Endothelial nitric oxide synthase gene polymorphism in women with idiopathic recurrent miscarriage

BACKGROUND: Lack of endothelium-derived nitric oxide is associated with vasospasm and vascular infarction. We investigated the relationship between idiopathic recurrent miscarriage and a polymorphism of the gene encoding endothelial nitric oxide synthase (NOS3). METHOD: In a prospective case-control study, 105 women with idiopathic recurrent miscarriage and 91 healthy controls were investigated. We used the polymerase chain reaction to identify the different alleles of a 27 base pair tandem repeat polymorphism in intron 4 of the NOS3 gene. RESULTS: The wild type B allele was identified on 329 out of 392 chromosomes (frequency 0.84). The polymorphic A allele was present on 63 chromosomes (frequency 0.16). The genotype frequencies were as follows: 68% (B/B), 31% (A/B) and.5% (A/A). The distribution of genotype frequencies was significantly different between the study and control groups for allele A/B heterozygotes (NOS3(A/B)) (36.7 versus 23.8%, P = 0.03, OR 1.6, 95% CI 1.1--3.8). Only one individual was homozygous for the A allele (NOS3(A/A)). She was in the study group. Between women with primary and secondary recurrent miscarriages, no statistically significant difference between the distribution of NOS3(A/B) genotypes (28 versus 34%) was observed. CONCLUSIONS: These data support a role for the NOS3 gene as a genetic determinant of the risk of idiopathic recurrent miscarriage.     

A polymorphism of the interleukin-6 gene promoter and idiopathic recurrent miscarriage

BACKGROUND: Cytokines have been described to play a major role in the pathogenesis of idiopathic recurrent miscarriage (IRM). We investigated the association between IRM and a polymorphism of the interleukin-6 (IL-6) gene and IL-6 serum levels. METHODS: In a prospective case-control study, we studied 161 women with IRM and 124 healthy controls. Peripheral venous puncture, DNA extraction and PCR were employed to genotype women for the presence of a polymorphism at position -174 in the promoter region of IL-6. Serum IL-6 levels were assessed by a commercially available ELISA. RESULTS: Allele frequencies among women with IRM and controls were 63.4 and 58.1% respectively for allele G (wild type), and 36.6 and 41.9% respectively for allele C (mutant). No association between allele C and the occurrence of IRM was found (odds ratio 0.8; 95% confidence interval = 0.57-1.12; P = NS). IL-6 serum levels were not significantly different between genotypes and between the study and control groups. CONCLUSIONS: This is the first report on an IL-6 polymorphism in IRM. Although known to alter IL-6 expression, the IL-6 polymorphism investigated was not associated with IRM and alterations in IL-6 serum levels in a Middle-European Caucasian population.     

Polymorphisms of the endothelial nitric oxide synthase gene in premenopausal women with polycystic ovary syndrome

Objectives

To investigate the association of two common genetic polymorphisms of the gene encoding for endothelial nitric oxide synthase (Nos3), the enzyme catalyzing the production of nitric oxide (NO), with occurrence of the polycystic ovary syndrome (PCOS).

Methods

In a prospective case–control study, we analyzed 2 polymorphisms of the Nos3 gene cluster (Nos 3 exon 7 Glu298Asp and 27-base pair repeat in intron 4 of Nos3) in a series of 210 premenopausal Caucasian women with PCOS and 171 healthy controls using pyrosequencing and PCR, respectively. Women completed a detailed questionnaire and underwent a peripheral venous puncture, ultrasonography, and a standardized oral glucose tolerance test (OGTT).

Results

Genotype frequencies were not significantly different among women with PCOS and controls for the exon 7Nos3 and the intron 4Nos3 polymorphism (p = 0.3 and 0.2, respectively).

Conclusions

In our series, two common polymorphisms of the Nos3 gene cluster were not associated with occurrence of PCOS.     

Endothelial nitric oxide synthase gene polymorphism in a cohort of Egyptian women with recurrent spontaneous miscarriage

The risk of miscarriage is enhanced by genetic and environmental factors. Several studies indicated that there was an association between endothelial nitric oxide synthase activity, implantation, and the maintenance of pregnancy. However, the results of these studies remain controversial. The aim of our study was to determine the association between recurrent spontaneous miscarriage (RSM) in Egyptian women and two common polymorphisms of the endothelial nitric oxide synthase (eNOS) gene: intron 4 and T-786C. The study was conducted on a total number of 100 participants, 50 patients with RSM and 50 age-matched healthy controls. Patients and controls were investigated for the two polymorphisms. Genotyping of the eNOS intron 4 polymorphisms was performed by PCR assay. Our results indicated that there was a significant difference in the frequencies of the genotypes between case and control groups for the polymorphisms in both intron 4 (odds ratio (OR) 5.09, 95 % confidence interval (CI) 2.091–12.396, P?<?0.001) and T-786C (OR 7.389, 95 % CI 3.043–17.942, P?<?0.001). In our study, the eNOS polymorphisms (in intron 4 and T-786C) were found to be significantly associated with increased risk of RSM in Egyptian women.     

Lack of consistent association between endothelial nitric oxide synthase gene polymorphisms, homocysteine levels and recurrent pregnancy loss in tunisian women

PROBLEM: Polymorphisms of the endothelial nitric oxide synthase (eNOS) gene have been associated with reduced vascular NO production or increased level of homocysteine, and evaluated as risk factors for recurrent pregnancy loss (RPL). Therefore, in this case-control study, we aimed to determine the effects of some eNOS functional polymorphisms: the 27-bp intron 4 repeat, the 894G/T of exon 7, and the promoter substitution -786T/C, in women with RPL. METHOD OF STUDY: We genotyped 350 patients with RPL and 200 healthy women by polymerase chain reaction (PCR) and restriction fragment length polymorphism-PCR (RFLP-PCR). The homocysteine total plasma concentrations (tHcy) were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: None of the eNOS polymorphisms-related alleles, genotypes, and haplotypes were associated with RPL. The tHcy were similar between patients and controls; no significant association between tHcy levels and eNOS genotypes could be evidenced. CONCLUSION: The present study identified a lack of association between eNOS gene polymorphisms, the risk of RPL and tHcy levels.     

一氧化氮合酶3基因多态性与复发性早期自然流产的关系

目的探讨一氧化氮合酶3(nitric oxide synthase 3,NOS3)基因第4内含子27bp数目可变串联重复序列(variable number of tandem repeat,VNTR)多态性和第7外显子894(G/T)多态性与复发性早期自然流产(recurrent early spontaneous abortion,RESA)的相关性。方法选取140例RESA患者和140名健康妇女,应用聚合酶链反应-琼脂糖凝胶电泳法检测NOS3基因第4内含子VNTR多态性,聚合酶链反应-限制性片段长度多态性分析技术检测第7外显子894(G/T)多态性。结果RESA组aa ba基因型频率和a等位基因频率与正常对照组相比差异有统计学意义(χ2=4.51,P<0.05;χ2=4.29,P<0.05)。与bb基因型相比,携带a等位基因的妇女与RESA显著相关(OR为1.8,95%CI:1.04~3.24)。RESA组TT GT基因型频率和T等位基因频率与正常对照组相比差异无统计学意义(χ2=1.16,P>0.05;χ2=1.12,P>0.05)。与GG基因型相比,携带T等位基因的妇女与RESA无相关。结论NOS3基因第4内含子27bp数目可变的串联重复序列多态性与复发性自然流产密切相关,NOS3基因第7外显子894G/T多态性与RESA无明显相关性,a等位基因是RESA重要的遗传易感基因。     

Haplotype analysis of the endothelial nitric oxide synthase gene in asthma

Nitric oxide (NO) is an important mediator of physiologic processes in the airways. Evidence exists that genetic factors affect NO formation and contribute to the pathophysiology of asthma. The aims of this study were to determine the endothelial NO synthase (eNOS) haplotypes in Czech asthmatics and control subjects and examine their relation to asthma. We analyzed a total of six polymorphisms. Two SNPs in the promoter (C-786T and C-691T), two variants in the introns (27-bp repeat in intron 4 and G11T in intron 23), and two others in the exons (C774T in exon 6 and G894T in exon 7) were genotyped in 610 subjects (asthma, n = 294; healthy controls, n = 316), and a case-control association study was conducted. No significant differences in allele or genotype frequencies for individual polymorphisms were observed between patients with asthma and controls after correction for multiple comparisons. Nevertheless, a G to T exchange in intron 23 was related with specific sensitization for feather (p = 0.008, p(corr) < 0.05). However, the common haplotype -786T/-691C/27-bp 5 repeat variant/774C/894G/11T was associated with lower risk of asthma (p = 0.001, p(corr) < 0.05, odds ratio = 0.58, 95% confidence interval = 0.46-0.73). These findings suggest that endothelial NOS variants may be one of the factors participating in protection or susceptibility to asthma in our population.     

Polymorphisms in the annexin A5 gene promoter in Japanese women with recurrent pregnancy loss

Recent findings have raised the possibility that polymorphisms within the annexin A5 gene (ANXA5) promoter contribute to the etiology of recurrent pregnancy loss (RPL). In our present study, 243 Japanese women who had suffered more than three fetal losses and a group of 119 fertile controls were genotyped for four ANXA5 gene promoter single-nucleotide polymorphisms (SNPs; SNP1-4: g.-467G >A, g.-448A>C, g.-422T>C, g.-373G>A) previously reported to be associated with this disorder. An additional two SNPs located within the 5'-untranslated region of the ANXA5 (SNP5 and 6: g.-302T>G, g.-1C>T) were also evaluated. Our case--control study revealed that the minor allele was significantly more frequent in the RPL group than controls for all six of these SNPs, among which SNP5 showed the highest significance (P= 0.002). As with the M2 haplotype for SNP1-4 (A-C-C-A) for a western population in previous reports, a haplotype comprising all of the minor alleles for SNP1-6 (A-C-C-A-G-T), the third major haplotype in the Japanese population, showed a significantly higher frequency in our current RPL subjects than in controls (P= 0.025). In addition, the second major haplotype (G-A-T-G-G-C) was found to confer a significant risk of RPL (P= 0.036), implicating SNP5 as a major risk determinant for this disease. Our present findings support the hypothesis that genomic variations within the ANXA5 gene upstream region impact upon the disease susceptibility to RPL. Our data indicate that SNP5 is a novel risk factor for this disease in the Japanese population.     

Prevalence of endothelial nitric oxide synthase gene polymorphisms in patients with atopic asthma

BACKGROUND: Asthma is a common multifactorial disease, the aetiology of which is attributable to both environmental and genetic factors. The endothelial nitric oxide synthase (NOS3) gene has been implicated in asthma pathogenesis. OBJECTIVE: This study investigated associations of 27 base-pair tandem repeat polymorphism in intron 4 and the Glu298Asp (G894T) variant of the NOS3 gene with atopic asthma in a Czech population. METHODS: Polymerase chain reaction was used to determine the NOS3 genotypes in subjects with atopic asthma (n = 163) and random controls (n = 209). RESULTS: The NOS3 allele or genotype distributions did not differ significantly between the control and asthma groups. However, the common genotype (bb) of the NOS3 polymorphism in intron 4 was found to be significantly associated with total IgE levels (P = 0.006), specific IgE levels for feathers (P = 0.0002) and a positive skin prick test for hay (P = 0.004). In one atopic patient, we identified an additional 27-bp repeat in the NOS3 gene (NOS3c), which occurred in heterozygous combination with the NOS3b allele (NOS3b/c genotype). In addition, we describe a new polymorphism (A5495G) in the NOS3 gene, which was in almost complete linkage disequilibrium with the NOS3 repeat polymorphism in intron 4. The Glu298Asp variant was not associated with asthma and/or related atopic phenotypes in our study. CONCLUSION: Neither the NOS3 'b' allele nor the NOS3 'b/b' genotype showed any general association with atopic asthma, but they were associated with atopy-related phenotypes. We conclude that the NOS3 gene polymorphisms may act as disease modifiers in atopic asthma phenotype in our population.     

Temporal and spatial relationships between lipopolysaccharide-induced expression of Fos, interleukin-1beta and inducible nitric oxide synthase in rat brain

Interleukin-1beta plays an important role in mediating central components of the host response to peripheral infection such as fever and neuroendocrine activation by acting in the brain. The present study assessed whether interleukin-1beta produced in the brain is relevant to neuronal activation and the fever response induced by intraperitoneal injection of bacterial lipopolysaccharide. The distributions of Fos protein, interleukin-1beta protein and inducible nitric oxide synthase messenger RNA, used as an anatomical indicator of interleukin-1beta bioactivity, were compared in brains of animals killed 2, 4 or 8 h after lipopolysaccharide (250 microg/kg) or saline injection. Saline did not induce interleukin-1beta or Fos immunoreactivity in the brain. Interleukin-1beta positive cells were found 2 h after lipopolysaccharide injection in circumventricular organs. Fos immunoreactivity at this time-point was not found in circumventricular organs, but in parenchymal structures such as the nucleus of the solitary tract, paraventricular hypothalamus and ventromedial preoptic area. Fos expression did occur in circumventricular organs only 8 h after lipopolysaccharide injection. This late pattern of Fos expression coincided with the rise in body temperature and the induction of inducible nitric oxide synthase messenger RNA. These data show that after peripheral lipopolysaccharide administration interleukin-1beta is synthesized and bioactive in circumventricular organs. Interleukin-1beta may activate local neurons that induce fever and neuroendocrine activation via projections to the ventromedial preoptic area and the nucleus of the solitary tract.     

Intron 4 polymorphism of the endothelial nitric oxide synthase eNOS gene and early microangiopathy in type 1 diabetes

Nitric oxide (NO) is an endogenous vasodilator involved in inflammatory and autoimmune response, and in the pathophysiology of diabetic vascular disease. Endothelium‐derived NO is formed from l ‐arginine by endothelial NO synthase (eNOS), and earlier studies have provided evidence for altered NO metabolism and impaired endothelial function in diabetes, probably due to polymorphisms in eNOS gene. In the present study we investigated the association of the eNOS gene intron 4 a/b VNTR polymorphism with diabetic microangiopathy in 61 young individuals with type 1 diabetes (T1D), 35 male and 26 female, aged 5.0–29.1 (mean 15.6) years, and followed up for 3.24–11.4 (mean 7.44) years. Ten patients (16.4%) had developed microalbuminuria, three hypertension and two retinopathy. Wild‐type b/b homozygosity for eNOS gene intron 4 VNTR was found in 37 (60.7%) and a/b polymorphism in 24 (39.3%). No significant relationship was demonstrated between eNOS gene intron 4 polymorphisms and microalbuminuria, hypertension or retinopathy in these young individuals. Our findings suggest that a/b polymorphism of the intron 4 eNOS gene is not associated with early onset diabetic microangiopathy.     

反复发热惊厥大鼠脑内NOS/NO体系的变化

目的研究一氧化氮合酶(NOS),一氧化氮(NO)体系与反复发热惊厥(febrile seizures,FS)的关系。方法采用热水浴诱导大鼠FS,隔日1次,每次大鼠进行热水浴的时间不超过5min,共10次。大鼠随机分为2组：正常对照组和发热组,后者又根据惊厥与否进一步分为发热对照组和反复FS组。用原位杂交法观察大脑皮层神经元型NOS(nNOS)mRNA的变化,用分光光度计检测大鼠脑组织及血浆中NO含量,用放射免疫法检测大鼠脑组织cGMP含量。结果在大脑皮层深层,FS组nNOS表达阳性的神经元明显增高,而发热对照组仅出现少量nNOS阳性神经元,正常对照组偶见nNOS阳性神经元;脑组织及血浆中NO含量各组间无统计学意义;FS组脑组织cGMP含量吸显高于正常对照组及高热对照组。结论大鼠反复FS后24h脑内nNOS mRNA表达增高,但此时NO不见增多,脑组织cGMP水平增高,可能由于其他途径调节所致。     

小凹蛋白与内皮型一氧化氮合成酶在人肝硬化组织中的表达及意义

目的检测人肝硬化组织中小凹蛋白(caveolin-1)、内皮型一氧化氮合成酶(eNOS)的细胞定位及蛋白表达水平的变化,探讨caveolin-1的表达对eNOS的影响。方法免疫组织化学染色检测30例肝硬化患者活检后肝组织和30例肝外伤、肝血管瘤患者正常肝组织中caveolin-1和eNOS的细胞定位。Western印迹检测caveolin-1和eNOS的蛋白表达水平变化。结果caveolin-1和eNOS均主要分布于肝窦内皮细胞中,caveolin-1在肝硬化组和对照组表达阳性率有差异,分别为87%和40%(P<0.05)。eNOS在肝硬化组和对照组表达阳性率有差异,分别为33%和66%(P<0.05)。caveolin-1在肝硬化组织中表达较正常肝组织中明显增强。eNOS在肝硬化组织中呈低水平表达,较正常肝组织中表达明显减少。结论肝硬化肝窦内皮细胞的损伤降低了eNOS的表达。caveolin-1的过表达促进eNOS-caveolin-1复合物的形成,加剧了门静脉高压症的发生。     

小凹蛋白与内皮型一氧化氮合成酶在人肝硬化组织中的表达及意义

目的 检测人肝硬化组织中小凹蛋白(caveolin-1)、内皮型一氧化氮合成酶(eNOS)的细胞定位及蛋白表达水平的变化,探讨caveolin-1的表达对eNOS的影响.方法 免疫组织化学染色检测30例肝硬化患者活检后肝组织和30例肝外伤、肝血管瘤患者正常肝组织中caveolin-1和eNOS的细胞定位.Western印迹检测caveolin-1和eNOS的蛋白表达水平变化.结果 caveolin-1和eNOS均主要分布于肝窦内皮细胞中,caveolin-1在肝硬化组和对照组表达阳性率有差异,分别为87%和40%(P＜0.05).eNOS在肝硬化组和对照组表达阳性率有差异,分别为33%和66%(P＜0.05).caveolin-1在肝硬化组织中表达较正常肝组织中明显增强.eNOS在肝硬化组织中呈低水平表达,较正常肝组织中表达明显减少.结论 肝硬化肝窦内皮细胞的损伤降低了eNOS的表达.caveolin-1的过表达促进eNOS-caveolin-1复合物的形成,加剧了门静脉高压症的发生.     

Association of plasma nitric oxide concentration and endothelial nitric oxide synthase T-786C gene polymorphism in coronary artery disease

Nitric oxide (NO) is synthesized from l-arginine by endothelium nitric oxide synthase (NOS3) and plays important roles in many physiologic and pathologic processes. NO involved in the pathogenesis of coronary atherosclerosis. In the present study we hypothesized that polymorphisms of NOS gene might be associated with increased risk of coronary artery disease (CAD) and plasma NO concentrations. The eNOS gene polymorphism was investigated in 241 unrelated CAD patients with positive coronary angiograms and 261 ages matched control subjects without a history of symptomatic CAD. The NOS3 gene polymorphisms were analyzed by RFLP. Plasma NO, lipid profile and other risk factors were also assessed. The genotype frequencies for T-786C polymorphism differed significantly between CAD patients and controls (p=0.041). The mean plasma NO(x) concentrations showed significant differences according to genotypes of T-786C polymorphism in total population only. The mean plasma NO(x) increased in those individuals that are homozygote for C allele in promoter compared with those individuals are heterozygote for this allele and homozygote for T allele in total population and Controls, but no in CAD patients. The present study provides evidences that T-786C polymorphism of the NOS3 gene is associated with CAD. T-786C polymorphism was not associated with increased plasma NO in CAD patients.     

Analysis of polymorphic sites in the promoter of the nitric oxide synthase 2 gene in Brazilian patients with leprosy

Leprosy is one of the most neglected infectious tropical diseases of the skin and the nerves caused by the intracellular pathogen Mycobacterium leprae. The inducible NOS isoform encoded by NOS2A plays a vital role in host defence against bacterial infections. The functional promoter polymorphisms in NOS2A are associated with various autoimmune and infectious diseases. We investigated the association of NOS2A variants with progression of leprosy in a Brazilian cohort including 221 clinically classified patients and 103 unrelated healthy controls. We observed a novel variant ss528838018A/G in the promoter region at position ?6558. The other functional variants were observed with low frequency of minor allele (<0.005). NOS2A promoter variant (?954G/C) was not observed in Brazilian populations, and the new observed promoter variant (ss528838018A/G) as well as other promoter variants were not associated with any clinical forms of leprosy in the Brazilian populations.