感染負荷、發炎基因多形性、代謝症候群與冠狀動脈疾病及急性冠心症危險性之研究

Infectious Burden, Inflammatory Genes Polymorphisms, Metabolic Syndrome and the Risk of Coronary Artery Disease and Acute Coronary Syndrome

戴道福(Dao-Fu Dai)

指導教授 ： 黃瑞仁

國立臺灣大學/醫學院/臨床醫學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.00097

摘要

背景: 愈來愈多證據顯示發炎因子在動脈粥狀硬化病程的重要性。不管慢性狹心症或急性冠狀動脈症候群的發生，都與慢性發炎反應有關。許多大規模的流行病學研究指出CRP、fibrinogen、TNFα、IL-1、IL-6、IL-10的血中濃度與急性冠心症的發生率及嚴重度是息息相關。另外，許多微生物因子例如Helicobacter pylori、Chlamydia pneumonia、Hepatitis A、B、C病毒的感染也曾被提出會增加動脈粥狀硬化的發生率。代謝症候群更是增加冠心病危險性的重要因子。在冠心病和急性冠狀症候群致病機轉中，基因和其他環境危險因子均扮演重要角色。因此，我們假定發炎因子的基因多形性和慢性感染會影響冠心病人的危險性，並進一步分析發炎基因多形性、感染負荷以及代謝症候群對冠心病生成以及急性冠心症的重要性。另外，我們將比較感染負荷與代謝症候群對發炎的貢獻。最後，分析影響預後的重要因子。
方法：從600位接受心導管病人的全血中萃取DNA，以PCR及RFLP以及Gene Scan分析其基因多形性(IL-1ß +3954 C/T, -511 C/T, -31 C/T, IL-1RA VNTR, CRP 1059G/C, CRP intron dinucleotide repeat)，同時分析血漿interleukin-1ß (ELISA)、CRP (immunonephelometry)、NT-proBNP(ELISA)濃度以及病原體的血清抗體 (Anti H.pylori IgG, anti HAV IgG, HBsAg, anti HCV)。臨床資料收集包括病人的基本資料、冠心病的傳統危險因子、代謝症候群因子(modified ATPIII criteria)、冠狀動脈硬化程度 (Gensini Score)以及病人的預後，包含心因性死亡、重大不良心血管事件 (Major Adverse Cardiovascular Events)、Target Vessel Revascularization (TVR)。病人追蹤期為8-26個月。統計分析採用genotype analysis、haplotype analysis 及multiple logistic regression 探討基因多形性與冠心病以及急性冠狀動脈症候群之相關性。此外，採用multiple linear regression探討感染因子及代謝因子對發炎反應以及冠狀動脈硬化程度的影響。最後，以Kaplan-Meier survival curve和Cox propotional hazard models分析病人的預後因子。
結果：IL-1ß基因多形性與冠狀動脈疾病並不相關。CRP1059基因型雖影響其血漿CRP濃度，同時、患有冠心病病人血漿CRP濃度較控制組高，但CRP 1059 G/C多形性與冠心病並無相關性。IL-1ß 基因多形性與急性冠狀動脈症候群有關聯。IL-1ß -31C對偶型和IL-1RA*2對偶型與急性冠狀動脈症候群有保護關聯 (protective association)。同時，IL-1ß -31C對偶型帶原者有較低的血漿IL-1ß和CRP濃度。單項病原體的血清抗體陽性(Anti H.pylori IgG, Anti HAV IgG, HBsAg, anti HCV)或感染負荷與冠狀動脈硬化程度亦無相關性。但是代謝症候群總分與血漿CRP濃度以及冠狀動脈硬化程度成正相關。Multiple linear regression 結果顯示，CRP血漿濃度及代謝症候群總分是冠狀動脈硬化程度(Gensini score)的重要決定子。另外，影響MACEs以及TVR的獨立預後因子包括糖尿病、血漿CRP濃度、血漿NT-proBNP濃度以及代謝症候群。

關鍵字

發炎基因多形性 ； 代謝症候群 ； 冠狀動脈疾病 ； 急性冠心症 ； 感染負荷

並列摘要

Background and Objectives:
The pathogenesis of atherosclerosis involves many inflammatory components. Several cytokines have been shown to play important roles in the atherosclerosis. Both epidemiological and in vitro studies have revealed the central position of inflammation in the pathogenesis of coronary artery disease (CAD) and acute coronary syndrome (ACS). However, the triggers of inflammation remain controversial. Several factors have been encountered such as genetic polymorphisms of inflammatory factors, oxidized LDL, infectious burden, etc. Both CAD and ACS are well-known as diseases with multifactorial causes, therefore, the interplay between genes and environmental factors are crucial in the pathogenesis of coronary atherosclerosis and the rupture of atheroma plaque, which causes ACS. Thus, we want to elucidate the effects of polymorphisms of inflammatory genes on the risks of CAD and ACS in our population. We choose interleukin-1 (IL-1) and C-reactive protein (CRP) as candidate genes in the setting of genetic case-control association studies. IL-1 is one of the most important cytokines in inflammatory process and CRP is one of the best markers in cardiovascular disease. Besides, we want to compare the impacts of infectious burden and metabolic abnormalities on the degree of inflammation and the severity of coronary atherosclerosis. Finally, we want to identify the prognostic factors of patients with CAD.

Methods
We recruited 600 patients undergoing coronary angiography in National Taiwan University Hospital from March 2002 to October 2003. Detailed characterizations of these patients, including demographic data, classical CAD risk factors and history of myocardial infarction, were recorded. Basic laboratory measurement such as complete blood count, blood biochemistry was performed. Clinical data analyses included the metabolic syndrome score, Gensini score, the proportion of major adverse cardiovascular events (MACEs) and target vessel revascularization (TVR) during the follow-up of 8-26 months after inclusion in our study. Metabolic syndrome score was defined as the number of metabolic abnormalities according to the criteria of the National Cholesterol Education Program – Adult Treatment Program III, with slight modification, abdominal circumference was changed to body mass index (BMI ≧25kg/m2). Gensini score was applied to assess the severity of coronary atherosclerosis. Follow-up was performed at the out-patient clinics with detailed questionnaire and MACEs were recorded. Patients with recurrent symptoms of CAD were hospitalized for follow-up coronary angiography and TVR was recorded. The laboratory works included the analysis of DNA and plasma. DNA was extracted from white blood cells and genotyping of IL-1ß +3954 C/T, -31 C/T, -511 C/T and CRP +1059G/C were performed with polymerase chain reaction (PCR) followed by restriction fragments length polymorphisms (RFLPs); genotyping of IL1RA VNTR was performed with PCR and gel electrophoresis; genotyping of CRP intron dinucleotide repeat was performed with PCR followed by gene scan. The plasma markers analysed included high sensitivity CRP (Dade Behring immunonephelometry), IL-1ßß (Quantikine immunoluminometer assay), NT-proBNP (Roche immunoassay), anti Helibacter pylori IgG (Behring enzygnost ELISA), anti HAV IgG, HBsAg and anti HCV IgG (Abbott microparticle ELISA). The statistical analysis was performed with STATA Intercooled 7.0. All continuous variables were presented as mean ± standard deviation and categorical variables were presented as proportion. The difference between cases and controls were examined by student `t test for continuous variables and chi-square test for categorical variables. One way analysis of variance was performed for comparison among groups. Multiple logistic regression was applied to adjust for the possible confounding factors, and multilple linear regression was used to identify the independent predictors of a continuous outcome. For haplotype analysis, we applied EH software to compare the estimate of haplotype frequency between cases and controls. Then we used SAS Genetics to calculate the haplotype estimates for the individuals and applied multiple logistic regression to adjust for the confounding factors. Kaplan-Meier survival curve and Cox proportional hazard model were applied to evaluate the independent prognostic factors for follow-up data.

Results
Inflammatory genes polymorphisms and CAD
Single locus analysis showed that genetic polymorphisms of IL-1ß, IL1RA and CRP were not associated with CAD (Table 3-5). Likewise, haplotype analysis also failed to show the association between these genes and CAD (Table 6), despite the facts that the genetic polymorphisms significantly affect the plasma level of CRP and IL-1ß (Table2).
Inflammatory genes polymorphisms and ACS
Single locus analysis showed that IL-1ß -31 C/T polymorphisms was associated with ACS. The genotype IL-1ß -31C>C had protective effect against ACS, OR=0.54, p=0.02 (Table 10). IL-1RN*2 also had protective association against ACS, OR=0.38, p=0.008 (Table 11). Likewise, haplotype analysis revealed that the haplotype frequencies of IL-1ß +3954/-511/-31 loci were significantly different in the distribution pattern between cases and controls (omnibus test, p<0.05), in which the estimated haplotype IL-1ß +3954C/-31C/-511C was significantly lower in cases than in controls (cases vs controls: 6.4% vs 12.7%, p=0.006).
Infectious burden versus metabolic syndrome on the degree of inflammation and the severity of coronary atherosclerosis
Neither the seropositivity to each pathogen nor the infectious score was associated with the plasma level of CRP and the severity of coronary atherosclerosis. After adjustment for sex, age, smoking and total cholesterol, higher MS score was an independent predictor of higher plasma CRP and higher Gensini score (p<0.001 for both) (Table 15-16). MS score and plasma CRP were also significantly associated with the risk of major adverse cardiovascular event, including cardiovascular mortality, myocardial infarction and stroke. (for one point increase in MS Score, OR:1.3, p=0.001; for 10 mg/L increase in plasma CRP, OR:1.4, p<0.001).
Plasma CRP and NT-proBNP as prognostic indicators of CAD
The combination of plasma CRP ≧ 3 mg/L and NT-proBNP ≧ 682 pg/mL was a strong prognostic indicator of CAD, in terms of MACEs and TVR (Figure 12-13). Cox regression showed the independent predictors for MACEs are: diabetes (HR:2.0, 95%CI: 1.0, 3.7, p=0.04), NT-proBNP (HR:5.4, 95% CI: 2.9, 10.0, p<0.001) and CRP (HR:2.1, 95%CI: 1.1, 4.1, p=0.03); for TVR: diabetes (HR:2.2, 95% CI:1.2,3.9,p=0.01), NT-proBNP (HR:3.0, 95% CI:1.8, 5.0,p<0.001) and CRP (HR:1.8, 95%CI:1.1, 2.8,p=0.02)

Conclusion
The genetic polymorphisms of IL-1 and CRP genes were not associated with CAD, though they affect the plasma concentration of CRP, which was an important predictor of atherosclerosis and future cardiovascular events. However, the polymorphisms of IL-1 gene were associated with ACS, in which inflammation plays major roles. The metabolic abnormalities had prominent effects on the degree of inflammation and the severity of coronary atherosclerosis, while the effects of infectious burdens on these factors were not observed in our study. For subjects with CAD, the combination of plasma CRP and NT-proBNP acts as strong prognostic indicator of MACEs and TVR.

並列關鍵字

Infectious Burden ； Acute Coronary Syndrome ； Coronary Artery Disease ； Metabolic Syndrome ； Inflammatory Genes Polymorphisms

參考文獻

Andreotti F, Porto I, Crea F, et al. Inflammatory gene polymorphisms and ischaemic heart disease: review of population association studies. Heart 2002;87:107-112

Auer J, Leitinger M, Berent R et al. Hepatitis A IgG seropositivity and coronary atherosclerosis assessed by angiography. Int J Cardiol. 2003;90:175-179.

Azzawi M, Hasleton P. Tumor necrosis factor alpha and the cardiovascular system; its role in cardiac allograft rejection and heart disease. Cardiovasc Res 1999;43:850-9

Basso F, Lowe GD, Humphries SE, et al. Interleukin-6 -174G/C polymorphism and risk of coronary heart disease in West of Scotland coronary prevention study (WOSCOPS). Arterioscler Thromb Vasc Biol 2002;22(4):599-604.

Berger P, McConell JP, Nunn M, et al. C-Reactive Protein levels are influenced by common IL-1 gene variations. Cytokine 2002; 17(4): 171-174.