The Relationship Between Polymorphisms In Endothelial Cell Nitric Oxide Synthase Gene And Ischemic Cerebrovascular Disease In Henan Han Population

BackgroundIschemic cerebrovascular disease,ICVD, which has high incidence, mortality, and disability, is a common and serious disease and now has become one of three most fatal diseases in the world. Although the accumulation of traditional risk factors often leads to ICVD, however, there is a population of patients without such risk factors who develop ICVD. New researchers has discovered that ICVD is related with both hereditary and environmental factors. Recently, many researchers focus on the predisposing genes of the related risk factors of cerebral infarction. Thus the eNOS gene is considered to be an important gene for ICVD.Nitric oxide (NO) synthesized by endothelial NO synthase plays a key in vascular regulation and atherosclerosis. Studies suggest the basal release of NO by the endothelium contributes to basal vascular tone and regulates blood flow and blood pressure. NO also inhibits the proliferation of smooth muscle cell. Furthemore, NO protects against platelet aggregation and inhibits platelet adhesion to vascular endothelium. In addition , NO inhibits leukocyte adhesion to endothelium. All of these processes are important events during atherogenesis. Dysfunction of this important mechanism may promote atherosclerosis.Several studies have reported polymorphism of the eNOS gene are associated with ischemic cerebral vascular diseases(IVCD). Among the reported polymorphyism of theeNOS gene, association of a variable number of tandem repeat ( VNTR) polymorphism inintron 4 of the eNOS gene with cardiovascular and ischemic stroke have been reported ,still the relationship between the VNTR polymorphism and ischemic stroke remains to becontrary. In order to study the relations of genetic risk factors with IVCD in Henan Hanpopulation, we performed a case-control study to determine whether the eNOS 4a/bpolymorphism are associated with risk of IVCDObjectiveTo determine whether a 27bp VNTR polymorphism in eNOS gene is associated with IVCDin Henan Han population.Methods1 Study population1.1 Patients We investigated 488 unrelated patients with IVCD ( 298 men and 190 women,mean age 60.58 years).All patients were selected from the department of neurology in Henan province hospital were enrolled from December 2004 to July 2006 , and all cases underwent head CT or MRI .The diagnosis of IVCD was defined according to the 1995 China criteria for ischemic cerebralvascular disease.1.2 Controls The 420 unrelated controls were selected from subjects in outpatient department who underwent regular check-up examinarion(234 men and 186 women,mean age 56. 28 years).The controls had no history of IVCD and family medical history of IVCD.All of them are Han population and without cancer, epilepsy and hepatic or renal diseases.2. Determination of a 27bp VNTR in eNOS gene polymorphismDNA was extracted from white cells,and stored at -20℃. eNOS4b/a genetype were determined by the polymerase chain reaction (PCR)with oligonucleotide primer (sense, 5' -AGGCCCTATGGTAGTGCCTT-3' ,Antisense 5' -TCTCTTAGTGCTGTGGTCAC-3' ) that flank the region of the 27bp direct repeat in intron 4 as described by wang,with minor modification. PCR products were separated by electrophoresis in 2% agarose gel. fragment were visualized under ultraviolet light after EB staining. Two alleles were found : A 420bp band indicated five repeats of the 27-bp(b allele), and a 393bp band indicated four repeated of the 27-bp(a allele). Three genetypes, namely a/b heterozygous, b/b homozygous,a/a homozygous, were identified.3 Statistical analysis:Genotype and allele frequencies were counted by the gene-counting method and comparedusing the x~2 analysis. Hardy-weinberg equilibrium was confirmed with the x~2 test .Allstatistical analysis were performed with SPSS 11.0 soft ware.Results:1. The genotype distribution of eNOS 4a/b polymorphism, was compatible with the Hardy-Weinberg equilibrium in the cases and controls.2. There are three kinds of genetypes of a 27bp VNTR in eNOS polymorphism,namely a/b heterozygous, b/b homozygous, a/a homozygous, were identified. The frequencies of the three genetypes were as follows: aa-type 2.3%,ab-typel8.4%,bb-type79.3% in patient group and aa-type 0.95%,ab-type 13.57%,bb-type 85.48% in control group and the a allele frequency was 11.5 % versus 7.7% in the two groups. The frequencies of the three genetypes were significantly different between patient groups and controls ( x~2=6.671, P<0.05).The frequency of a allele was significantly higher in patient groups than that in controls(x~2=7.168, P<0.05).3. Among the normal subjects,the frequency of these three gene mutation in the Henan Han population is significantly lower than the other races of Asian and European. There were possibly exists ethic or geographic diference in a 27bp VNTR in eNOS polymorphisms.Conclusion1. There is association between polymorphisms of eNOS VNTR 4a/b gene and ischemic cerebrovascular disease. The eNOS 4a/b mutation may be an independent risk factor.2. There are eNOS 4a/b polymorphism in Henan Han population.We found significant differences of these three gene polymorphism among Chinese and Asian , European. There may be diversity among diffenent races and regions.