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

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.