血管紧张素原基因核心启动子区域突变与藏族原发性高血压的关联分析

目的 寻找血管紧张素原(angiotensinogen, AGT)基因核心启动子区域存在的突变,分析该突变在中国西藏人群中的分布以及与原发性高血压的关联.方法 以藏族103例原发性高血压患者和82名健康受试者为研究对象进行病例-对照研究.用聚合酶链反应-单链构象多态性(polymerase chain reaction/single strand conformation polymorphism, PCR/SSCP)分析和自动荧光测序方法,对AGT基因核心启动子区域DNA序列进行突变分析;用聚合酶链反应-限制性片段长度多态性(polymerase chain reaction/restriction fragment length polymorphism, PCR-RFLP)方法分析AGT基因(-6)位点多态性.结果 PCR/SSCP分析发现,AGT基因转录起始位点上游(-20)位存在A→C突变,统计分析显示,藏族正常人群与高血压人群中该位点A等位基因均有较高的发生频率(0.9175,0.9124),突变位点多态性分布无统计学差异(P＞0.8).AGT基因转录起始位点上游(-6)位点存在A→G突变,在藏族正常人群中等位基因A和G分布频率分别为0.780和0.220,原发性高血压群体中它们的分布频率分别为0.626和0.374,两者之间存在差异(P＜0.025).结论 (1)藏族群体中AGT基因(-20)A等位基因有较高的分布频率;(2)AGT基因(-6)G等位基因在藏族高血压患者群体中发生频率较高,可能是藏族原发性高血压的遗传易感因子.     

血管紧张素原基因的六种单核苷酸多态与原发性高血压的相关性

目的　研究血管紧张素原 (angiotensinogen,AGT)基因 6个位点的单核苷酸多态及其构成的单倍型与中国汉族人原发性高血压的相关性。方法　采用多重SNa Pshot反应 ,在 185例原发性高血压患者和185名健康对照者中 ,对 AGT基因启动子区域的 G- 2 17A、G- 15 2 A、A- 2 0 C、G- 6 A及第 2外显子的T174 M和 M2 35 T多态进行基因分型。结果　 6种单核苷酸多态的基因型分布及其等位基因频率在原发性高血压组和对照组中差异无显著性 (P>0 .0 5 )。单倍型分析提示由 - 15 2 A,- 2 0 C,- 6 A和 2 35 T等位基因构成的 H4单倍型在原发性高血压组中明显增加 ,与对照组相比差异有显著性 (P<0 .0 5 )。结论　AGT基因G- 15 2 A,A- 2 0 C,G- 6 A和 M2 35 T多态可能对中国汉族人原发性高血压的发病起了重要作用。     

Effect of Genetic Polymorphism of ALOX15 on Aspirin-Exacerbated Respiratory Disease

Background: Aspirin-exacerbated respiratory disease (AERD) is a clinical syndrome associated with chronic inflammation in the airways coincident with chronic rhinitis, sinusitis, recurrent polyposis and asthma. Eosinophils are the key inflammatory cells in the development of AERD. AERD has been attributed to abnormalities of the arachidonic acid metabolism, but the pathogenesis of AERD is not fully understood. Our aim was to investigate the genetic contribution of the arachidonate 15-lipoxygenase gene (ALOX15) to the development of AERD. Methods: We enrolled 171 patients with AERD, 229 patients with aspirin-tolerant asthma, and 195 normal healthy controls in a Korean population. Three polymorphisms (-427G/A, -272C/A, -217G/C) in the promoter region of ALOX15 were genotyped. The functional variability of the promoter polymorphisms were analyzed by luciferase reporter activity assay. Result: No significant difference in the genotype frequency of the ALOX15 genetic polymorphism was found. Peripheral total eosinophil count was significantly higher in the patients carrying the GG genotype of the -427G/A polymorphism (p = 0.016). Similarly, the patients carrying haplotype 1 (ht1) (GCG) of -427G/A, -272C/A and -217G/C showed a significantly higher total eosinophil count compared to the other haplotypes (p = 0.008) in the AERD group. The promoter activity of the ht1 (GCG) construct was significantly higher compared to that of the ht3 (AGG) construct in A549 and U937 cells (both p < 0.001). Conclusion: These results suggest that the promoter polymorphisms of the ALOX15 gene affect ALOX15 activity leading to increased eosinophil infiltration in AERD patients.     

血管紧张素原基因单倍型与原发性高血压的关联研究

目的 研究中国人血管紧张素原（angiotensinogen,AGT)基因多态性及单倍型与原发性高血压的关系。方法 在335例高血压患者与196名血压正常人中采用PCR－限制性酶切片段长度多态性法检测血管紧张素原基因的多态性，同时用最大期望值方法进行两位点连锁不平衡和三位点的单倍型分析。结果 在M235T和A－20C，M235T和A－6G，A－20C和A－6G位点观察到了连锁不平衡（P＜10^-4)。病例－对照检验显示T235等位基因频率在高血压组中高于对照组，但所有单倍型频率分布在高血压组和正常对照组间差异无显著性。结论 受检人群中AGT基因各单倍型与高血压未见关联，但AGT基因T235位点可能是一种重要的高血压风险因子。     

一氧化氮合酶2A基因启动子-969(G→C)多态性与肝硬化门静脉高压的相关性

目的探讨一氧化氮合酶2A(nitricoxidesynthase2A,NOS2A)基因启动子-969(G→C)多态性与肝硬化门静脉高压的相关性。方法采用病例对照和聚合酶链反应-限制性片段长度多态性技术,检测106例乙肝后肝硬化患者和108名健康对照者NOS2A基因启动子-969(G→C)多态性,比较等位基因及基因型频率;应用逆转录-聚合酶链反应和Western印迹技术,检测肝硬化组织NOS2AmRNA和蛋白表达。构建NOS2A基因启动子-969(G→C)多态性荧光素酶重组质粒,瞬时转染,分析多态性的功能。结果在门静脉高压症组C等位基因和GC基因型频率为16.9%、33.8%,比正常对照组高(8.8%、17.6%),差异有显著性(P<0.05,OR=2.42),相关分析呈正相关(r=0.18)。单纯肝硬化组与对照组的C等位基因和GC基因型频率相比,差异无显著性(P>0.05)。C等位基因携带者比G等位基因携带者肝硬化组织NOS2AmRNA及蛋白表达明显增强,GC基因型启动子的活性显著升高。Logistic多元逻辑回归分析显示这一多态性是形成门静脉高压症新的独立危险因素。结论NOS2A基因启动子-969(G→C)多态性与门静脉高压症相关,导致启动子活性增强,是形成门静脉高压症的新的危险因素。     

ACE、AGT基因联合多态性与高血压合并脑梗塞相关

目的探讨北京地区汉族人群血管紧张素转化酶(ACE)基因插入/缺失(I/D)多态性与血管紧张素原(AGT)基因CD235Met-Thr变异(M235T)与高血压合并脑梗塞发生的关系。方法分别用PCR法、突变基因分离聚合酶链反应(MS-PCR)法检测664例高血压合并脑梗塞患者(CI),678例单纯高血压患者(EH)和716例对照者(C)的ACE基因I/D多态性及AGT基因M235T多态性,分析两个基因多态性分布与高血压合并脑梗塞发病的相关性。结果 CI组ACE-DD和AGT-TT基因型频率(分别为0.309和0.643)均显著高于C组(分别为0.203和0.543,P<0.001)和EH组(分别为0.217和0.569,P<0.01)。CI组与C组比较,ACE-DD、AGT-TT联合基因型OR值(2.547,95%CI:1.919～3.382)明显高于ACE-DD单基因型(1.759,95%CI:1.376～2.248)和AGT-TT单基因型(1.515,95%CI:1.220～1.880)。结论 ACE-DD和AGT-TT基因型与北京地区汉族人群单纯高血压发病无相关性,但与高血压合并脑梗塞发病显著相关,并且A...     

Functional interaction between -629C/A, -971G/A and -1337C/T polymorphisms in the CETP gene is a major determinant of promoter activity and plasma CETP concentration in the REGRESS Study

Cholesteryl ester transfer protein (CETP) plays a key role in the determination of high-density lipoprotein (HDL) levels via its action on intravascular HDL metabolism. The TaqIB polymorphism of the CETP gene is associated with plasma CETP and high-density lipoprotein cholesterol (HDL-C) levels and with premature coronary artery disease. Such associations appear to result from linkage disequilibrium between TaqIB and other functional polymorphisms. To date, only one functional promoter variant, which may explain the effects of TaqIB, has been identified at position -629 in the CETP gene. Here we describe a C/T polymorphism located at position -1337 in the human CETP gene (C allele frequency: 0.684), which is significantly associated with plasma HDL-C and CETP levels (P=0.0001 and P<0.0001, respectively). Transient transfection of a reporter gene construct containing the CETP promoter from -1707/+28 in liver cells (HepG2) revealed that the -1337T allele was expressed to a significantly lower degree (-34%, P<0.0001) than the -1337C allele. In addition, we clearly demonstrated that the -971G/A polymorphism is functional and that its functionality is intimately linked to the presence of the -1337 site. In vitro evaluation of potential interaction between -1337C/T and other functional variants of the CETP gene (-971G/A and -629C/A) demonstrated that these three functional CETP promoter polymorphisms can interact together to determine the overall activity of the CETP gene and thus contribute significantly to variation in plasma CETP mass concentration.     

血管紧张素系统基因多态性与原发性高血压的相关研究

目的：探讨中国人血管紧张素原（angiotensinogen,AGT)基因的蛋白产物M235T、血管紧张素Ⅱ-I型受体（AT1R)基因的蛋白产物A1166C以及血管紧线素转换酶（angiotensin convertingenzyme,ACE)基因I／D多态性与高血压病（hypertension,HT)的关系。方法：用PCR以及PCR加酶解方法检测了161例HT患者及134名健康人（normotensive controls,NT)ACEI/D基因多态性、AGTM235T及AT1RA1166C突变,并检测了血清ACE活性。结果：HT组ACEI／D基因多态性等位基因频率I为0．571,D为0．429,等位基因频率及基因型频率与NT组比较差异无显著性（P＞0．05）;＜60岁HT组D等位基因频率（0．457)显著高于NT组（0．358,P＜0．05）。HT组与NT组的AGT235T分别为0．813及0．832,两组间差异无显著性。AT1RA1166C的C等位基因频率HT组为0．021,NT组为0．053,两组间差异无显著性;但在＜60岁NT组AGTM235T显著高于NT组。两组中均发现ACE基因型与血清ACE活性相关。HT组DD－TT及ID－TT联合基因型显著高于对照组。结论：D等位基因及AGT235T对于HT早期发病可能有重要意义,DD－TT及ID－TT基因型人群可能是高血压发病的高危人群。     

A search for cyclophilin-A gene (PPIA) variation and its contribution to the risk of atherosclerosis and myocardial infarction

Cyclophilin A is secreted by vascular smooth muscle cells in response to inflammatory stimuli, and could thus contribute to atherosclerosis. We hypothesized that the genetic variation at the cyclophilin A gene (PPIA) could affect the risk for developing atherosclerosis and myocardial infarction. This study included 250 myocardial infarction patients (all male and < 60 years; 95% are smokers). All these cases had at least one atherosclerotic diseased coronary vessel. DNA was obtained from patients and from 250 healthy controls. The variation at the PPIA gene was determined in the patients through single-strand conformation analysis and direct sequencing of seven polymerase chain reaction fragments. Allele and genotype frequencies were compared between patients and controls. The effect of a promoter polymorphism (-11 G/C) on gene expression was in vitro analysed with luciferase-reporter assays. We found two common polymorphisms in the PPIA promoter (-11 G/C) and the 5' non-translated (+36 G/A) regions. Cells transfected with luciferase-plasmids containing the -11 G had significantly higher luciferase activity. Genotype frequencies for these polymorphisms did not differ between patients and controls. In conclusion, we reported a functional variant in the PPIA promoter. However, the PPIA variation did not significantly contribute to the risk of suffering from myocardial infarction among patients with atherosclerotic diseased vessels.     

Exclusion of angiotensinogen gene in molecular basis of human hypertension: sibpair linkage and association analyses in Australian anglo-caucasians.

Linkage with essential hypertension has been claimed for a microsatellite marker near the angiotensinogen gene (AGT; chromosome 1q42), as has association for the AGT variants M235T, G(-6)A and A(-20)C. To more rigorously evaluate AGT as a candidate gene for hypertension we performed sibpair analysis with multiple microsatellite markers surrounding this locus and using more sophisticated analysis programs. We also performed an association study of the AGT variants in unrelated subjects with a strong family history (two affected parents). For the linkage study, single and multiplex polymerase chain reaction (PCRs) and automated genescan analysis were conducted on DNA from 175 Australian Anglo-Celtic Caucasian hypertensives for the following markers: D1S2880-(2.1 cM)-D1S213-(2.8 cM)-D1S251-(6.5 cM)-AGT-(2.0 cM) -D1S235. Statistical evaluation of genotype data by nonparametric methods resulted in the following scores: Single-point analysis - SPLINK, P > 0.18; APM method, P > 0.25; ASPEX, MLOD < 0.28; SIB-PAIR, P > 0. 24; Multipoint analysis - MAPMAKER/SIBS, MLOD < 0.24; GENEHUNTER, P > 0.35. Exclusion scores of Lod -4.1 to -5.1 were obtained for these markers using MAPMAKER/SIBS for a lambda(s) of 1.6. The association study of G(-6)A, A(-20)C and M235T variants in 111 hypertensives with strong family history and 190 normotensives with no family history showed significant linkage disequilibrium between particular haplotypes, but we could find no association with hypertension. The present study therefore excludes AGT in the etiology of hypertension, at least in the population of Australian Anglo-Celtic Caucasians studied.     

Identification of a type 1 diabetes-associated CD4 promoter haplotype with high constitutive activity

CD4 is a candidate gene in autoimmune diseases, including Type 1 diabetes mellitus (T1DM), because the CD4 receptor is crucial for appropriate antigen responses of CD4(+) T cells. We previously found linkage between a CD4-1188(TTTTC)(5-14) promoter polymorphism and T1DM. In the present study, we screened the human CD4 promoter for mutations and identified three frequent single nucleotide polymorphisms (SNPs): CD4-181C/G, CD4-521C/G and CD4-1050T/C. The SNPs are in strong linkage disequilibrium (LD) and association with the CD4-1188(TTTTC)(5-14) alleles, and we observed nine CD4 promoter haplotypes, of which four are frequent. We genotyped the SNPs in 253 Danish T1DM families (1129 individuals) and found evidence for linkage and association of a CD4 (A4(-1188)T(-1050)G(-521)C(-181)) haplotype to T1DM. In reporter studies, we show that (1) the T1DM-associated CD4 haplotype encodes high constitutive promoter activity and (2) the CD4-181G variant encodes higher stimulated promoter activity than the CD4-181C variant. This difference is in part neutralized in the frequently occurring CD4 promoter haplotypes by the more upstream genetic variants. Thus, we report functional impact of a novel CD4-181C/G SNP on stimulated CD4 promoter activity and the identification of a novel CD4 haplotype with high constitutive promoter activity that is linked and associated with T1DM.