eNOS基因G894T多态性与深静脉血栓形成的相关性研究

目的探讨内皮细胞型一氧化氮合酶（eNOS）基因G894T多态性与深静脉血栓形成（DVT）的相关关系。方法利用聚合酶链反应和限制性片段长度多态性（PCR-RFLP）方法,检测103例DVT患者与250例正常对照一氧化氮合酶（eNOS）基因G894T基因多态,并加以对照分析。结果DVT组GG、GT和TT基因型频率分别为83.5%,14.56%和1.94%;对照组的基因型频率分别为89.6%,10.0%和0.4%。两组基因型频率与等位基因频率比较,差异无统计学意义（P〉0.05）。结论eNOS基因G894T多态可能不是河南汉族人群深静脉血栓形成的独立危险因素。     

内皮型一氧化氮合酶基因G894T多态性与青海省汉族妊娠高血压综合征的相关性

目的探讨内皮型一氧化氮合酶(eNOS)G894T基因多态性与青海省汉族妊娠高血压综合征(PIH)的相关性。方法采用限制性片段长度多态性聚合酶链反应(PCR-RFLP)方法对138名妊娠高血压综合征患者和135名正常孕妇e NOS基因G894T多态性进行分型,并测序验证。结果妊娠高血压综合征组基因型频率野生型(GG)、杂合子型(GT)和突变纯合子型(TT)分别为17.4%、82.6%、0;对照组分别为0、95.6%、4.4%,两组基因型频率分布有差异(P0.05)。妊娠高血压综合征组e NOS等位基因频率G、T分别为58.7%、41.3%;对照组分别为47.8%、52.2%,两组比较差异有统计学意义(P0.05),妊娠高血压综合征组G基因频率高于对照组。结论e NOS基因G894T多态性可能与青海省汉族妊娠高血压综合征有关,G等位基因可能为妊娠高血压综合征的易感基因(OR=1.229,95%CI:1.048~1.441),T等位基因可能为妊娠高血压综合征的保护基因。携带GG基因型的孕妇可作为青海省妊娠高血压综合征的易感人群。     

MTHFR基因和eNOS基因多态性与妊娠高血压综合征发病的相关性研究

目的探讨内皮型一氧化氮合酶（endothelial nitricoxide synthase,eNOS）基因第7外显子G894T突变和N5,N10-亚甲基四氢叶酸还原酶（methyenetetrahydrofolate reductase,MTHFR）基因C677T突变与潍坊地区汉族人妊娠高血压综合征（妊高征）发病的关系。方法应用PCR-RFLP方法,对92例妊高征患者（妊高征组）和89例正常妊娠妇女（对照组）的eNOS基因G894T突变和MTHFR基因C677T突变进行检测。结果妊高征组eNOS基因Glu/Glu、Glu/Asp、Asp/Asp基因型频率分别为71.7%、28.3%、0.0%,MTHFR基因CC、CT、TT基因型频率分别为21.8%、40.2%、38.0%。妊高征患者MTHFR基因TT基因型频率（38.0%）显著高于对照组（18.0%）（P〈0.05）,而CT基因型频率妊高征组（40.2%）显著低于对照组（61.8%）（P〈0.05）,携带TT基因型个体发生妊高征的风险增加2.80倍。eNOS基因型和等位基因频率两组比较差异均无显著性（P〉0.05）。结论MTHFR基因TT基因型能增加妊高征的患病风险,eNOS基因G894T突变与妊高征发病无关。     

汉族人群一氧化氮合酶基因三个位点单核苷酸多态性与静息心率相关性研究

目的研究汉族人群的一氧化氮合酶（nitric oxide synthase，NOS）基因NOS3-922A／G和NOS3 894G／T以及NOS2-1173C／T3个位点的单核苷酸多态性（single nucleotide polymorphisms，SNP）与静息心率的相关性。方法随机选择自然人群个体211名为研究对象，获取其静脉血白细胞基因组DNA。用等位基因特异性引物PCR技术检测NOS3-922A／G、NOS3 894G／T、NOS2-1173C／T的SNP。结果NOS3-922A／G的AA、AG、GG，NOS3 894G／T的GG、GT和TT与NOS2-1173C／T的CC、CT和TT各基因型频率分布，均符合Hardy-Weinberg平衡（P〉0．05）。NOS3-922A／G各等位基因AA、AG、GG静息心率比较，发现携带从等位基因者静息心率较GG者高，差异有统计学意义（P〈0．01）。NOS3 894G／T各等位基因静息心率比较，发现携带GG等位基因者静息心率较TT者高，差异有统计学意义（P〈0．05）。NOS2-1173C／T各等位基因的静息心率比较，差异均无统计学意义（P〉0．05）。结论NOS3-922A／G与NOS3 894G／TSNP突变型其静息心率较野生型降低，提示上述位点SNP可能与其静息心率有相关性。     

eNOS基因和MTHFR基因多态性与子痫前期发病关系的研究

目的探讨内皮型一氧化氮合酶(eNOS)基因第7外显子G894T突变和N5,N10-亚甲基四氢叶酸还原酶(MTHFR)基因C677T突变与子痫前期的关系。方法应用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术,对53例子痫前期患者(子痫前期组)和49例正常妊娠妇女(对照组)的eNOS基因G894T突变和MTHFR基因C677T突变进行检测。结果子痫前期组eNOS基因Glu/Glu、Glu/Asp、Asp/Asp基因型频率分别为71.7%、28.3%、0.0%;MTH-FR基因CC、CT、TT基因型频率分别为22.7%、39.6%、37.7%。对照组eNOS基因Glu/Glu、Glu/Asp、Asp/Asp基因型频率分别为83.7%、16.3%、0.0%;MTHFR基因CC、CT、TT基因型频率分别为20.4%、61.2%、18.4%。子痫前期患者TT基因型频率(37.7%)显著高于对照组(18.4%)(P<0.05),而CT基因型频率子痫前期组(39.6%)显著低于对照组(61.2%)(P<0.05),而eNOS基因型和等位基因频率两组比较差异均无显著性(P>0.05)。携带TT基因型个体发生子痫前期的风险增加2.69。结论eNOS基因G894T突变与子痫前期发病无关;MTHFR基因TT基因型能增加子痫前期的患病风险;eNOS基因和MTHFR基因在子痫前期发病中无协同作用。     

江苏汉族人群XRCC1基因的遗传多态性研究

目的探讨江苏省汉族正常人群中X线修复交叉互补基因1（XRCC1）常见的两个单核苷酸多态（SNPs）C26304T和G27466A的遗传分布特点。方法采用聚合酶链反应（PCR）及限制性片段长度多态（RFLP）方法分析江苏省扬中地区511名健康汉族人的XRCC1基因C26304T和G27466A的基因多态性。结果511名江苏汉族人的XRCC1 C26304T基因型CC、CT、TT的频率分别为45．8％、42．7％和11．5％,等位基因C、T的频率分别为67．1％和32．9％。G27466A基因型GG、GA、AA的频率分别为68．9％、29．0％和2．1％,等位基因G、A的频率分别为83．4％和16．6％。江苏人群的C26304T基因型频率和等位基因频率分布与浙江、台湾人群均无明显差异（P〉0．05）,但与意大利人、美国白人、美国黑人的差异具有显著性（P〈0．05）。江苏人群的G27466A基因型频率和等位基因频率分布与浙江人、台湾人、意大利人、美国白人、美国黑人的差异均具有显著性（P〈0．05）。结论本研究揭示了江苏汉族人群XRCC1基因C26304T和G27466A的等位基因频率和基因型频率分布特点;证实了C26304T和G27466A位点的等位基因和基因型频率存在种族、地区差异。     

湖北汉族人群TIM-3基因多态性与变应性哮喘的相关性

目的分析湖北地区汉族人群T细胞免疫球蛋白域粘蛋白域蛋白-3（T cell simmunoglobulin domain and mucin domain protein-3，TIM-3）启动子区-1541C／T和-574G／T单核苷酸多态性，探讨其与支气管哮喘易感性之间的关系。方法分别采用聚合酶链反应-限制性片段长度多态性和等位基因特异性聚合酶链反应检测湖北地区153例哮喘患者和130名健康者TIM-3启动子区-1541C／T和-574G／T的单核苷酸多态性，计算基因型和等位基因频率。结果（1）湖北地区健康人群TIM-3启动子区-1541位CC、CT和TT基因型频率分别是0．961、0．039和0，而哮喘人群其频率分别为0．935、0．065、0，其基因型和等位基因频率均与对照组差异无统计学意义（P=0．314，P=0．321）；（2）湖北地区健康人群TIM-3启动子区-574位GG、GT和TT基因型频率分别是0．992、0．008和0，而哮喘人群频率分别为0．941、0．059、0，其基因型和等位基因频率均与对照组差异有统计学意义（P=0．046，P=0．048）。结论湖北汉族人群TIM-3启动子区存在多态性变异，其中-574G／T单核苷酸多态性可能与湖北地区汉族成人变应性哮喘易感性有关。     

HIF1A基因两个位点多态性与夏尔巴人群高原低氧适应关系的研究

目的研究西藏高原夏尔巴人群缺氧诱导因子1α（hypoxia-inducible factor 1，alpha subunit；HIF1A）基因（HIF1A）第12外显子1772（C→T）、1790（G→A）多态性与高原低氧适应相关性。方法选取世居西藏高原夏尔巴族148人及广东汉族健康个体90人的血样，提取白细胞基因组DNA，聚合酶链反应-限制性片段长度多态性（polymerase chain reaction-restriction fragment length polymorphism，PCR-RFLP）检测HIF1A基因第12外显子1772（C→T）、1790（G→A）的单核苷酸多态性，分析其基因多态性特征。结果1772（C→T）CC、CT和TT基因型频率在夏尔巴人组与汉族对照组分别为14．19％和16．67％、39．19％和41．11％、46．62％和42．22％，两组间比较差异无统计学意义；1790（G→A）GG、GA和AA基因型频率在夏尔巴人组与汉族对照组分别为57．43％和75．56％、37．84％和21．11％、4．73％和3．33％，夏尔巴人组的GG基因型频率较汉族对照组的低（P〈0．01），而GA基因型频率高于汉族对照组（P〈0．01）。组合基因型分布，夏尔巴人CC＋GA、CT＋AA、TT＋GA和TT＋AA的组合基因型频率高于汉族组。结论HIFIA基因1790（G—A）多态性与夏尔巴人群高原低氧适应存在相关性，GA、AA基因型可能对低氧适应有利，值得进一步深入探讨。     

巨噬细胞移动抑制因子-173G/C多态性与冠心病的相关性研究

目的研究巨噬细胞移动抑制因子（macrophage migration inhibitory factor, MIF）基因MIF 5′非翻译区-173G／C多态性在中国南方汉族人群中的分布及与冠心病的相关性。方法采用聚合酶链反应-限制性片段长度多态性技术，对汉族138例冠心病患者及163名正常人群MIF基因-173G／C位点进行研究，对于限制酶酶切结果再进行DNA测序鉴定。结果冠心病患者与正常对照中只检出-173GG和-173GC基因型，均未检出-173CC基因型。正常人群和冠心病患者的MIF-173G等位基因频率分别为0．966和0．917，MIF-173C等位基因频率分别为0．034和0．083，冠心病患者MIF-173GC基因型频率（0．167）明显高于对照组（0．068）（OR：2．764，95％CI：1．295～5．899；P=0．007）。结论MIF基因-173G／C位点多态性与冠心病有关，C等位基因可能是汉族人群冠心病的易感性标志。     

蒙古族冠心病人群与MTHFRC677T等5个相关基因和基因多态性的相关性研究

目的针对内蒙古蒙古族人群的MTHFRC677T、eNOSG894T、ADRBlG1165C、GNB3C825T、ACEI／D、AGTM235T、CYP11B2基因C-344T多态性进行多因素分析,探讨蒙古族人群冠心病人群的遗传易感因素。方法应用PCR—RFLP技术及Sequenom系统检测患者与116名冠心病患者的和156名健康对照组MTHFRC677T、eNOSG894T、ADRB1G1165C、GNB3C825T、ACEI／D、AGTM235T、CYP11B2基因C-344T多态性。结果冠心病组和对照组在年龄、收缩压、舒张压等方面存在统计学差异P〈0．05,eNOS基因G894T位点GT基因型与GG基因型在单因素及多因素分析中均有统计学差异,单因素分析中P值为0．023,多因素分析中P值为0．024,OR=2．405,95％可信区间为1．119～4．807。结论eNOS基因G894T位点GT基因型可能是蒙古族人群患冠心病的易感基因位点。     

The Glu-298→Asp (894G→T) mutation at exon 7 of the endothelial nitric oxide synthase gene and coronary artery disease

We examined associations between the endothelial nitric oxide synthase (eNOS) gene Glu-298-->Asp (894G-->T) mutation and the occurrence and severity of angiographically defined coronary artery disease (CAD). eNOS mediates basal vascular wall nitric oxide production, and altered nitric oxide production has been implicated in atherosclerosis. The newly identified eNOS Glu-298-->Asp mutation in exon 7 is common and likely to be functional. It was found to be associated with myocardial infarction (MI) in Japanese but not in whites. We genotyped 763 white Australians undergoing coronary angiography for the eNOS Glu-298-->Asp mutation. The frequencies of the eNOS GG, TG and TT genotypes were 47.8%, 41.2% and 11.0% in men and 45.2%, 41.1% and 13.7% in women with CAD, and were not significantly different from those without CAD (43.2%, 40.7% and 16.0%, P=0.423 in men; 40.2%, 48.1% and 11.7%, P=0.582 in women). The mutation was also not associated with MI (P=0.469 in males; P=0.389 in females) or with the number of significantly stenosed vessels (P=0.954; P=0.734). The "T" allele frequency (32.5%) was much greater than that reported for the Japanese population (7.8% in controls and 10.0% in MI patients). In conclusion, the eNOS Glu-298-->Asp mutation is common, occurring with an allele frequency of 32.5%, but is not associated with either the occurrence or severity of CAD in the Australian population or with other established coronary risk factors assessed in our study. The mutation is significantly more frequent in the Australian than in the Japanese.     

The 894T allele of endothelial nitric oxide synthase gene is related to left ventricular mass in African Americans with high-normal blood pressure

BACKGROUND AND OBJECTIVES: The 894T allele in exon 7 of the endothelial nitric oxide synthase (eNOS) gene has been inconsistently associated with hypertension in different racial groups. Because high-normal blood pressure (BP) confers an increased risk for the development of hypertension and other cardiovascular disorders, including left ventricular hypertrophy (LVH), we tested the hypothesis that the allelic variation (894T) in the eNOS gene would directly correlate with alterations in LV mass (LVM) in individuals with high-normal BP. METHODS: Genotype distribution of G894T was compared between 20 African Americans (10 females/10 males) with high-normal BP (systolic BP of 130-139 and/or diastolic BP of 85-89 mmHg) and 64 counterparts (37 females/27 males) with normal BP (<130/85 mmHg). Echocardiographic LVM was calculated (Devereux formula) and indexed to body surface area to define the presence of LVH (LVMI >134/110 g/m2 for men/women). RESULTS: For the entire group, the 894T allelic frequencies (15, 48%) and G894T genotype distributions were consistent with the Hardy-Weinberg equilibrium expectations (estimated disequilibrium coefficient = 0.0118, P=0.40). LVMI was significantly higher in homozygous carriers (TT) of the rare 894T allele (n = 3 females/0 males) than in heterozygous GT (n = 13 females/7 males) and individuals bearing the GG (n=34 females/27 males) variant (124 +/- 70 vs. 82 +/- 24 and 82 +/- 19 g/m2, respectively, P < 0.05). The observed relationship between eNOS 894T allele and LVMI was restricted to individuals with high-normal BP (r = 0.94, P = 0.03) but not in those with normal BP (r = 0.39, P =0.64), by analysis of variance (ANOVA) after adjusting for age, gender, body mass index, smoking and systolic BP. CONCLUSION: These findings, not previously described, provide important preliminary evidence to suggest an increased susceptibility to LVH in African Americans who carry the 894T variant of the eNOS gene and have high-normal blood pressure.     

Effects of aerobic exercise on the blood pressure, oxidative stress and eNOS gene polymorphism in pre-hypertensive older people

The polymorphisms of endothelial nitric oxide synthase (eNOS) are associated with reduced eNOS activity. Aerobic exercise training (AEX) may influence resting nitric oxide (NO) production, oxidative stress and blood pressure. The purpose of this study was to investigate the effect of AEX on the relationship among blood pressure, eNOS gene polymorphism and oxidative stress in pre-hypertensive older people. 118 pre-hypertensive subjects (59 ± 6 years) had blood samples collected after a 12 h overnight fast for assessing plasma NO metabolites (NOx) assays, thiobarbituric acid reactive substances (T-BARS) and superoxide dismutase activity (ecSOD). eNOS polymorphism (T-786C and G-894T) was done by standard PCR methods. All people were divided according to the genotype results (G1: TT/GG, G2: TT/GT + TT, G3: TC + CC/GG, G4: TC + CC/GT + TT). All parameters were measured before and after 6 months of AEX (70% of VO_2 max). At baseline, no difference was found in systolic and diastolic blood pressure, ecSOD and T-BARS activity. Plasma NOx levels were significantly different between G1 (19 ± 1 μM) and G4 (14.2 ± 0.6 μM) and between G2 (20.1 ± 1.7 μM) and G4 (14.2 ± 0.6 μM). Therefore, reduced NOx concentration in G4 group occurred only when the polymorphisms were associated, suggesting that these results are more related to genetic factors than NO-scavenging effect. After AEX, the G4 increased NOx values (17.2 ± 1.2 μM) and decreased blood pressure. G1, G3 and G4 decreased T-BARS levels. These results suggest the AEX can modulate the NOx concentration, eNOS activity and the relationship among eNOS gene polymorphism, oxidative stress and blood pressure especially in C (T-786C) and T (G-894T) allele carriers.     

A novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C > T, eNOS +894 G > T and - eNOS -786 T > C variants among Malaysian Malays

ABSTRACT: BACKGROUND: Hyperhomocysteinemia as a consequence of the MTHFR 677 C > T variant is associated with cardiovascular disease and stroke. Another factor than can potentially contribute to these disorders is a depleted nitric oxide level, which can be due to the presence of eNOS +894 G > T and eNOS 786 T > C variants that make an individual more susceptible to endothelial dysfunction. A number of genotyping methods have been developed to investigate these variants. However, simultaneous detection methods using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis are still lacking. In this study, a novel multiplex PCR-RFLP method for the simultaneous detection of MTHFR 677 C > T and eNOS +894 G > T and eNOS 786 T > C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C > T and eNOS +894 G > T and eNOS 786 T > C variants were genotyped using the novel multiplex PCR-RFLP and confirmed by DNA sequencing as well as snpBLAST. Allele frequencies of MTHFR 677 C > T and eNOS +894 G > T and eNOS 786 T > C were calculated using the Hardy Weinberg equation. METHODS: The 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primer pair was designed using Primer 3 Software version 0.4.0 and validated against the BLAST database. The primer specificity, functionality and annealing temperature were tested using uniplex PCR methods that were later combined into a single multiplex PCR. Restriction Fragment Length Polymorphism (RFLP) was performed in three separate tubes followed by agarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing. RESULTS: The allele frequencies for MTHFR 677 C > T were 0.89 (C allele) and 0.11 (T allele); for eNOS +894 G > T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for eNOS 786 T > C, the allele frequencies were 0.87 (T allele) and 0.13 (C allele). CONCLUSIONS: Our PCR-RFLP method is a simple, cost-effective and time-saving method. It can be used to successfully genotype subjects for the MTHFR 677 C > T and eNOS +894 G > T and eNOS 786 T > C variants simultaneously with 100 % concordance from DNA sequencing data. This method can be routinely used for rapid investigation of the MTHFR 677 C > T and eNOS +894 G > T and eNOS 786 T > C variants.     

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。     

Endothelial nitric oxide synthase gene polymorphisms in Fabry's disease

The gene encoding endothelial nitric oxide synthase (eNOS) is involved in abnormalities in nitric oxide (NO) synthesis that mediates functional damage of vascular cells, especially of endothelial cells (ECs), a common characteristic in cardiovascular diseases. In Fabry's disease, the characteristic mutation in the alpha-galactosidase A (alpha-gal A) gene induces large deposits of glycosphingolipids, particularly concentrated in ECs, a process associated with endothelial dysfunction. To determine whether in addition to alpha-gal A gene mutations, eNOS genetic variations are implicated in this process, we examined the genotypes of the missense Glu298Asp (G894T) variant in exon 7 and 27-bp tandem repeats in intron 4 (4b/a) in 19 patients with Fabry's disease, and 39 normal volunteers. The results showed that both varials have a significant association with Fabry's disease. The frequencies of mutant Glu/Asp + Asp/Asp genotypes and Asp allele are significantly higher in Fabry's disease (68.4%, p = 0.044, and 47.4%, p = 0.022, respectively) than in controls (46.7% and 25%, respectively). The frequencies of eNOS 4b/a polymorphisms are also significantly different in Fabry's disease when compared to controls. The mutant 4b/a + 4a/a genotype frequencies are 55.5% (p = 0.032) and 4a allele 27.8% (p = 0.05) compared with controls (23.1% and 12.8%, respectively). These results indicate that more than half of the patients with Fabry's disease carry the Glu298Asp variant ( approximately 68%) and/or the 4b/a polymorphism ( approximately 55%). To the best of our knowledge, this is the first report showing an influence of eNOS gene polymorphisms in patients with Fabry's disease.     

Endothelial nitric oxide synthase as a potential susceptibility gene in the pathogenesis of emphysema in alpha1-antitrypsin deficiency

A role for endothelial nitric oxide synthase (NOS3) in the susceptibility of individuals with alpha1-antitrypsin (alpha1AT) deficiency to destructive lung disease was evaluated. Six polymorphic sites were identified within the NOS3 gene (i.e., -924A/G, -788C/T, -691C/T, 774C/T, 894G/T, and 1998C/G). The genotype distribution was determined in 339 patients and 94 control individuals. Frequency of the 774T allele in severely affected individuals was 0.417 versus 0.269 in control subjects (P = 0.018), whereas the 894T allele frequency was 0.427 versus 0.280 in control subjects (P = 0.024). Patients with less severe lung disease had the 774T and 894T allele frequencies of 0.289 and 0.344, respectively, similar to frequencies in a control group (P > 0.3). No direct correlation between pulmonary function and five other NOS3 polymorphisms was observed. Thus, functional allelic variants that are in linkage disequilibrium with the 774C/T and 894G/T may be present in the specified genomic area. These data are consistent with a modulatory role for NOS3 in destructive lung disease associated with alpha1AT deficiency.