs and CHD risk was estimated by computing odds ratios (ORs) and 95% confidence intervals (CIs) from the multivariate logistic regression analyses. An unconditional logistic model was used to adjust for multiple cardiovascular risk factors. The probability level accepted for significance was P<0.05. The significance of multiplicative interactions between the selected SNPs and covariates was determined by the likelihood ratio test using the logistic regression model. All data analyses were carried out with the statistical analysis software package SPSS12.0 (SPSS Inc).
General Characteristics of the Subjects
The general characteristics of the study subjects are presented in Table 1. The traditional CHD risk factors such as hypertension, diabetes, BMI, smoking, and family history of CHD were significantly different between the cases and controls. However, total cholesterol levels were significantly lower in cases than in controls, which could be the result of cholesterol-lowering medication in the patients after diagnosis (4.4±1.1 mmol/L versus 5.1±1.3 mmol/L, P<0.01). The proportion of subjects reported taking cholesterol-lowering medications such as a statin in the cases and controls in our study were 67.1% and 0.3%, respectively.
Relation of the SNPs and CHD Risk
The observed genotype frequencies of the 2 SNPs on chromosome 9p21 were in Hardy-Weinberg equilibrium among the controls (P=0.63 and 0.64, respectively). As shown in Table 2, univariate analyses indicated that the 2 SNPs rs2383206 and rs2383207 were significantly associated with CHD. The risk genotypes of GG were found in both SNPs, with odds ratio (OR) were 1.29 (95% CI 1.05 to 1.58) and 1.40 (95% CI 1.09 to 1.80), respectively. In multivariate analyses, after adjusting for conventional CHD risk factors such as age, gender, smoking, BMI, hypertension, diabetes, and family history of CHD, only the GG genotype of rs2383207 had a significant association with CHD (OR=1.52, 95% CI 1.13 to 2.04). When we examined MI separately, the association with this SNP became even stronger (OR of MI for the GG genotype=2.64, 95% CI 1.35 to 5.17; P=0.005). For the risk allele G of the both SNPs, the univariate ORs of CHD were 1.14 (95% CI 1.03 to 1.27) and 1.26 (95% CI 1.12 to 1.41), respectively (Table 2).
We conducted stratified analysis for the two SNPs (Table 3). GG carriers of two SNPs (rs2383206 and rs2383207) had higher risk in males (OR=1.57, 95% CI 1.14 to 2.16 and OR=1.96, 95% CI 1.32 to 2.90, respectively), younger than 60-year-old subjects (OR=1.35, 95% CI 1.00 to 1.84 and OR=1.58, 95% CI 1.02 to 2.47, respectively), smokers (OR=1.55, 95% CI 1.10 to 2.20 and OR=2.03, 95% CI 1.34 to 3.08, respectively), and BMI ≥25 kg/m2 subjects (OR=1.52, 95% CI 1.00 to 2.33 and OR=2.39, 95% CI 1.41 to 4.05, respectively). Furthermore, when multiplicative interaction was tested for each possible pair of these 2 SNPs, we found significant interactions between rs2383207 and gender (P=0.018) and smoking (P=0.037).
We evaluated cumulative effects of allele G of the SNP rs2383207 and family history of CHD (Table 4). Subjects who carried 3 risk factors, including 2 risk alleles G and family history of CHD had an OR of 4.59 (95% CI 2.52 to 8.37) for CHD, as compared with subjects who carried none of the risk factors, after adjusting for other conventional risk factors. The P for trend of this cumulative effect was 1.0×10−6.
Our large case-control study not only replicated the findings of the SNPs on chromosome 9p21 that were associated with CHD established previously by genome-wide association studies, but also provided several novel findings that were relevant to this locus. Our results showed that the genotype GG of the SNP rs2383207 was associated with increased overall risk of CHD. Especially among males, young subjects (age ≤60), smokers, and overweight patients (BMI ≥25 kg/m2), the genotypes GG of the 2 SNPs rs2383206 and rs2383207 were strongly associated with higher risk of CHD. It is possible that genetic factors would exert a greater influence in younger persons and males, and smoking and obesity may exacerbate the influence of the genetic factors. Significant interactions were found between the traditional CHD risk factors (gender and smoking) and rs2383207 in our study. These findings support the notion that many genes, each with a relatively small effect, work in combination with other modifier genes and environmental factors.5
In addition, we found that the risk of CHD for the subjects with two G alleles of rs2383207 and family history was increased by 4.59-fold. Thus, it is possible to use the combined information from the risk SNPs at 9p21 and family history to assess an individual patient’s risk of CHD.
Significant associations of the 2 SNPs on chromosome 9p21 with CHD have been replicated in multiple populations.7–9,12–14 Helgadottir et al conducted GWAS in 4587 MI cases and 12769 controls and found that variant rs2383207 was associated with MI with an OR of 1.64 for the risk genotype. They also observed the corresponding risk was doubled for early onset MI cases.7 McPherson et al found the homozygote of rs2383206 was associated with a 30% to 40% increased risk of CHD in 6 independent samples (n>23 000 participants).8 Recently, an Italian case-control study replicated the association between the SNPs on 9p21 (including rs2383206 and rs2383207) and the MI risk in 416 MI cases and 308 non-MI controls.13 In our study, rs2383207 were more strongly and robustly associated with CHD than rs2383206 after multivariate adjustment.
The region on chromosome 9p21 which showed an association with the CHD in our or previous studies is located in 2 blocks of strong intrablock and interblock LD.9 HapMap data suggest that the 5 SNPs (rs2383206, rs2383207, rs10757274, rs10757278, and rs1333049) are in 1 block, and the D′ between each of the 5 SNPs was 1.0 in Chinese Han (CHB) origin. For example, rs2383206 captures rs10757274, rs10757278, and rs1333049 in the HapMap CHB population (D′=1.0, r2>0.90). Thus, the 2 SNPs (rs2383206 and rs2383207) are sufficient to tag the region in our population.
The frequencies of the risk allele G of rs2383206 and rs2383207 are different between the Chinese Han population and European population. The data from the HapMap show that the rs2383206-G allele differs from 0.53 in Europeans to 0.46 in Han Chinese, and the rs2383207-G allele differs from 0.53 in Europeans to 0.62 in Han Chinese. Our findings suggest that rs2383207 has stronger association with CHD than the other SNP on chromosome 9p21 in Chinese Han population.
Few studies have examined the associations between SNPs on chromosome 9p21 and CHD risk in East Asian populations. Recently, 2 case-control studies reported a significant association of the same 9p21 locus with CHD in Korean and Japanese populations.12,14 It is interesting to note that SNP rs2383207 had a stronger association than other SNPs (including rs2383206) in the Korean population,12 which is consistent with our results.
The region of chromosome 9p21 contained the coding sequences of gene for 2 cyclin-dependent kinase inhibitors, CDKN2A and CDKN2B, which played an important role in the regulation of the cell cycle and would be implicated, through their role in transforming growth factor (TGF)-β–induced growth inhibition, in the pathogenesis of atherosclerosis.15–17 However, the most strongly associated SNPs lie considerably upstream of these genes, and the SNP rs2383207 is about 100 kb upstream of them. McPherson et al resequenced the coding regions of the 2 genes most proximal to the risk locus and found no explanation of the CHD risk associated with this locus.8 However, the same region has recently been associated with increased susceptibility to type 2 diabetes.18–20 These results suggest that the region of 9p21 plays a role in multiple complex diseases. Further studies should focus on the identification of the underlying mechanism at this locus of CHD. In addition, the interactions between gender, smoking, and overweight with these SNPs need to be replicated in other populations.
In conclusion, we have demonstrated that the SNP rs2383207 on chromosome 9p21 is significantly associated with CHD in Chinese Han population. The risk allele of the SNP rs2383207 plus family history of CHD have a cumulative, significant association with CHD.
We are particularly grateful to all volunteers for participating in the present study and to the medical personnel of Tongji Hospital, Union Hospital, Wugang Hospital and WT’s laboratory in Wuhan city, Hubei Province, China for their kind assistance in collecting the data and samples.
Sources of Funding
This work was supported by research funds from the National Natural Scientific Foundation of China (30430590 and 30525031).
L.Z. and X.Z. contributed equally to this study.
Original received May 26, 2008; final version accepted August 20, 2008.
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