A Population-based Study Of The NOS Gene Family Polymorphism For Ischemic Stroke In A Chinese Population

Ischemic stroke is an acute neurologic event resulting from vascular occlusion, which leads to death of neural tissue of the brain and resulting in loss of motor, sensory and/or cognitive function. It is the second cause of death and the leading cause of disability in China. Ischemic stroke accounts for approximately 80% of all strokes. Aspirin and anticoagulants are used as preventative therapy, but estimates indicate that the incidence of the disease still increased in this decade. No medical treatment is approved for the treatment of ischemic stroke other than tPA, a thrombolytic factor, which has to be administered within 3 hours after stroke. Only 1% to 2% of stroke patients meet the criteria for treatment with this thrombolytic agent. Therefore, the fundermental strategy to solve the problem is taking the effective measures to prevent the disease. Ischemic stroke is a complex disease caused by genetic and enviromental factors. In which genetic factors play an important role.Investigations demonstrated that the concordance rate is higher for monozygotic twins than dizygotic twins. A positive history of paternal or maternal stroke significantly increased the risk of stroke in offspring. The medical treatment mode in the 21st century is converting from diagnosis- therapy mode to early warning-precaution mode, accordingly if we can identify the susceptible individual, then perform the individual precaution and therapy, the incidence of the ischemic stroke may decrease and the human health may improve.Single nucleotide polymorphism(SNP) is a molecule polymorphism resulting from single nucleotide insertion, substitution or depletion. The majority of SNPs are the genitic marker of diallele, thus they are easy to be detected. The final version of the sequence map of Human Genome project covered about 99% DNA sequence of the whole genome. However, the analysis of the differences among individuals found that 90% of the sequence are identical in all individuals, only 10% of the sequence are variant. Among which variable number of tandem repeats presents about 10%, SNPs present about 1%, but the variants of SNPs contribute to the differences of the phenotype among individuals. Therefore, investigating these variants systematically in the whole genome, theoretically, can reveal any of the common phenotype, then reveal the susceptibility and the medicine sensitivity of the complex diseases. So, the genetic research of the complex disease is being the hot spot of lots of governments, institutes and medicine enterprises.Before the genome-wide association analysis is used widespreadly, the sporadic population-based candidate gene association analysis is the main method to screening the susceptible gene of the complex disease. In the past, the research of the etiology of the ischemic stroke obtained some achievements, involving①lipid metabolism-related genes;②inflammation reaction-related genes;③renin-angiotensin system correlated genes;④eNOS gene;⑤PDE4D gene. But the results of these researches are hard to duplicated. Artherosclerosis (AS) is the main etiology of the ischemic stroke, and the proteins which the members of NOS gene family (NOS3, NOS1, NOS2A) encode are all related to the process of AS.NOS3 gene encodes endothelial nitric oxide synthase (eNOS), eNOS is mainly expressed in the vascular endothelial cell, which catalyzes L-arginine to generate nitric oxide (NO) and citrulline. Endothelial -derived NO relaxes vascular smooth muscle, mediates vascular tone and blood pressure, inhibits aggregation of platelets and white cells, prevents the oxidation of low-density lipoprotein cholesterol (LDL), reduces vascular smooth muscle cell proliferation, thus NO plays a central role in inhibiting artherosclerosis. NOS3 gene is an important candidate gene in the genetic research of the ischemic stroke. Many studies investigate the association of the NOS3 gene polymorphism with ischemic stroke, but the results were mostly paradoxical. NOS1 gene encodes neuronal nitric oxide (nNOS), nNOS is mainly expressed in the neuron and nitrergic fiber. The nitregic fibers dominate the adventitia of brain and some periphery artery. Neuronal-derived NO is the main resource of NO in arteriole, which inhibits the arteriosclerosis. NOS2A gene encodes inducible nitric oxide (iNOS). Macrophages activation penetrates the whole processes of AS, and iNOS is one of the important inflammation mediators produced by macrophages. Inducible-derived NO promotes the AS in its early, developed and advanced processes. In the early period iNOS facilitated the oxygenized modification of LDL infiltrated to the endothelium; in the developed period iNOS promotes the inflammation reaction of the vessel wall, promotes the vascular smooth muscle cell proliferation and differentiation; in the advanced period iNOS promotes the vascular smooth muscle cell apoptosis, which leads to unstable plaque and ischemic stroke event. The members of the NOS gene family are not only associated with AS, and then ischemic stroke in the biological function, but also have homology of the DNA sequence. They were potential candidate genes for ischemic stroke. But there is no systemic research involving in the association of NOS gene family polymorphisms with ischemic stroke, and in the association of NOS1, NOS2A gene polymorphisms with ischemic stroke. Therefore, the present study investigates the relationship of NOS gene family polymorphisms with ischemic stroke to elucidate their genetic mechanism of the disease.Different pathogenesis may reside in the arteriosclerotic thrombotic cerebral infarction due to large or middle vessel occlusion and in the lacunar infarction due to small vessel occlusion. Angiostenosis is the intermedial phenotype of the ischemic stroke, then cerebral infarction with angiostenosis is the exacter phenotype of arteriosclerotic thrombotic cerebral infarction. Therefore we investigate the association of NOS gene family polymorphisms with arteriosclerotic thrombotic cerebral infarction, lacunar infarction and cerebral infarction with angiostenosis.Evidences showed that NOS3 gene polymorphism is related to hypertension and coronary artery disease (CAD); NOS2A gene polymorphism is related to hypertension and the severity of angina; the activity of NOS1 gene plays a key role in the long-term adjustment of arterial blood pressure. Therefore, we investigate the association between NOS gene family polymorphisms with the risk factors of ischemic stroke such as hypertension, CAD and diabetes mellitus.Evidence indicated that mice with hypercholesteremia had dereased expression of endothelial NOS1, then leading to vascular endothelial cell damage. Overexpression of iNOS results in increased production of oxygen free radical in carotid arteries of rabbits with hypercholesteremia. Therefore, we investigate the association of NOS gene family polymorphisms with the level of plasma total cholesterol, plasma total triglyceride and body mass index by quantitative trait analysis.Materials and methods1. SubjectsA total of 605 unrelated patients and 313 unrelated control subjects were recruited in this study. These 605 patients (430 males and 175 females), aged 60.14±11.65 years, were consecutively hospitalized at the First Hospital of Jilin University, Changchun, China, during the period between 2005 and 2006. Diagnosis of stroke was made independently by at least two well-trained neurologists based on strict neurological examination, computed tomography, or magnetic resonnance imaging. Detailed information regarding their medical history, medication and clinical presentation was taken through either their close relatives or themselves. The clinical materials include sex, age, body mass index, lipid profile, presence of diabetes, hypertension, cigarette smoking status and alcohol resistance, electrocardiogram and so on . Some patients were examed by ultrasound of brain blood vessel and neck blood vessel, and their condition of angiostenosis had been recorded. The ischemic stroke group was divided into four subgroups:(1) arteriosclerotic cerebral infarction(resulting from big artety and middle artery occlusion), 118 cases; (2)lacunar infarction(resulting from small artery occlusion), 385 cases; (3)mixed cerebral infarction(arteriosclerotic cerebral infarction with lacunar infarction), 102 cases; (4)Ischemic stroke with angiostenosis, 127 cases. The 235 unrelated control subjects (186 males and 127 females), aged 59.17±9.15 years, were healthy individals, coming from Medical Examination Center of the First Hospital of Jilin University. The control group were well matched in age and sex.All the subjects gave written informed consent for the genetic analysis. The whole blood samples were taken from them for extraction of genomic DNA. The present study has been approved by the ethics committee of Jilin University, Changchun, China.2. Genotyping of SNPsFive single nucleotide polymorphisms (SNPs) were selected for genotyping with PCR-based restriction fragment length polymorphism (RFLP) analysis.3. Statistical analysisThe Hardy-Weinberg equilibrium for genotypic distributions was tested using the chi-square (X2) goodness-of-fit test. Allelic association and LD degree were tested by the cocaphase, quantitative trait analysis was tested by the Qtaphase, they were built-in program of the UNPHASED program (Dudbridge, 2003) and genotypic association was tested by the SPSS program for windows 15.0. Logistic regression analysis was used to assess the association of the polymorphisms with the disease and with other tradiational risk factors. Results and Conclusions1. Analysis of the frequency of genotypic and allelic association of the NOS1, NOS3, NOS2A genes with the ischemic stroke:The frequency of genotype GG of rs9658281 of NOS1 gene was significantly higher in ischemic stoke group than controls (P=0.021, OR=1.473). The frequcncy of G allele of rs9658281 of NOS1 gene was significantly higher in ischemic stroke group than controls (P=0.048, OR=1.362), especially in female patients. After adjustment of the traditional risk factors by logistic regression, the P value was 0.023. We postulated that the NOS1 gene was associated with ischemic stroke.2. Analysis of the frequency of genotypic and allelic association of the NOS1, NOS3, NOS2A genes with the ischemic stroke subgroups:(1) After delaminated the ischemic stoke group into subgroups, we found that the frequency of GG genotype of rs9658281 of NOS1 gene was significantly higher in lacunar infarction subtype than in controls(P=0.015, OR=1.579). The frequcncy of G allele of rs9658281 of NOS1 gene was significantly higher in lacunar infarction than in controls (P=0.042, OR=1.422), especially in female patients. After adjustment of the traditional risk factors by logistic regression, the P value was 0.014. We postulated that the NOS1 gene was associated with lacunar infarction.3. Analysis of the frequency of genotypic and allelic association of the NOS1, NOS3, NOS2A genes with the ischemic stroke risk factor, such as hypertension, CAD and diabetes:(1) The frequency of genotype GG of rs1800780 of NOS3 gene was significantly higher in ischemic stoke with hypertension than in that without it (P=0.042, df=2). The frequcncy of G allele of rs1800780 of NOS3 gene was significantly higher in ischemic stroke with hypertension than in that without it (P=0.019, OR=1.431). These results suggested that the NOS3 gene was associated with hypertension.(2) The frequency of genotype AC+CC of rs28944190 of NOS2A gene was significantly higher in ischemic stoke with CAD than in that without such a disease (P=0.007, OR=1.957). The frequcncy of C allele of rs28944190 of NOS2A gene was significantly higher in ischemic stroke with CAD than in that without it (P=0.026, OR=1.560). These results suggested that the NOS2A gene was associated with CAD.4. Analysis of the frequency of allelic association of the NOS1, NOS3, NOS2A genes with quantitative traits, such as plasma total cholesterol, plasma triglyceride, body mass index etc.:(1) rs9658281 polymorphism of NOS1 gene was associated with plasma total cholesterol significantly. We postulated that people carrying G allele had poor protection against hypercholesteremia causing vascular endothelium injury. So it was easy to result in the occurrence of atherosclerosis and ischemic stroke.(2) rs28944190 polymorphism of NOS2A gene was significantly associated with plasma total cholesterol. We postulated that the people carrying A allele may have higher iNOS gene expression, so iNOS and hypercholesteremia synergy result in functional impairment of vessel endothelium.(3) rs28944190 polymorphism of NOS2A gene was significantly associated with plasma total triglyceride. We guessed that NOS2A gene and hypertriglyceridemia may have synergy on ischemic stroke The present study involvs the front topic of the post human genome era. The results of the study will conduce to elucidate the genetic mechanisms of ischemic stroke and its subgroups, the risk factor of ischemic stroke, such as hypertension, CAD and diabetes to investigate the differences of ischemic stoke in hypercholesteremia or hypertriglyceridemia among individuals. It provides new clues of the early precaution, new drug development and individualized theraphy of the ischemic stroke on the level of the molecule.