38 Shi et al Original Article Association between MCP-1 gene polymorphism and acute coronary syndrome in a Han ethnic Chinese cohort Gan-wei Shi 1, Guo-ping He 1, Gao-jun Cai 1, Chuan-ping Qi 2, Lei Gao 1, Dan-dan Shen 1, Zhi-hong Qian 1, Lian-hong Xu 2 1 Department of Cardiology, Affiliated Wujin Hospital of Jiangsu University, Changzhou 213002, China 2 Department of Central Laboratory, Affiliated Wujin Hospital of Jiangsu University, Changzhou 213002, China Corresponding Author: Guo-ping He, Email: guopinghe6@sina.com BACKGROUND: Acute coronary syndrome (ACS) is a multifactorial disease influenced by environmental and genetic factors. A growing number of human epidemiological studies have suggested that the chemokine, monocyte chemo-attractant protein-1 (MCP-1) are involved in atherosclerosis and ACS, but genetic evidence is contradictory. We investigated the A-2518G polymorphism of the MCP-1 gene in a Han Chinese cohort of patients with acute coronary heart disease. METHODS: The study comprised 484 patients and 346 healthy controls. This polymorphism was determined by using the polymerase chain reaction restriction fragment length polymorphism method. RESULTS: Our data showed that frequency distribution of the genotype (AA: 16.12%, AG: 51.86% and GG: 32.02% in cases; AA:15.32%, AG: 53.47% and GG: 31.21% in controls; P was 0.762 and 0.914, respectively;) and G allele genotype (57.95% in cases; 57.95% in controls; P was 1.000) were not signifi cantly different between the patients and controls. CONCLUSION: This study showed that there is no significant association between A-2518G polymorphism of the MCP-1 gene and ACS in the Han Chinese population. KEY WORDS: Monocyte chemoattractant protein-1; Acute coronary syndrome; Polymorphism World J Emerg Med 2013;4(1):38 42 DOI: 10.5847/ wjem.j.1920 8642.2013.01.007 INTRODUCTION Acute coronary syndrome (ACS) is an atherosclerosisassociated complication, a leading cause of mortality and disability around the world. [1] It is a multi-factorial disease influenced by environmental and multiple genetic factors. [2 4] The most important risk factors for ACS are hypercholesterolemia, high blood pressure, diabetes, obesity, smoking and family history of coronary heart disease. Atherosclerosis of the coronary arteries is the predominant mechanism of ACS. In recent years, atherosclerosis has been recognized as a disease of arterial inflammation that arises from migratory monocytes and T-lymphocytes. [5 7] Monocyte chemoattractant protein-1 (MCP-1), also known as CCL2, is a CC-type chemokine that is thought to be responsible for monocytes and T-lymphocytes recruitment in inflammatory conditions. In several animal models, the evidence suggest that MCP-1 is involved in the pathogenesis of atherosclerosis. [8 11] Several human epidemiological studies have also suggested links between MCP-1 levels and atherosclerotic disease. Higher MCP-1 levels have been associated with increased risks of myocardial infarction (MI), sudden death, coronary angioplasty, and stent restenosis. [12 16] MCP-1 levels are also elevated in the systemic circulation of patients with acute coronary syndrome. [17 19] There is genetic evidence in man to support a functional role for MCP-1 in atherogenesis. [20] The SNP 2013 World Journal of Emergency Medicine
39 2518G (alternatively 2578G) in the regulatory region of MCP-1, which causes increased promoter activity, is associated with elevated, circulating levels of MCP-1 [21,22] and increased risk of myocardial infarction. [21,23] However, the results of the role of the MCP-1 gene polymorphism and its plasma levels in MI are controversial. In the Czech population, a study [24] showed that MCP-1 gene A-2518G polymorphism was not associated with MI. Hence a casecontrol study design was adopted in the present study to explore the possible involvement of MCP-1 in MI in a Han Chinese population. METHODS Study population The subjects were divided into two groups: a case group (ACS patients) and a control group. Between June 2005 and May 2009, a total of 484 ACS patients (336 men and 148 women) were recruited from the Affiliated Wujin Hospital of Jiangsu University which is a public health system hospital in the city of Changzhou, China. The control group consisted of 346 subjects (175 men and 171 women), selected from inpatients at the same hospital, who were proved to be free from coronary heart disease by angiography. The study was approved by the Ethical Committee of the Affiliated Wujin Hospital of Jiangsu University. Definitions MI diagnosis was based on the diagnostic criteria of American Heart Association /Ameyican College of Cardiology (AHA/ACC) for 2002. Arterial hypertension was considered to be present if the patient had a history of high blood pressure or presented with blood pressure values above 140/90 mmhg one week after the event. High blood pressure was also considered in patients receiving anti-hypertensive medication. Diabetes mellitus was considered to be present when fasting blood glucose concentrations were over 126 mg/dl or if a history of diabetes was reported (including patients receiving diabetes medication). Smoking was defined as the smoking of 100 or more cigarettes during patient's lifetime. Measurement of lipid level Venous blood was drawn from fasting subjects with 0.1% EDTA. Plasma was separated from the blood sample by centrifugation and stored at 80 C until measurement. The plasma concentrations of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG) were determined by enzyme methods using a chemistry analyzer. Analysis of polymorphism Peripheral venous blood (2 ml) was drawn from participants and stored in EDTA tubes for no longer than a week at 4 C. Genomic DNA was extracted from white blood cells by a phenol-chlorofrom method. Genotyping of the MCP-1 gene was studied by the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method. The primers used for MCP-1 were forward 5 -TCTCTCACGCCAGCACTGACC-3 and reverse 5 -GAGTGTTCACATAGGC TTCTG-3. [22] The PCR was performed in a total reaction volume of 25 μl, containing approximately 50 ng of DNA, 2.5 pmol of each primer, and Premix Ex Taq Hot Start Version (TaKaRa) 12.5 μl, under the following conditions: an initial denaturation at 95 C for 5 minutes; then, 30 cycle of denaturation at 95 C for 40 seconds, 60 C for 30 seconds, 72 C for 40 seconds; and a final extension at 72 C for 10 minutes. Amplification with the primers produced a 234 bp product. Digestion with PvuII (TaKaRa, Dalian, China) yielded 159 and 75 bp fragments when G is at position 2518. The digestion products were analyzed on a 2.5% agarose gel stained with ethidium bromide. Statistical analysis All statistical analyses were carried out using SPSS 17.0 software. Allelic and genotypic frequencies were obtained by direct counting. Evaluation of Hardy-Weinberg equilibrium was performed by comparing observed and expected genotypes, using the Chi-square test. The Chi-square test was used to examine differences between cases and controls when two nominal variables were compared, and unpaired Student's t test was used to compare the mean values of measurement variables. The association between the MCP-1 polymorphism and risk of MI was estimated by odds-ratios and 95% confidence intervals. Multiple logistic regression analysis, applied to adjust conventional risk factors, was performed to investigate the independent role of the MCP-1 gene polymorphism. A P-value of less than 0.05 was considered statistically significant. RESULTS Clinical characteristics of the study subjects The principal clinical characteristics of all study
40 Shi et al subjects are shown in Table 1. In the present study, significant unmatched age, male/female ratio, and the frequencies of DM, hypertension, LDL-C, and smoking burden were found between the MI and control groups, as the age of the MI group (65.6 ±11.2 years) was almost 9 years older than that of the controls (56.4±10.5 years), and the males were 69.39% in the MI group and 50.91% in the control group. No significant differences were found between patients and controls in the serum levels of TC, TG, and HDL-C. MCP-1 gene A-2518 G polymorphism Regarding the MCP-1 polymorphism, after digestion of the 234 bp fragment obtained by PCR, three genotypes were distinguished (Figure 1): homozygous AA (234 bp), heterozygous AG (234 bp, 159 bp and 75 bp), and homozygous GG (159 bp and 75 bp). The PCR product was further confirmed (Figure 2). Table 1. Characteristics of the study group Variables Control group ACS group (n=346) (n=484) P value Age (y) 59.73±8.82 65.31±11.24 <0.001 Males (n, %) 175 (50.58) 336 (69.42) <0.001 Smoking (n, %) 49 (14.16) 178 (36.78) <0.001 Hypertension (n, %) 148 (42.77) 213 (44.01) 0.776 DM (n, %) 32 (9.25) 78 (16.12) <0.001 TC (mmol/l) 4.47±0.97 4.65±1.10 0.022 TG (mmol/l) 1.67±1.12 1.74±1.17 0.471 HDL (mmol/l) 1.11±0.33 1.10±0.29 0.617 LDL (mmol/l) 2.65±0.65 2.86±0.84 <0.001 Values are means for quantitative variables, and number(%) for qualitative variables; TG: triglycerides; TC: total cholesterol; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol. Frequency distribution of polymorphism The genotype and allele frequencies of the MCP-1 gene -2518 G/A polymorphism and their associations with ACS are presented in Table 2. These data are consistent with the distribution predicted by the Hardy- Weinberg equilibrium. The prevalence of GG genotype in patients with ACS was not significantly different from that in the controls (32.02% vs. 31.21%, P=0.914). Similarly, the prevalence of AG (51.86% vs. 53.47%, P=0.762) genotypes was not significantly different. The G allele frequency was not slightly higher in the controls than the patients with ACS. Table 2. The polymorphism distribution of the MCP-1 gene A-2518G in the patients with ACS (n, %) Variables ACS Controls (n=484) (n=346) P χ 2 OR (95%CI) Genotypes AA 78 (16.12) 53 (15.32) AG 251 (51.86) 185 (53.47) 0.762 0.161 1.085 (0.729 1.614) GG 155 (32.02) 108 (31.21) 0.914 0.013 1.025 (0.669 1.571) Allele frequencies A 407 (42.05) 291 (42.05) 1.000 0.000 1.000 (0.820 1.218) G 561 (57.95) 401 (57.95) 50 60 AA genotype 40 50 60 bp bp 234 159 75 500 300 200 100 GG genotype 40 50 60 1 2 3 4 5 6 7 8 9 10 11 12 13 Figure 1. Determination of the A-2518G genotype by PCR amplification and restriction analysis. The AA homozygote was not recognized by the PvuII enzyme, and only showed one 234 bp band (lanes 1, 3, 8, 9). The AG heterozygote had three bands, with sizes of 234, 159 and 75 bp (lanes 4, 5, 11, 12). The GG heterozygote had two bands, with sizes of 159 and 75 bp (lanes 2, 6, 7, 10). Lane 13: DNA marker. AG genotype Figure 2. The sequencing chromatogram of the MCP-1 gene A-2518G Polymorphism.
41 DISCUSSION The results of the study showed that the MCP- 1 gene A-2518G polymorphism was not significantly associated with ACS in the study population. Rovin and coworkers [22] found that a novel genotype made up of MCP-1 gene promoter polymorphisms (-2518G) was associated with MCP-1 production. But they did not study the association between the MCP-1 polymorphism and coronary artery disease. However, it is important to mention that the G/A polymorphism demonstrates racial heterogeneity. The G allele frequency is increased in Asians (47%) and Mexicans (47%) compared with Caucasians (29%) and African Americans (22%). Our data revealed that the prevalence of the G allele (61.17%) in control subjects was more frequent than in Asian and Mexican people. Thus it is important to determine if there is a relationship between racial differences in the MI and racial variations in the frequency of this polymorphism. Evidence has shown that the MCP-1 gene polymorphism might be significantly related to atherosclerotic lesions and acute vascular injury, [21,23] but their results are often inconsistent and sometimes even contradictory. [24,25] The reported positive association between the MCP-1 gene A-2518G polymorphism and MI in the Framingham Heart Study was based on 1 797 study subjects, including 107 MI subjects. Hence it was statistically powerful (P<0.05) to conclude that the G genotype increased risk for MI, [21] unlike the result in our study. Another study [25] suggested a significant but not independent association between -2518A/G of MCP-1 and MI in Tunisian population, indicating that MCP-1 may exert its effect on MI by established risk factors, such as age, smoking, hypertension and hypercholesterolemia. In the Czech population, the G genotype was not associated with the development of MI. [24] Clearly, the true effect of the MCP-1 gene A-2518G variant on the development of MI has remained elusive so far. Although the present study was difficult to compare our results with those published elsewhere, several points should be considered when interpreting the apparent discrepancy. The failure to repeat the association may be due to several causes: heterogeneity across races and ethnicities, environmental and additional genetic factors, and differences in study designs. The most likely explanation is genetic heterogeneity across races and ethnicities. In addition, a failure to recognize gene-gene and gene-environment interactions exerts a critical influence on the precise correlation between genes and ACS. It is possible that the MCP-1 variant conferred a modestly increased or reduced risk that might be added, amplified or even overcome by other acquired environmental and/or additional genetic factors. The controversial results can be attributed to the selection bias of samples, mismatches between patients and controls, and small sample sizes. No association between the MCP-1 gene A-2518G polymerphism and ACS was likely, because of the relatively small sample size in the present study. Furthermore, the average age at onset of ACS in the patients in our study was greater than that in the Framingham Heart Study. Several limitations remain in the present study. First, the control subjects were not healthy volunteers representing the general population. They had various atypical symptoms. However, the controls were confirmed to be free from coronary heart disease by angiography. Second, since the study subjects were not recruited prospectively, a survival bias could not be excluded. Third, most of ACS risk factors such as age, male/female ratio, smoking burden, the frequencies of DM, hypertension, and LDL-C are significant between the patients and controls. These discrepancies may also have been a source of bias. Finally, the intermediate phenotypes of the polymorphisms were not studied so that our report did not provide data on potential functionality of the MCP-1 polymorphism. Thus, this study only revealed the association between the MCP-1 polymorphism and ACS. In conclusion, the findings of this study suggest that the MCP-1 gene A-2518G variant has a more marked prevalence in Chinese than in western people and it is not associated with ACS. Obviously, the association of the MCP-1 gene A-2518G polymorphism with ACS awaits further investigations. Large studies based on different populations should be conducted to further elucidate the contribution of polymorphism to cardiovascular diseases. ACKNOWLEDGEMENT The authors thank the staff of the Wujin Hospital of Jiangsu University for their assistance in collection and analysis of the data. Funding: This work was supported by a grant from Jiangsu University (JCY20080065). Ethical approval: The study was approved by the Ethical Committee of Wujin Hospital Affiliated to Jiangsu University. Conflicts of interest: None. Contributors: Shi GW proposed the study, analyzed the data and wrote the first drafts. All authors contributed to the design and interpretation of the study and to further drafts.
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