Supplementary Material for: A Case-Control Study of the Association of the Polymorphisms of MTHFR and APOE with Risk Factors and the Severity of Coronary Artery Disease

Objectives: To explore the association between single-nucleotide polymorphisms (SNPs) in MTHFR and APOE and the risk of CAD and, more importantly, the severity of CAD and the profile of serum lipids, we performed a case-control study in a Chinese Han population. Methods: A total of 1,207 cases of consecutive CAD-suspected inpatients were recruited, and 406 CAD cases and 231 non-CAD controls were enrolled for the final analysis after screening for exclusion criteria. All subjects had undergone coronary angiography, and the severity of CAD was evaluated by 2 cardiologists according to the Gensini scores. The genotypes of MTHFR and APOEwere detected using real-time PCR, and then verified by Sanger sequencing. Environmental risk factors, such as age, sex, smoking, alcohol consumption, hypertension, diabetes, dyslipidemia, and BMI were collected. Statistical analyses (the χ2 test, binary logistic regression analysis, and ordinal polytomous logistic regression analysis) were performed with SPSS v16.0. Results: The genotypes ofall the subjects included in the CAD and non-CAD groups in this study were successfully detected, with an agreement of 100% with Sanger sequencing. The distributions of genotypes CT and TT at MTHFR C667T were higher in CAD cases than in non-CAD controls (OR 1.99, 95% CI 1.34–2.95; OR 1.77, 95% CI 1.18–2.67; p < 0.05), whereas genotype AC at MTHFR A1298Cwas lower in CAD cases (OR 0.71, 95% CI 0.50–1.02; p < 0.05). A significant association was observed in genotypes CT and TT at MTHFR C667T and the risk of CAD (OR 1.44, 95% CI 1.27–3.67; OR 1.56, 95% CI 0.88–2.78; p < 0.05). Both genotypes and alleles of APOE were comparable in the CAD cases and non-CAD controls (p > 0.05). The genotype TT at MTHFR C667T and ε4+ at APOE were more likely to be found in the CAD subgroup with a Gensini score ≥72 (p = 0.040 and p = 0.028, respectively). Meanwhile, in the patients with genotype TT,a higher level of serum Hcy was detected, while genotype ε4+ patients possessed higher levels of serum apolipoprotein E (ApoE) and low-density lipoprotein cholesterol (LDL-C) than other genotypes. Conclusion: This study revealed that the SNP site of MTHFR C667Tis associatedwith the risk of CAD in this Chinese Han population. In addition, the genotypes of TT in MTHFR C667T and ε4+in APOE may increase the severity of CAD, and higher Hcy, LDL-C, and ApoE levels may be involved in this pathogenic process.研究目的：本研究旨在探讨亚甲基四氢叶酸还原酶（MTHFR）与载脂蛋白E（APOE）的单核苷酸多态性（single-nucleotide polymorphisms, SNPs）与冠状动脉粥样硬化性心脏病（CAD）发病风险的关联，更重要的是分析其与CAD严重程度及血清血脂谱的相关性，故在中国汉族人群中开展此项病例对照研究。 研究方法：本研究共纳入1207例疑似CAD的连续住院患者，经排除标准筛选后，最终纳入406例CAD患者与231例非CAD对照者进行最终分析。所有受试者均接受冠状动脉造影检查，由2名心内科医师根据Gensini评分评估CAD严重程度。采用实时荧光定量PCR（real-time PCR）检测MTHFR与APOE的基因型，并通过桑格测序（Sanger sequencing）进行验证。收集受试者的年龄、性别、吸烟、饮酒、高血压、糖尿病、血脂异常及体质量指数（BMI）等环境危险因素信息。采用SPSS v16.0软件开展χ²检验、二元logistic回归分析及有序多分类logistic回归分析等统计学处理。 研究结果：本研究纳入的CAD组与非CAD组所有受试者的基因型均成功检出，且与Sanger测序结果的一致性达100%。MTHFR C667T位点的CT、TT基因型在CAD患者中的分布频率高于非CAD对照者（OR=1.99，95%CI：1.34~2.95；OR=1.77，95%CI：1.18~2.67；均p<0.05），而MTHFR A1298C位点的AC基因型在CAD患者中分布频率更低（OR=0.71，95%CI：0.50~1.02；p<0.05）。MTHFR C667T位点的CT、TT基因型与CAD发病风险显著相关（OR=1.44，95%CI：1.27~3.67；OR=1.56，95%CI：0.88~2.78；p<0.05）。APOE的基因型与等位基因频率在CAD患者与非CAD对照者间均无显著差异（均p>0.05）。在Gensini评分≥72的CAD亚组中，MTHFR C667T位点TT基因型与APOE ε4+基因型的检出率更高（分别为p=0.040与p=0.028）。同时，携带MTHFR C667T位点TT基因型的患者血清同型半胱氨酸（Hcy）水平更高，而携带APOE ε4+基因型的患者血清载脂蛋白E（ApoE）与低密度脂蛋白胆固醇（LDL-C）水平均高于其他基因型患者。 研究结论：本研究显示，在中国汉族人群中，MTHFR C667T位点的单核苷酸多态性与CAD发病风险相关。此外，MTHFR C667T位点TT基因型与APOE ε4+基因型可能会增加CAD的严重程度，而血清高Hcy、LDL-C及ApoE水平可能参与了这一病理进程。