Identification Of Polymorphism Of INOS Gene Associated With Essential Hypertension

Identification of polymorphism of iNOS gene Associated with Essential HypertensionIntroductionEssential hypertension (EH) is a common complex disease and its pathogenesis remains elusive. The cardiovascular and cerebrovascular diseases caused by the essential hypertension have been the most important cause of death in both developed and developing courtries.As all know, Nitric oxide (NO), a potent vasodilator, the smallest known bioactive product of mammalian cells, can be produced by three enzyme, nNOS, eNOS and iNOS in most cell types, iNOS exoression is induced in various tissues, including some relevant to the cardiovascular systems, cardiac and vascular smooth muscle, renal tubules, and afferent arteriole. The iNOS gene spans --～37 kb and contains 26 exons. The control of expression of iNOS, which is complex, is for the most part at the transcriptional level.To determine the relationship of genetic variation in the regulatory region of the iNOS gene with the essential hypertension in Chinese population, the affected sib-pair study and case-control study were used. Meanwhile, a series of experiments were performed to identify the functional consequences of the variant that modify the susceptibility to essential hypertension. To our knowledge, this is the first description of iNOS-1026C/A association with the essential hypertension and the first functional identification for this SNP site.Materials and MethodsMaterials:1. Human Hepatoma Bel cell 2. Reagents for STR detection3. Reagents for real-time PCR4. Reagents for gene cloning and luciferse assay.5. Reagents for Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (CHIP).6. Reagents for Western blotting.Methods:1. All subjects of 67 families were Han Chinese in origin and selected from the high prevalent region of hypertension in Liaoning province northeastern China. At the region of 17qcen-17q 11.1 and 17q 21-17q 22 at human chromosome 17, two microsatellite markers of D17S1878 and D17S932 were selected, respectively. The polymorphism of D17S1878 and D17S932 sites were genotyped with Genetic Analyzer and GeneScan Software. Discordant sib pair analysis and affected sib pair analysis were used in linkage analysis.2. 610 subjects were selected for case-control study randomly from high prevalent region of hypertension in Liaoning province northeastern China. The polymorphism of iNOS-1026C/A site was detected by real-time PCR. After association analysis, non-conditional Logistic analysis was used to determine the relationship between the SNP and essential hypertension excluding the interaction of the other risk factors.3. Two different constructs containing -1026C/A were generated by PCR amplification and cloning into pGL3-Basic. Transient transfection was performed in Bel cells using liposome. A luciferase activity assay was undertaken.4. EMSA and ChIP were performed to identify the transcription factor binding to the iNOS-1026C/A. The specific antibody against to YY1 was added to reaction for indentifying the specific responsive element.5. The LPS with different concentrations was used to stimulate YY1, and the western blotting was performed to detect the expression of the YY1 protein. Results1. In the family-based studies, the analysis for subject characteristics suggested that the elder, the drinker and irascible persons were more liability to hypertension. Meanwhile, BMI, TG in the hypertensive affected sibs were higher than those in the normotensive sibs, significantly. The similar results were acquired in the case-control studies. The age, TG and BMI were risk factors in this population.2. Sixty-seven pedigrees were analyzed with affected sib pair analysis, the t values of the D17S1878 and D17S932 were 1.88 and 3.95(P＜0.05), respectively. These results suggested that the transitivity consistency of the D17S1878 and D17S932 sites were higher than the expected (25%).3. In the case-control study, there was significant association between the iNOS-1026C/A and essential hypertension. After the non-conditional Logistic analysis, genotype -1026AA was one of the independent risk factors for EH (OR=0.129, 95% CI 0.053～0.318), and it would be a protective factor for EH.4. The different constructs with -1026C/A were detected in reporter activity, the transcription activity of the -1026A was higher than that of the -1026C.5. Putative YY1 transcription factor binding element in iNOS gene on -1026C/A was identified by EMSA, showing that -1026C probe could form protein-DNA complex with nuclear extracts from Bel cells, while -1026A couldn't. Meanwhile, this binding to YY1 under in vivo conditions could be confirmed by the ChIR6. In the luciferase assay and EMSA, it could be indentified that the binding affinity of the YY1 responsive element to YY1 could be changed by the LPS. Meanwhile, the expressing quantity of the YY1 could be increased by LPS using Western blotting.Conclusions1. In our study population, some predisposing genes for the essential hypertension were located at the region of 17qcen: 17q 11.1 and 17q 21-17q 22. 2. The polymorphism of iNOS—1026C/A site was associated with the essential hypertension. The genotype AA would be the protective factor for the essential hypertension.3. -1026C/A polymorphism in iNOS promoter altered the binding affinity of YY1 to responsive element, and led to decreased transcriptional activity of iNOS promoter with -1026C compared with -1026A.4. LPS could stimulate the expression of the transcription factor YY1 and enhance binding affinity of YY1 to responsive element to increase iNOS promoter activity.