Article Type: Regular Manuscript

Methylenetetrahydrofolate reductase C667T polymorphism is associated with increased risk of coronary artery disease in a Chinese population

Weiqiang Chena,1, Kun Huab,1, Haiyong Guc, Jian Zhanga,*, Lina Wang d, e* a

Department of Cardiovascular Medicine, Cardiovascular Clinical College of Tianjin Medical University & TEDA International Cardiovascular Hospital, Tianjin 300457, China; b

State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100037, People’s Republic of China; c

Department of Cardiothoracic Surgery, Affiliated People’s Hospital of Jiangsu University, Zhenjiang 212002, China;

d

Key Laboratory of Environmental Medicine Engineering, Ministry of Education, Department of Epidemiology & Biostatistics, School of Public Health, Southeast University, Nanjing, China; e

School of Biological Science and Medical Engineering, Southeast University, Nanjing, China.

1

These authors contributed equally to this study.

*

Correspondence to: Department of Cardiovascular Medicine, Cardiovascular Clinical College of Tianjin Medical University & TEDA International Cardiovascular Hospital, Tianjin 300457, China, Jian Zhang: Email address [email protected] and Lina

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/sji.12215 This article is protected by copyright. All rights reserved.

Wang, Key Laboratory of Environmental Medicine Engineering, Ministry of Education, Department of Epidemiology & Biostatistics, School of Public Health, Southeast University, 87 Ding Jiaqiao Rd., Nanjing 210009, China; E-Mail: [email protected].

Running title: Polymorphisms of MTHFR and coronary artery disease risk

Key words: MTHFR; polymorphisms; cardiovascular disease; molecular epidemiology

Abbreviations: methylenetetrahydrofolate reductase, MTHFR; linkage disequilibrium, LD; odds ratio, OR; confidential interval, CI; single nucleotide polymorphisms, SNPs; coronary artery disease, CAD.

Grant support: This study was supported in part by the National Natural Science Foundation of China (No. 81101889, 30901230, 30900630).

Condensed abstract: We conducted a hospital based case–control and replication study to evaluate the association between methylenetetrahydrofolate reductase rs1801133 C/T, matrix metalloproteinases-2, tumor necrosis factor-α, macrophage migration inhibitory factor rs755622 G/C and cyclin D1 rs678653 G/C single nucleotide polymorphism (SNP) and the susceptibility of coronary artery disease (CAD). Our results demonstrated that MTHFR rs1801133 C/T SNP might be associated with CAD risk.

Acknowledgements: We appreciate all patients who participated in this study.

Abstract Coronary artery disease (CAD) is a complex disease resulting from a combination of environmental and genetic factors. We hypothesized polymorphisms in methylenetetrahydrofolate reductase (MTHFR) rs1801133 C/T, matrix This article is protected by copyright. All rights reserved.

metalloproteinases (MMPs)-2, tumor necrosis factor (TNF)-α, macrophage migration inhibitory factor (MIF) rs755622 G/C and cyclin D1 (CCND1) rs678653 G/C contribute to CAD susceptibility. We examined the association between the five polymorphisms and the risk of CAD in a Chinese population of 435 CAD patients and 480 controls. Genotyping was performed using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF-MS). When the MTHFR rs1801133 CC homozygote genotype was used as the reference group, the TT or CT/TT genotypes were associated with a significantly increased risk for CAD. The CT heterozygote genotype was not associated with the risk for CAD. Logistic regression analyses revealed that MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, MIF rs755622 G/C and CCND1 rs678653 G/C polymorphisms were not associated with the risk of CAD. These findings suggest that MTHFR rs1801133 C/T polymorphism is associated with CAD development. Future larger studies with other ethnic populations are required to confirm current findings.

Introduction Homocysteine is an independent risk factor for atherosclerosis and thrombosis [1]. An elevated level of total plasma homocysteine (tHcy) has been associated with risk of coronary artery disease (CAD) [2]. Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of methylation and located in 1p36.3. It is a methyl donor and catalyzes reduction of 5, 10-methylene-tetrahydrofolate to 5-methyltetra hydrofolate [3]. Functional polymorphisms of MTHFR may lead to attenuate of 5-methyl tetrahydrofolic acid to induce a decrease of the conversion of homocysteine to methionine [4]. MTHFR rs1801133 C>T (MTHFR C667T) mutation results in an alanine to valine substitution and a reduction in enzyme activity, causes reduced enzyme activity and elevated concentrations of homocysteine [5].

Matrix metalloproteinases (MMPs)-2, also known as gelatinase, is involved in inflammation and immunity, as well as degradation and remodeling of the extracellular matrix [6]. Experiments in animal models showed that homocysteine induces elastolysis in arterial media through the activation of MMP-2. The MMP-2 gene is located on chromosome 16q13, with 13 exons and 12 introns. The MMP-2 -1306 C/T (rs243865) SNP is located in the CCACC box of the Sp1-binding site, and the T allele is associated with greatly reduced promoter activity [7]. It is likely that the CC genotype is associated with high transcription levels and enzyme activity of

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MMP-2, while the -1306T allele produces lower MMP-2 protein levels in individuals carrying the CT or TT genotype. Tumor necrosis factor (TNF)-α is able to induce the expression of MMP-2 in blood and inflamed synovial tissue [8, 9]. The TNF-α gene is located on chromosome 6. The TNF-α SNP -308 A/G (rs1800629) in the promoter region has been intensively studied [10], with the A allele being associated with higher levels of TNF transcription [11, 12]. In lipopolysaccharide-driven responses, MIF is also a potent promoter of the expression of other cytokines such as TNF-α, interleukin (IL)-1β, IL-6, IL-8, and prostaglandin E2 [13]. Human macrophage migration inhibitory factor (MIF) is located on chromosome 22q11.2 [14]. The MIF -173 G/C (rs755622) polymorphism is located in the promoter region, and has been shown to influence MIF promoter activity in T lymphoblast cell lines [15]. MIF -173 G/C polymorphism was identified and shown to be functional in vitro and in vivo [16]. The serum MIF levels are also significantly higher in patients with the MIF -173 C allele than patients with the MIF -173 GG genotype [17]. MIF can up-regulate the expression of cyclin D1 (CCND1) to activate cyclin dependent kinases 4/6 by activation of mitogen-activated protein kinase [18]. CCND1 plays a critical role in the G1 to S-phase cell cycle transition [19, 20]. The CCND1 gene, CCND1, is located on chromosome 11q13. CCND1 rs678653 G/C is on the 3′-untranslated region and well studied. Genetic variations MTHFR rs1801133 C/T, MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, MIF rs755622 G/C and CCND1 rs678653 G/C polymorphisms may contribute to the development of CAD. Therefore, we performed a hospital-based case–control study to genotype a cohort of 435 CAD patients and 480 controls from a Chinese population.

Materials and methods Ethical approval of the study protocol The study was approved by the Ethical Committee of Tianjin Medical University. We have complied with the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects and/or animals. Each subject was interviewed. All subjects provided written informed consent to be included in the study.

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Patients and controls This case–control study included 435 consecutive patients with CAD from Tianjin TEDA International Cardiovascular Hospital between November 2011 and July 2012 and 480 CAD-free controls, the controls were inpatients to excluding CAD by quantitative coronary angiography (QCA) in Tianjin TEDA International Cardiovascular Hospital as previously described [21]. All 435 CAD patients and 480 inpatients received QCA with a Cardiovascular Measurement System (Philips Integris and Philips Allura Xper) shortly after being admitted to the hospital, and coronary angiograms were analyzed by two experienced interventional cardiologists. CAD patients were defined as having angiographic coronary stenosis of at least 50% lumen reduction in at least one major epicardial coronary artery. Exclusion criteria were patients who not meeting CAD diagnosis standard after QCA and inability to give written informed consent. The 480 CAD-free inpatients were considered as controls. The control pathologies were luminal narrowing 6 months were considered to be alcohol drinkers. Diabetes mellitus was defined as self-reported diabetes mellitus or nonfasting glucose levels ≥ 11.1 mmol/L (200 mg/dL). Hypertension was defined as blood pressure level exceeding 140/90 mmHg or use of antihypertensive therapy.

Isolation of DNA and genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) An approximately 2-ml venous blood sample was collected from each subject. Blood samples were collected using vacutainers and transferred to test tubes containing ethylenediamine tetra-acetic acid (EDTA). Genomic DNA was isolated from whole blood using the QIAamp DNA Blood Mini Kit (Qiagen, Germany). DNA quality and quantity were assessed by a UV spectrophotometer at 260 nm and 280 nm and all DNA samples were visualized in gel array, the quality of DNA is high in more than 99.5% samples (Fig. 1). Genotyping was done by MALDI-TOF MS using the MassARRAY system (Sequenom, San Diego, CA, USA) as previously described (Fig. 2A-2E) [22]. For quality control, repeated analyses were undertaken on 10% of randomly selected samples with high DNA quality. Furthermore, 50 DNA samples were randomly selected to confirm the MTHFR rs1801133 C/T genotyping results by direct sequencing (Fig. 3). This article is protected by copyright. All rights reserved.

Statistical analyses Differences in demographics, variables, and genotypes of the MTHFR rs1801133 C/T, MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, MIF rs755622 G/C and CCND1 rs678653 G/C polymorphisms were evaluated using a chi-squared test. The associations between the five SNPs genotypes and risk of CAD were estimated by computing odds ratios (ORs) and 95% confidence intervals (CIs) using logistic regression analyses, and by using crude ORs. The Hardy–Weinberg equilibrium (HWE) was tested by a goodness-of-fit chi-squared test to compare the observed genotype frequencies to the expected frequencies among controls. All statistical analyses were done with SAS software (version 9.1.3; SAS Institute, Cary, NC, USA).

Results Characteristics of the study population The demographic and clinical characteristics of all subjects are summarized in Table 1. Subjects were adequately matched for age (p = 0.383) and sex (p = 0.398) for CAD cases and controls in Tianjin. The average body mass index (BMI) was not significantly different between CAD cases and the controls. For the family disease history, 98 (22.5%) cases had family history of CAD which was significantly higher than that of controls (13.1%), there were more hypertension and diabetes mellitus in CAD cases than in controls. No significantly differences occurred for hyperlipidemia between CAD cases and controls. There were more smokers in cases than in the controls. The level of fasting blood glucose, uric acid, serum creatinine, triglyceride and fibrinogen in CAD cases were higher than those of controls, while the level of high density lipoprotein of CAD cases was significantly lower than that of controls. Among 915 DNA samples (435 CAD patients and 480 controls in Tianjin), MTHFR rs1801133 C>T was successful in 424 (97.5%) CAD cases and 476 (99.2%) controls (Table 2). The genotyping success rate of five SNPs is ranging from 98.03% to 99.45% in all 915 samples (Table 2). The concordance rates of repeated analyses were 100% (50/50).

Associations between MTHFR rs1801133 C>T MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, MIF rs755622 G/C and CCND1 rs678653 G/C polymorphisms and risk of CAD The genotype frequencies of the MTHFR rs1801133 C>T polymorphism were 23.1% (CC), 49.3% (CT) and 27.6% (TT) in CAD patients, and 29.6% (CC), 46.4% (CT) This article is protected by copyright. All rights reserved.

and 23.9% (TT) in controls (p = 0.077) (Table 3). When the MTHFR rs1801133 CC homozygote genotype was used as the reference group, the TT or CT/TT genotypes were associated with a significantly increased risk for CAD (TT vs. CC: adjusted OR = 1.47, 95% CI = 1.01–2.14, p = 0.046; CT/TT vs. CC: adjusted OR = 1.39, 95% CI = 1.02–1.89, p = 0.039). The CT heterozygote genotype was associated with a borderline significantly increased risk for CAD (adjusted OR = 1.36, 95% CI = 0.99–1.87, p = 0.059). In the recessive model, when the MTHFR rs1801133 CC/CT genotypes were used as the reference group, the TT homozygote genotype was not associated with the risk for CAD (adjusted OR = 1.22, 95% CI = 0.89–1.66, p = 0.219) (Table 3). None of the MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, MIF rs755622 G/C and CCND1 rs678653 G/C polymorphisms achieved a significant difference in the genotype distributions between cases and controls. Logistic regression analyses revealed that MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, MIF rs755622 G/C and CCND1 rs678653 G/C polymorphisms were not associated with the risk of CAD (Table 3).

Discussion We determined the association between the MTHFR rs1801133 C/T, MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, MIF rs755622 G/C and CCND1 rs678653 G/C polymorphisms and the risk of CAD in a Chinese population. We found that the MTHFR rs1801133 C>T polymorphism may be associated with an increased risk of CAD. The MTHFR genetic variation resulted in 5-methyltetrahydrofolate reduction and homocysteine amassing, which made the methyl donor of the methionine dys-synthesis. Elevated plasma levels of homocysteine promote endothelial damage and adhesion of leukocytes to the endothelial surface, and would be an indicator of continuing tissue damage and an enhancer of inflammatory vascular wall thickening, rather than an initiator of atherosclerosis [23]. Homocysteine contributes to oxidative stress and endothelial damage [24], is also considered to be a risk factor for CAD, and has been shown to be increased in persons with CAD. MTHFR rs1801133 CT/TT causes reduced enzyme activity and elevated concentrations of homocysteine [5], which may be related with increased CAD risk. Genetic polymorphisms often vary between ethnic groups. In the present study, which included 480 controls, we reported that the allele frequency of MTHFR

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rs1801133 C>T (0.472 and 0.448) was similar to that reported in other Chinese populations (0.439). But the allele frequency is significantly higher than that of European population (0.237) and Sub-Saharan African population (0.110) (http://www.ncbi.nlm.nih.gov/SNP, http://hapmap.ncbi.nlm.nih.gov/). Using Power and Sample Size Calculation (PS, version 3.0, 2009, http://biostat.mc.vanderbilt.edu/twiki/bin/view/Main/PowerSampleSize), considering MTHFR rs1801133 C>T mutant alleles in the control group, the OR, and the sizes of the CAD and control samples, the power of our analysis (α = 0.05) was 0.820 in 424 CAD cases and 476 controls with adjusted OR 1.47. Several limitations of the present study must be noted. First, this was a hospital-based case–control study. The controls were inpatients received QCA and excluding CAD, and selection bias was unavoidable because the subjects were not fully representative of the general population. Second, based on functional considerations, the polymorphisms investigated may not offer a comprehensive view of the genetic variability of MTHFR, so further fine mapping studies are warranted. Third, a single case–control and replication study is insufficient to fully interpret the relationship between the MTHFR rs1801133 C>T polymorphism and an individual’s susceptibility to CAD because the number of patients evaluated is limited. Studies of larger numbers of subjects are required to confirm our findings. Finally, environmental factors differ between Chinese and other populations. Because the risk of CAD is probably influenced by gene–gene and gene–environment interactions, the MTHFR gene may be associated with different degrees of genetic risk in different ethnic groups and under different environmental circumstances. In conclusion, the present study provides evidence that the MTHFR rs1801133 C>T polymorphism is associated with an increased risk of CAD. However, further studies are required to confirm our results.

Conflict of interest: None of the authors has any potential financial conflict of interest related to this manuscript.

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Table 1 The distributions of the selected variables in the CAD cases and controls CAD

Controls

(n = 435)

(n = 480)

Mean age, y

61.30 (±9.71)

60.79 (±7.54)

0.383

Woman, %

155 (35.6)

184 (38.3)

0.398

Mean BMI, kg/m2

25.99 (±3.28)

26.24 (±2.75)

0.222

Family history of CAD, %

98 (22.5)

63 (13.1)

Methylenetetrahydrofolate reductase C667T polymorphism is associated with increased risk of coronary artery disease in a Chinese population.

Coronary artery disease (CAD) is a complex disease resulting from a combination of environmental and genetic factors. We hypothesized that polymorphis...
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