| BackgroundIschemic stroke (IS) is a multi-factorial disease, which is related to both the genetic and environmental factors. Gene-gene and gene-environmental interaction makes great contribution to the risk of IS. With the advance of Human Genome Project, it has become more and more popular in medical fields to clarify the pathogenesis of IS at genetic level, especially IS. In recent years, studies have suggested that endothelial nitric oxide synthase (eNOS), growth hormone(GH) and insulin-like growth factor-I receptor (IGF-1R) gene, which can significantly affect gene expression, have been associated with IS and coronary heart disease (CHD). However, reports are extremely rare presently on the association between the above-mentioned genes and IS in the Han nationality of China, and the interaction between the gene polymorphisms and environmental factors.Objectives1. To explore the association between the T-786C, A-922G and G894T polymorphism in eNOS gene and IS.2. To explore and assess the possible interaction effects between eNOS gene polymorphisms and environmental factors.3. To explore the association between GH1 T1663A gene polymorphism and IS, in addition, to assess the interaction between GH1 T1663A gene polymorphism and IS.4. To explore the possible association between IGF-1R gene G/A (rs2229765), A/G (rs951715) and A/G (rs2593053) polymorphisms and IS.5. To explore and assess the possible interaction effects between the IGF-1R gene polymorphisms and environmental factors.6. Classification tree model was applied to build the risk model for IS.MethodsCandidate genes and case-control study were used to determine the possible the association between gene and IS. A 1:1 matched case-control study was performed. 309 cases were those onset IS patients registered in two general hospitals in Shenzhen. The controls were selected by the same gender and ethnic group, and each pair's ages were permitted to differ within 5 years. The cases and controls were interviewed using the same questionnaire, and the blood samples were drawn in terms of the same conditions and standards. Gene polymorphisms were determined by using Taqman MGB genotyping assay. Univariate test and multiple logistic regression models were used to explore the association between the above-mentioned genes polymorphisms and IS. Haplotype analyses of these polymorphisms were performed using PHASE2.0 software. Additionally, the interaction between genes and environmental risk factors were assessed by multivariate logistic regression model. The odds ratio values (OR) was calculated by using regression model to determine the addition effects among different factors and measure the interaction. Finally, Classification tree model was applied to build up the risk model for IS.Results1. Univariate logistic regression demonstrated that risk factors of IS included income, education, body mass index (BMI), WHR, smoking, hypertension, diabetes mellitus (DM), negative events and TG levels (triglyceride). In multivariate logistic model, smoking and hypertension were positively associated with IS, with OR=5.42 (95%CI: 2.00~14.63) and OR=3.51 (95%CI: 1.83~6.71) respectively, while moderate physical training and history of tea-drinking were inversely associated with IS, with OR=0.10 (95%CI: 0.05~0.22) and OR=0.25 (95%CI: 0.12~0.55), respectively.2. For eNOS T-786c polymorphism, there were no significant difference in the distributions of genotypes between two groups (P=0.132). Stratified by sex, the p values for the genotypes of the above mentioned polymorphism was 0.053 in male. Conditional logistic regression revealed that the CC genotype of eNOS was associated with IS (OR=3.819, P=0.029). After adjustment for confounding factors, eNOS CC genotype was still significant associated with IS (OR=4.533, P=0.047). For eNOS A-922G polymorphism, the frequency of eNOS -922 G allele was significant higher in the patients than the controls (12.14% vs 8.09%, P=0.018). Linear tendency test showed the risk for development of IS raised with increasing G allele (chi-square value=4.886, P=0.027). After adjustment for confounding factors, eNOS A-922G polymorphism was significant associated with IS (OR=2.156, P=0.029).3. There were no significant difference in the distributions of allele and genotypes in GH1 gene T1663A polymorphism between two groups (P=0.124 and 0.358, respectively.). Multiple logistic regressions revealed that the GH T1663A polymorphism may not be an additional risk factor for the development of IS.4. For IGF-R G/A (rs2229765) polymorphism, the frequency of IGF-1R A allele was significant higher in the patients than the controls (50.00% vs 31.93%, P=0.001). After adjustment for confounding factors, AA genotype was significant associated with an increased risk of developing IS with the GG genotype as reference genotype (OR=1.992, P=0.015). For IGF-R A/G (rs951715) polymorphism, the frequency of IGF-1R G allele was significant higher in the patients than the controls (56.15% vs 43.85%, P=0.000). Compared with AA genotype, AG genotype was significant associated with an increased risk of developing IS without adjusting for other confounding factors (OR=2.201, P=0.000). After adjustment for confounding factors, AG genotype was significant associated with an increased risk of developing IS (OR=2.381, P=0.000).5. The positive additive interactions were found between eNOS gene T-786C polymorphism and family history of hypertension, DM, family history of DM and smoking, synergy Index (S) were 3.76, 3.10, 4.22 and 1.63, respectively. The interactions analysis between A-922G polymorphism and environmental factors indicated that a negative additive interaction was found between A-922G and alcohol drinking (S=0.43), and the S for family history of hypertension, family history of DM and smoking were 2.46, 3.24 and 1.99, respectively. No interactions between IGF-1R and environmental factors were found.6. Classification tree model was applied to build up the risk model for IS, and the model had four stratum. Six explanatory variations were screened out in our model. The indexes of the screening test were used to evaluate the fitness of the model. The results revealed that sensitivity and specificity and Youden index were 76.70%, 81.88% and 58.58%, respectively.Conclusions: 1. The classical risk factors are still main reasons of patients with IS in the Han population of Shenzhen city. Therefore it is an important measure to prevent IS in community population to propose healthy life style including proper exercise, control of high blood pressure, high blood fat and weight.2. The T-786C, A-922G polymorphism in eNOS gene and G/A (rs2229765) and A/G (rs951715) polymorphisms in IGF-1R are all significant associated with the hereditary susceptibility of IS in the Han population of Shenzhen city.3. There are obvious interactions between T-786C and A-922G polymorphisms in eNOS gene and environmental factors such as smoking, alcohol drinking, hypertension, diabetes and family history vascular diseases.4. Classification tree model can properly predict the occurrences of IS.5. IS is caused by the interactions between many minor genes and environmental risk factors. It is very important to study their correlation to classify the cause and pathogenesis of IS.
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