The Relationship Between G894T Polymorphism Of Endothelial Nitric Oxide Synthase Gene And Spontaneous Aneurismal Subarachnoid Hemorrhage

BackgroundIntracranial aneurysms (IA) are saccular dilatations of IA that occur most commonly at arterial bifurcations. Aneurysms are only rarely symptomatic unless they ruptured, which typically results in subarachnoid hemorrhage,It is a kind of critical disease with high mortality and morbidity. As the development of minimally invasive surgery and endovascular coiling technique,the morbidity and mortality of patients with ruptured aneurysm have decreased rapidly in recent years. However,the overall outcome of intracranial aneurysms is still not perfect. Because, there is limited understanding to date, about the biological mechanisms associated with the pathogenesis, growth, and rupture of intracranial aneurysms. At present, a fairly broad consensus that genetic and environmental factors contribute to the formation, growth, and rupture of intracranial aneurysms. However, investigations for genetic markers have not been successful in isolating a mutant gene that is directly related to aneurysm formation or weakening of the vessel wall . Different specific genetic factors that affect architecture and function of all layers of the vessel wall may be important for the formation, growth, and rupture of intracranial aneurysms.Nitric oxide (NO) is synthesized from L-arginine by nitric oxide synthase (NOS). Three isoforms of nitric oxide synthase have been described. They are, endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), and immunological nitric oxide synthase (iNOS). Basal cerebral vascular tone requires continuous release of NO by eNOS. It has been suggested that eNOS has both haemodynamic and structural roles. Experimental data from eNOS knockout mice models demonstrated that luminal remodelling is impaired in the absence of eNOS, suggesting that NO derived from eNOS, in addition to its role as a vasodilatator,may have a role in controlling vessel wall architecture. eNOS gene locates in 7q35-7q36,which have 26 extrons and 25 introns. The eNOS gene is proposed as the susceptible gene for some vascular diseases including coronary artery disease, carotid atherosclerosis, hypertension, and abdominal aortic aneurysm. Polymorphisms of the eNOS gene may influence the expression and functional activity of the enzyme. The relationship between subarachnoid haemorrhage (SAH) due to intracranial aneurysm rupture and polymorphism of the eNOS gene is controversial. In this study, we investigated a possible association between aneurysmal SAH and G894T(Glu298Asp) polymorphism in exon 7 of the endothelial nitric oxide synthase gene.Purpose1. To investigate the genotype distribution and allele frequency of the eNOS gene G894T polymorphism of healthy population and aSAH patients, and assess the relationship between eNOS genotype and the onset of aSAH:2. To explore the influence of the genotype distribution and allele frequency of the eNOS gene G894T polymorphism on the sizes of ruptured aneurysms:3. To confirm the relationship between the genotype distribution and allele frequency of the eNOS gene G894T polymorphism and the outcome of aSAH:Methods:The subjects of this case-control study consisted of 58 aSAH patients and 67 normal people .All aSAH patients were verified by DSA /CTA or operation. All members of control group were recruited after the exclusion of aSAH history. Obtain the venous blood of all the members and extract the genomic DNA. Based on the gene sequence of eNOS genes,certain gene regions include interested single nucleotide polymorphisms were selected and related primers were designed .After primers synthesis the polymerase chain reactions were performed to amplify those interested gene regions .The PCR products were confirmed by the 2% agarose gel electrophoresis ,those qualified PCR products were digested with the restriction enzyme and sequenced. Genotype and allele frequency were calculated, Chi square test were used to compare the difference between aSAH and control group to analyze the significance. The patients' characteristics, Hunt-Hess clinical grading score, and Fisher's grading score and GOS grade after 3 months were also recorded .Logistic regression analysis was used to analyze the relationship between eNOS genotype and outcome of patients with aSAH.Results1. There kinds of genetypes and two kinds of alleles were detected in this test. Among the normal subject,the frequencies of the GG,GT and TT genotypes in exon 7 of the eNOS gene were 0.672,0.224 and 0.104,respectively. G and T allele frequencies were 0.784 and 0.216 respectively. Among the aSAH group,the frequencies of the GG,GT and TT genotypes were 0.466,0.293 and 0.241,respectively. G and T allele frequencies were 0.612 and 0.388 respectively.2. Significant differences between patient and control subjects in both genotype distribution and allele frequency were observed for eNOS G894T polymorphism. eNOS GT+TT genotype and T allele were significantly more common in the patient group compared to the control group.3. No significant differences in the distributions of the eNOS G894T genotypes were found with regard to the sizes of ruptured aneurysms. 4. High Hunt-Hess score ,poor Fisher's grade and GT+TT genetype were significantly associated with the outcome of aSAH. Multiple logistic regression analysis showed the eNOS GT+TT genotype was independently associated with an unfavorable outcome (GOS grade 3-5) of aSAH.Conclusion1. Polymorphism in exon 7 of the endothelial nitric oxide synthase gene G894T seems to be a possible risk factor for the onset of aneurismal subarachnoid hemorrhage.2. No significant differences in the polymorphism in exon 7 of the eNOS gene were found with regard to the sizes of ruptured aneurysms.3. Polymorphism in exon 7 of the eNOS gene is a risk factor for outcome of aSAH and GT+TT genetype is was independently associated with unfavorable outcome of aSAH.