DDAH1和ALDH2基因多态性与高血压及冠心病易感性的关联关系及机制研究
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摘要
研究背景
原发性高血压或高血压病由于其高发病率和高死亡率,是目前影响人类健康的主要常见疾病之一。大量的研究业已证实,高血压病是由环境与遗传因素共同作用所致的多基因疾病。
血管内皮在维持心血管系统的稳态中发挥极其重要的作用,内皮功能受损是包括高血压病在内的多种心血管疾病的早期表现和共同的病理生理特征。NO是维持血管内皮功能最重要的一种介质之一,由L-精氨酸在一氧化氮合酶(NOS)的催化作用下生成,具有血管舒张作用。
非对称二甲基精氨酸(Asymmetric Dimethylarginine, ADMA)是一种内源性NOS抑制剂,能竞争性地抑制NO的生成。人体内生成的ADMA 80%经二甲基精氨酸二甲胺水解酶(包括DDAH1和DDAH2两种亚型)水解代谢而失活。因此,体内DDAH酶活性的高低可通过影响ADMA的代谢,从而影响NOS的活性和NO的水平。遗传因素可能是影响体内DDAH酶活性的因素之一。由遗传因素所导致的个体间DDAH酶活性的差异可能通过影响ADMA的体内浓度,从而影响机体对高血压病的易感性。本文通过病例-对照研究,探讨DDAH1基因多态性与中国汉族人群高血压病遗传易感性的关系。
方法
1.候选单核苷酸多态性(single nucleotide polymorphism, SNP)位点的选择与基因分型
应用NCBI数据库中的数据资源,对中国人群DDAH1基因的单核苷酸多态性(single nucleotide polymorphism, SNP)进行登记,通过连锁不平衡分析,明确该基因区域SNP位点间的连锁不平衡情况,选择待分型的标签SNP (haplotype tag SNP, htSNP)。同时,随机选取20个无亲缘关系的高血压患者,通过PCR产物测序,对该基因所有外显子、外显子-内含子交界以及2kb范围内的启动子区进行重新测序和SNP发掘,寻找新的标签SNP。应用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)的分析方法,在1318例原发性高血压患者和1005例健康人群中对DDAH1基因的标签SNP进行基因分型,并通过应用高效液相色谱-质谱联用法测定病例和对照人群血浆ADMA的水平。
2.荧光素酶报告基因实验对SNP的功能进行验证
对于3’端未翻译区(3'-untranslated region,3'-UTR)出现阳性关联的SNP, PCR扩增含不同等位基因的3'-UTR区段并克隆入荧光素酶报告基因质粒,以pRL-SV40载体为内参,两种质粒混合并转染人脐静脉内皮细胞株HUVECs。双荧光素酶报告实验体系测定荧光素酶活性,分析和比较含不同等位的3'-UTR序列的转录活性。
3.培养的人脐静脉内皮细胞观察SNP的功能
分离和培养人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs), PCR-RFLP对脐静脉组织DDAH1 SNP位点进行基因分型,第4代内皮细胞于24孔板中培养24小时,ELISA法测定细胞内及培养基中ADMA的浓度和DDAH1的活性；应用放线菌酮处理内皮细胞,测定药物处理前(0小时)及药物处理后4、8、12、16、24和36小时DDAHl mRNA表达水平,比较DDAH1不同基因型个体HUVECs DDAH活性、ADMA水平、DDAH1 mRNA表达水平及半衰期的异同。
结果
(1)通过在20个无关中国个体中对DDAH1基因进行重新测序,总共发现了46个SNP,其中有10个为新发现的SNP。所有的46个SNP均不改变DDAH1的氨基酸结构,其中12个位于3'-UTR区。
(2)病例和对照人群中rs233113 A/T、rs3087894 C/G和rs233112A/G多态性位点基因型分布存在显著的差异(P值分别为0.003、0.003和0.042),且rs3087894 C/G和rs233113 A/T多态性呈完全连锁不平衡(D'=1,r2=1)；病例和对照人群中rs3813600 C/T多态位点基因型分布差异不明显(p=0.073)。
(3)通过对高血压的风险因素进行校正,Logistic回归分析结果显示,携带rs233113 T(或rs3087894 G)等位的个体患原发性高血压病的风险显著降低(比值比=0.747,95%可信区间：0.618-0.904,P=0.005); rs233112GG基因型也可降低高血压病的发病风险(比值比=0.772；95%可信区间：0.607-0.983,p=0.035)；而rs3813600多态位点与高血压病的发病风险不相关(p>0.05)。
(4)病例组人群血浆ADMA的浓度显著高于对照人群组(p<0.001)。在对照人群中,携带rs233113 T等位的个体血浆中ADMA浓度显著高于基因型为AA的个体(p=0.016),而在病例人群中,rs233113 TT基因型个体血浆ADMA浓度与rs233113 AA基因型个体相比有降低的趋势(p=0.089)；对照组中携带rs3813600 T等位的个体血浆ADMA的浓度显著低于该位点基因型为CC的个体(p=0.033),rs233112GG基因型个体血浆ADMA的浓度显著高于该位点为AA基因型的个体(p=0.007)；而在病例组中,rs3813600位点携带T等位的个体血浆ADMA的浓度显著高于该位点为CC基因型的个体(p=0.048); rs233112GG基因型个体血浆ADMA的浓度显著低于该位点为AA基因型的个体(p=0.036)。
(5)携带rs233113 T等位的pGL3质粒荧光素酶活性显著高于携带rs233113 A等位的质粒；与rs233113 (A/T)多态性呈完全连锁不平衡的SNP rs3087894 (C/G)/rs233115 (C/T)/rs233114 (A/C)构成的两种单倍型(rs3087894C-rs233115C-rs233114A和rs3087894G-rs233115T-rs233114C)间报告基因的活性无显著差别。
(6)携带rs233113 T等位的HUVECs (n=4)细胞内DDAH的活性显著高于基因型为rs233113 AA的HUVECs (n=5, p=0.039),而细胞内ADMA的浓度显著低于rs233113 AA基因型HUVECs(p=0.037)。放线菌酮处理前及处理后4小时和8小时,携带rs233113T等位的HUVECs (n=4)内DDAH1 mRNA表达水平显著高于基因型为rs233113 AA的HUVECs (n=5,p<0.05)。与基因型为rs233113AA的HUVECs (n=5)相比,携带rs233113 T等位的HUVECs (n=4)内DDAH1 mRNA消除半衰期有缩短的趋势(t1/2分别为17.3±3.4h和51.0±31.5h,p=0.073)。
结论
DDAH1基因rs233113A/T和rs233112A/G多态性与中国长沙地区汉族人群原发性高血压的遗传易感性相关,rs233113A/T多态性可通过增加报告基因的活性、DDAH1 mRNA表达和活性,从而降低体内ADMA的水平,降低原发性高血压的易感性。
研究背景
冠心病是人类死亡的主要原因并已成为世界主要卫生问题之一。流行病学调查资料表明,冠心病是由环境因素和遗传因素相互作用所导致的多基因疾病。
血管内皮在维持心血管系统的稳态中起极其重要的作用,内皮功能受损是包括冠心病在内的多种心血管疾病的早期表现。NO能够调控内皮功能且能反映内皮功能的健康状况,其生成减少或活性降低在动脉粥样硬化(Atherosclerosis, AS)和冠心病的发生发展中起到关键作用。
非对称二甲基精氨酸(ADMA)是一种内源性NOS抑制剂,能竞争性的抑制NO的生成。研究表明,血浆ADMA水平和冠心病病变程度间呈强关联。此外,冠心病患者体内活性氧(Reactive oxygen species, ROS)水平显著增加,氧化应激在AS和冠心病的发生发展中起重要作用。研究表明,病理生理浓度的ADMA也可通过增加培养的人脐静脉内皮细胞和脂肪细胞细胞内ROS的生成,从而参与AS的发生发展。
人体血浆中的ADMA主要经二甲基精氨酸二甲胺水解酶1和2(Dimethylarginine Dimethylaminohydrolases, DDAH1和DDAH2)水解代谢而失活。研究表明,遗传因素可能是影响体内DDAH酶活性的因素之一。因此,由遗传因素所导致的个体间DDAH酶活性的差异可能通过影响ADMA的体内浓度以及NO的水平,从而影响机体对冠心病的易感性。本文通过病例-对照研究,探讨DDAH1基因多态性与中国汉族人群冠心病遗传易感性的关系。
方法
1.病例对照标本候选SNP位点基因分型
应用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)的分析方法,在544例冠心病患者和992例健康对照人群中对DDAH1基因的标签SNP进行基因分型。
2.荧光素酶报告基因实验对SNP的功能进行验证
PCR扩增含不同等位基因的3’-UTR区段并克隆入pGL3-Control质粒中,以pRL-SV40载体为内参,两种质粒以及microRNA 218(miR-218)模拟物共同转染人脐静脉内皮细胞株HUVECs。双荧光素酶报告实验测定荧光素酶活性,分析和比较含不同等位的3’-UTR序列在不同浓度的miR-218模拟物处理下的报告基因活性。
结果
(1)冠心病病例组和对照组中rs233113位点AA、AT和TT基因型频率分别为53.5%、37.1%、9.4%和47.3%、43.3%、9.4%,病例组rs233113 AA基因型频率显著高于对照组(P=0.020)。病例组和对照组中rs233112位点AA、AG和GG基因型频率分别为32.8%、50.6%、16.6%和28.9%、49.9%、21.2%,病例组rs233112GG基因型频率显著低于对照组(P=0.036)。
(2)通过对冠心病的风险因素进行校正,Logistic回归分析结果显示,携带rs233113 T等位的个体患冠心病的风险显著降低(OR=0.503,95%CI:0.316-0.801, P=0.004),而携带rs233112 A等位的个体患冠心病的风险显著增加(OR=1.969,95%CI:1.040-3.728,P=0.037)。
(3)冠心病病例组人群血浆ADMA的浓度显著高于对照人群组(p<0.05)；病例组携带rs233113 T等位的个体血浆中ADMA浓度略低于AA基因型个体；携带rs233112 G等位的个体血浆中ADMA浓度显著低于基因型为AA的个体(p=0.024)。
(4)携带rs233112 A等位的质粒的荧光素酶活性显著高于携带rs233112 G等位的质粒(p<0.05), miR-218 (2.5nM-20nM)可显著降低含rs233112 A质粒的荧光素酶活性,而对携带rs233112 G等位的质粒荧光素酶活性无影响。
结论
DDAH1基因rs233113A/T多态性和rs233112A/G多态性可分别降低和增加中国长沙地区汉族人群冠心病的发病风险,rs233112多态位点可影响报告基因活性和miR-218的结合,可能是影响冠心病易感性的功能性SNP位点。
研究背景
冠心病是一种由环境因素和遗传因素相互作用所导致的复杂性多因素疾病。大量的研究结果表明,氧化应激和活性氧(ROS)在动脉粥样硬化(AS)及其并发症的发生发展中起到重要作用。ROS可导致多种活性醛如4-羟基壬烯醛(4-HNE)等生成增加,而活性醛类可进一步加重氧化应激,引起血管内皮功能障碍,促进AS的病理生理过程。
人类线粒体醛脱氢酶(ALDH2)参与多种活性醛类的代谢灭活,其中包括4-HNE。ALDH2基因的第12号外显子上存在功能性单核苷酸序列多态性rs671多态,该SNP使ALDH2多肽第504位的谷氨酸被赖氨酸取代,从而导致ALDH2酶活性降低,代谢乙醛等活性醛的能力下降。
非对称二甲基精氨酸(ADMA)是一种内源性的一氧化氮合酶(NOS)抑制剂,它能够降低NO的生物利用度而导致内皮功能障碍,因此ADMA被视为一种新的冠心病和AS的危险因子。ADMA在体内主要是由二甲基精氨酸二甲胺水解酶(DDAH1和DDAH2)代谢灭活。在培养的内皮细胞中,4-HNE能通过与DDAH1形成Michael加合物而剂量依赖性地抑制]DDAH1活性,进而增加ADMA产生并降低NO生成。
本实验在中国人群中研究了ALDH2 rs671多态性是否与冠心病发病风险相关,并研究该多态是否可影响DDAH1的mRNA表达和ADMA的浓度,从DDAH/ADMA系统探讨其影响冠心病易感性的可能机制。
方法
1.病例对照标本候选SNP位点基因分型
收集冠心病和性别年龄相匹配的健康对照人群血液标本。应用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)的分析方法,对所有病例和对照人群ALDH2基因rs671多态性进行基因分型。
2. ALDH2 rs671多态性对原代人脐静脉内皮细胞血管紧张素Ⅱ处理前后DDAH1/ADMA系统的影响
取11根人脐带进行ALDH2基因分型,分离培养人脐静脉内皮细胞HUVECs。第4代细胞用不同浓度(10-7至10-5mol/L)的血管紧张素Ⅱ处理24小时。应用实时定量PCR法检测细胞内DDAH1 mRNA表达水平。应用高效液相色谱-质谱联用法检测细胞内及培养基中ADMA的水平。
结果
(1)冠心病病例组和对照组中rs671位点GG、GA和AA基因型频率分别为52.5%、41.0%、6.5%和65.0%、30.1%、4.9%,病例组rs671 GA+AA基因型频率显著高于对照组(χ2=13.807,P=0.0002)。通过对冠心病风险因素进行校正,Logistic回归分析结果显示,在总人群中,携带rs671 A等位的个体患冠心病的风险显著增高(OR=1.85,95%CI：1.38-2.49,P=0.00005)。
(2)在病例和对照人群中,不饮酒人群携带rs671 A等位个体的频率均显著高于饮酒人群(病例组中分别为53.7%和28.8%,P=1.1×10-5；对照组中分别为38.8%和22.5%,P=0.003)；不饮酒人群中病例组携带rs671 A等位个体的频率(53.7%)显著高于对照组人群(38.8%,P=0.00012)。通过对冠心病的风险因素进行校正,Logistic回归分析结果显示,携带rs671 A等位可显著增加不饮酒人群冠心病的发病风险(OR=1.95,95%CI:1.40-2.70, P=0.00007),但不影响饮酒人群冠心病的发病风险(OR= 1.56,95%CI:0.77-3.18, P=0.22)。
(3)在不饮酒健康人群,rs671 AA纯合子个体血清高密度脂蛋白(HDL-C)浓度(1.20±0.22 mmol/L, n=22)显著低于GG纯合子个体(1.39±0.36 mmol/L, n=212, P=0.015)。
(4)基因型为rs671 GG的HUVECs (n=6) DDAH1 mRNA表达水平显著高于基因型为rs671 GA的HUVECs (n=5) (p＜0.05),而细胞内ADMA水平显著低于基因型为GA的HUVECs (p＜0.05)。高浓度的血管紧张素Ⅱ(10-5mol/L)可显著降低基因型为rs671 GG的HUVECs中DDAH1 mRNA表达,升高该基因型HUVECs细胞内ADMA浓度,但对基因型为GA的HUVECs DDAH1 mRNA表达和ADMA浓度影响不明显(p>0.05)。
结论
ALDH2基因rs671多态性可能通过降低HDL-C水平和HUVECs内DDAH1的mRNA表达,升高HUVECs内ADMA的水平,从而增加不饮酒的中国长沙地区汉族人群冠心病的发病风险。
Background
Essential hypertension (EH) is one of the most important global health problems beacause of its high prevalence and high mortality. EH is a multigenic disease with components of environmental and genetic factors, genetic factors may account for about 30%-60% of blood pressure (BP) variance.
The vascular endothelium plays an important role in the maintainance of the homeostasis of cardiovascular system. Endothelial dysfunction is the early characterization of cardiovascular diseases including EH. Nitric oxide (NO) is one of the most important mediators that are involved in the maintainance of vascular endothelium function. NO is an endogeneous vasodilator, and is synthesized from L-arginine catalyzed by NO synthase (NOS).
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NOS which can competitively inhibit NO production. About 80% of ADMA in plasma is metabolized and inactivated by dimethylarginine dimethylaminohydrolases (DDAH1 and DDAH2). Therefore, variation in the DDAH activity may influence NO level through affecting plasma ADMA level and NOS activity, and consequently influence EH predisposition. This study was designed to evaluate whether genetic polymorphisms in DDAH1 is associated with hypertension susceptibility by a case-control study.
METHODS
1. Genotyping the candidate SNPs in cases and controls
By using the public data deposited in the NCBI database, single nucleotide polymorphisms (SNPs) at DDAH1 locus in Beijing Chinese Han were recorded. After analysis of linkage disequilibrium (LD) and establishment of haplotypes, haplotype tag SNPs (htSNPs) were selected for further study. DNA discovery was also carried out by resequencing all exons, exon-intron boundaries, and 2kb region upstream the transcriptional start site in the DDAH1 promoter in 20 randomly selected EH patients. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to genotype the candidate SNPs. A case-control study of 1318 newly diagonised EH patients and 1005 normotensive controls of Han nationality recruited from Hunan province was carried out.
2. Functional analysis of DDAH1 SNPs by luciferase reporter gene assay system
For the positively associated SNPs in 3'-untranslated region (3'-UTR) of DDAH1,3'-UTR encompassing the SNPs were cloned to downstream of pGL3-Control vectors. The vectors were then transfected into human umbilical vein endothelial cells (HUVECs) by using pRL-SV40 vector as an internal standard. Dual-luciferase reporter assay system was used to determine the luciferase activity.
3. Verifying the function of SNPs in HUVECs
HUVECs were isolated from 9 human umbilical cords, DDAH1 SNPs were genotyped, and the cells were cultured in EGM-2 medium. Cells of the fourth passage were cultured for 24 hours, and then cycloheximide (CHX) were added. ADMA in lysate and cell medium and DDAH1 activity were determined by ELISA. RNA were extracted just before and at 4h,8h,12h,16h,24h, and 36h, respectively after CHX treatment. DDAH1 mRNA expression were determined by real-time quatitative PCR.
RESULTS
(1) A total of 46 SNPs were idenfied at the DDAH1 locus in 20 Chinese EH patients,10 of the SNPs were newly discovered. None of the SNPs lead to change in the amino acid sequence of DDAH1, and 12 of the SNPs were located in 3'-UTR.
(2) Significant difference in genotype distribution for rs233113 A/T, rs3087894 C/G and rs233112 A/G polymorphisms were observed between EH cases and controls (p values were 0.003,0.003, and 0.042, respectively). The rs233113 A/T and rs3087894 C/G polymorphisms were in complete linkage disequilibrium (D'=1, r2=1). No difference in genotype distribution of the rs3813600 C/T polymorphism was observed between cases and controls (p=0.073).
(3) When adjusted by EH risk factors, results of unconditional logistic regression analysis showed that carriers of the rs233113 T (or rs3087894 G) allele showed significantly decreased risk for EH (odds ratio [OR]=0.747,95% confidence interval [CI]:0.618-0.904, p=0.005). The rs233112 GG was also associated with decreased risk for EH (OR=0.772, 95%CI:0.607-0.983,p=0.035). No association between rs3813600 polymorphism and EH risk was observed.
(4) EH cases showed significantly higher plasma ADMA level than controls (p<0.001). In controls, carriers of the rs233113 T allele showed significantly higher mean plasma ADMA level as compared with individuals with the rs233113 AA genotype, carriers of the rs3813600 T allele showed significantly lower mean plasma ADMA level as compared with individuals with the rs3813600 CC genotype, carriers of the rs233112 G allele showed significantly higher mean plasma ADMA level as compared with individuals with the rs233112 AA genotype. While in EH cases, carriers of the rs3813600 T allele showed significantly higher mean plasma ADMA level as compared with individuals with the rs3813600 CC genotype, individuals genotyped as rs233112 GG showed significantly lower mean plasma ADMA level as compared with individuals with the rs233112 AA genotype.
(5)pGL3 vectors bearing the rs233113 T allele showed significantly higher luciferase activity than those bearing the rs233113 A allele. No difference in luciferase activity was observed between bearing the rs3087894 C-rs233115 C-rs233114 A and rs3087894 G-rs233115 T-rs233114Calleles.
(6) HUVECs carrying the rs233113 T allele showed significantly higher DDAH1 activity and lower intracellular ADMA levels as compared those with the rs233113 AA genotype. HUVECs carrying the rs233113 T allele showed significantly higher DDAH1 mRNA expression levels as compared those with the rs233113 AA genotype before (0 hour) and at 4 hour and 8 hour after CHX treatment (p<0.05, respectively). As compared HUVECs with the rs233113 AA genotype, half-life time of DDAH1 mRNA in HUVECs bearing the rs233113 T allele trended to be decreased (17.3±3.4 h vs 51.0±31.5 h,p=0.073).
CONCLUSION
DDAH1 rs233113 A/T and rs233112 A/G genetic polymorphisms are associated with EH susceptibility in Chinese. The rs233113 A/T polymorphism can increase the activity of reporter gene and increase DDAHl mRNA expression.
Background
Coronary artery disease (CAD) has become a leading cause of death and a major health problem worldwide. Epidemiological studies have indicated that CAD is a complex, multifactorial disease with both genetic and environmental components.
The vascular endothelium plays an important role in maintaining the homeostasis of cardiovascular system. Endothelial dysfunction is the early characterization of many cardiovascular diseases including CAD. Nitric oxide (NO) plays important roles in the regulation of endothelium function. Decrease in NO bioavailability can lead to endothelial dysfunction, and thus contribute to the initiation and progression of atherosclerosis (AS) and CAD.
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NOS which can competitively inhibit NO production. A strong relationship between ADMA plasma levels and CAD severity was reported. Oxidative stress and ROS also play important roles in the pathogenesis of AS. Pathophysiological concentration of ADMA can increase production of intracellular reactive oxygen species (ROS) in cultured HUVECs and adipocytes.
About 80% of ADMA in plasma is metabolized and inactivated by dimethylarginine dimethylaminohydrolases (DDAH1 and DDAH2). Therefore, variation in the DDAH activity may influence plasma ADMA level and NO level, and consequently influence CAD predisposition. This study was designed to evaluate whether genetic polymorphisms in DDAH1 is associated with CAD susceptibility by a case-control study.
METHODS
1. Genotyping the candidate SNPs in case-control samples
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to genotype the candidate SNPs. A case-control study of 544 newly diagonised CAD patients and 992 healthy controls of Han nationality recruited from Hunan province was carried out.
2. Verifying the function of SNPs by luciferase reporter gene experiment
3'-UTR encompassing the SNPs of interesting were cloned to downstream of pGL3-Control vectors. The vectors were then transfected into human umbilical vein endothelial cells (HUVECs) by using pRL-SV40 vector as an internal standard. MicroRNA 218 (miR-218) mimic were also co-transfected in combination with the vectors. Dual-luciferase reporter assay system was used to determine the luciferase activity.
RESULTS
(1) Genotype frequencies for DDAH1 rs233113 AA、AT and TT genotypes were 53.5%、37.1% and 9.4%, respectively, in cases, and 47.3%、43.3% and 9.4%, respectively, in controls. Significant difference in genotype distribution of rs233113 polymorphism was observed (P=0.020) between cases and controls. Genotype frequencies for DDAH1 rs233112 AA、AG and GG genotypes were 32.8%、50.6% and 16.6%, respectively, in cases, and 28.9%, 49.9% and 21.2%, respectively, in controls. Significant difference in genotype distribution of rs233112 polymorphism was observed (P=0.036) between cases and controls.
(2) When adjusted by CAD risk factors, results of unconditional logistic regression analysis showed that carriers of the rs233113 T allele showed significantly decreased CAD risk (OR=0.503,95%CI: 0.316-0.801, P=0.004), while carriers of the rs233112 A allele showed significantly increased risk for CAD (OR=1.969,95%CI:1.040-3.728, P=0.037).
(3) CAD cases showed significantly higher plasma ADMA level than controls (p<0.05). Carriers of the rs233113 T allele showed lower mean plasma ADMA level as compared with individuals with the rs233113 AA genotype, carriers of the rs233112 G allele showed significantly lower mean plasma ADMA level as compared with individuals with the rs233112 AA genotype (P=0.024).
(4) The pGL3 vectors bearing the rs233112 A allele showed significantly higher luciferase activity than those bearing the rs233112 G allele. When treated with microRNA 218 mimic, luciferase activity decreased significantly in vectors bearing the rs233112 A allele but not in vectors bearing the rs233112 G allele.
CONCLUSION
DDAH1 rs233113 A/T and rs233112 A/G genetic polymorphisms are associated with decreased and increased risk for CAD, respectively, in Chinese population. The rs233112 A/G genetic polymorphism may act through affecting miR-218 binding.
Background
Coronary artery disease (CAD) is a complex, multifactorial disease with both genetic and environmental components. A growing body of evidence suggests that oxidative stress and reactive oxygen species (ROS) play an important role in the pathogenesis of atherosclerosis (AS) and its complications. ROS can increase the production of active aldehydes, such as 4-hydroxy-2-nonenal (4-HNE).4-HNE is also implicated in atherosclerosis through induction of ROS generation and endothelial barrier dysfunction.
Decrease in NO bioavailability can lead to endothelial dysfunction, and thus contribute to the initiation and progression of atherosclerosis. An increase in plasma concentration of the endogenous inhibitor of nitric oxide synthase, asymmetric dimethylarginine (ADMA), is regarded as a novel independent cardiovascular risk factor. Dimethylarginine dimethylaminohydrolases 1 and 2 (DDAH1 and DDAH2) are two enzymes responsible for the metabolism of ADMA. In cultured vascular endothelial cells,4-HNE can form Michael adducts with DDAH1, decrease DDAH1 activity, increase ADMA formation, and decrease NO generation.
Mitochondrial aldehyde dehydrogenase (ALDH2) is an enzyme responsible for the detoxification of aldehydes such as 4-HNE. A common Glu504Lys polymorphism (rs671) of ALDH2, which accounts for decreased ALDH2 activity, is common in Asian population. In the present study, we investigated the association of ALDH2 rs671 polymorphism with CAD susceptibility and the possible mechanism in Chinese population.
METHODS
1. Genotyping the candidate SNP in case-control samples
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to genotype the candidate SNP. A case-control study of 417 newly diagonised CAD patients and 448 healthy controls of Han nationality recruited from Hunan province was carried out.
2. Effect of ALDH2 rs671 on DDAH1/ADMA system in HUVECs
HUVECs from 11 human umbilical cords were genotyped, cultured and treated with angiotensin II (Ang II,10-7-10-5 mol/L). DDAH1 mRNA expression was detected by real-time PCR. ADMA in lysate and cell medium was determined by high performance liquid chromatography-mass spectrometry (HPLC-MS).
RESULTS
(1) Genotype frequencies for ALDH2 rs671 GG、GA and AA genotypes were 52.5%、41.0% and 6.5%, respectively, in cases, and 65.0%、30.1% and 4.9%, respectively, in controls. Significant difference in genotype distribution of rs671 polymorphism was observed (P=0.0002) between cases and controls. When adjusted by CAD risk factors, results of unconditional logistic regression analysis showed that carriers of the rs671 A allele were associated significantly with increased CAD susceptibility in overall subjects (P=0.00005).
(2) In both cases and controls, carriers of the rs671 A allele were significantly overrepresented in non-drinkers than in drinkers (53.7% vs 28.8%, non-drinkers vs drinkers, p=1.1×10-5 for cases; 38.8% vs 22.5%, non-drinkers vs drinkers,p=0.003 for controls). Carriers of the rs671 A allele were also significantly overrepresented in non-drinking patients with CAD (53.7%) than in non-drinking controls (38.8%) (p=0.00012). After adjustment for CAD risk factors, carriers of the rs671 A allele were at an increased risk of CAD among non-drinkers (OR=1.95,95% CI: 1.40-2.70,p=0.00007) but not among drinkers (OR=1.56,95%CI: 0.77-3.18,p=0.22).
(3) In non-drinking controls, rs671 AA homozygotes showed significantly lower serum concentration of high density lipoprotein cholesterol (HDL-C) than GG homozygotes.
(4) HUVECs from rs671 GG homozygotes showed significantly higher DDAH1 mRNA expression and lower intracellular ADMA levels than those from GA heterozygotes. High concentration of Ang II (10-5 mol/L) decreased DDAH1 mRNA expression and increased intracellular ADMA concentration in HUVECs with the rs671 GG genotype more obviously. CONCLUSION
ALDH2 rs671 polymorphism is associated with an increased risk of CAD in Chinese non-drinkers possibly through influencing HDL-C levels and endothelial ADMA levels.
原发性高血压或高血压病由于其高发病率和高死亡率,是目前影响人类健康的主要常见疾病之一。大量的研究业已证实,高血压病是由环境与遗传因素共同作用所致的多基因疾病。
血管内皮在维持心血管系统的稳态中发挥极其重要的作用,内皮功能受损是包括高血压病在内的多种心血管疾病的早期表现和共同的病理生理特征。NO是维持血管内皮功能最重要的一种介质之一,由L-精氨酸在一氧化氮合酶(NOS)的催化作用下生成,具有血管舒张作用。
非对称二甲基精氨酸(Asymmetric Dimethylarginine, ADMA)是一种内源性NOS抑制剂,能竞争性地抑制NO的生成。人体内生成的ADMA 80%经二甲基精氨酸二甲胺水解酶(包括DDAH1和DDAH2两种亚型)水解代谢而失活。因此,体内DDAH酶活性的高低可通过影响ADMA的代谢,从而影响NOS的活性和NO的水平。遗传因素可能是影响体内DDAH酶活性的因素之一。由遗传因素所导致的个体间DDAH酶活性的差异可能通过影响ADMA的体内浓度,从而影响机体对高血压病的易感性。本文通过病例-对照研究,探讨DDAH1基因多态性与中国汉族人群高血压病遗传易感性的关系。
方法
1.候选单核苷酸多态性(single nucleotide polymorphism, SNP)位点的选择与基因分型
应用NCBI数据库中的数据资源,对中国人群DDAH1基因的单核苷酸多态性(single nucleotide polymorphism, SNP)进行登记,通过连锁不平衡分析,明确该基因区域SNP位点间的连锁不平衡情况,选择待分型的标签SNP (haplotype tag SNP, htSNP)。同时,随机选取20个无亲缘关系的高血压患者,通过PCR产物测序,对该基因所有外显子、外显子-内含子交界以及2kb范围内的启动子区进行重新测序和SNP发掘,寻找新的标签SNP。应用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)的分析方法,在1318例原发性高血压患者和1005例健康人群中对DDAH1基因的标签SNP进行基因分型,并通过应用高效液相色谱-质谱联用法测定病例和对照人群血浆ADMA的水平。
2.荧光素酶报告基因实验对SNP的功能进行验证
对于3’端未翻译区(3'-untranslated region,3'-UTR)出现阳性关联的SNP, PCR扩增含不同等位基因的3'-UTR区段并克隆入荧光素酶报告基因质粒,以pRL-SV40载体为内参,两种质粒混合并转染人脐静脉内皮细胞株HUVECs。双荧光素酶报告实验体系测定荧光素酶活性,分析和比较含不同等位的3'-UTR序列的转录活性。
3.培养的人脐静脉内皮细胞观察SNP的功能
分离和培养人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs), PCR-RFLP对脐静脉组织DDAH1 SNP位点进行基因分型,第4代内皮细胞于24孔板中培养24小时,ELISA法测定细胞内及培养基中ADMA的浓度和DDAH1的活性；应用放线菌酮处理内皮细胞,测定药物处理前(0小时)及药物处理后4、8、12、16、24和36小时DDAHl mRNA表达水平,比较DDAH1不同基因型个体HUVECs DDAH活性、ADMA水平、DDAH1 mRNA表达水平及半衰期的异同。
结果
(1)通过在20个无关中国个体中对DDAH1基因进行重新测序,总共发现了46个SNP,其中有10个为新发现的SNP。所有的46个SNP均不改变DDAH1的氨基酸结构,其中12个位于3'-UTR区。
(2)病例和对照人群中rs233113 A/T、rs3087894 C/G和rs233112A/G多态性位点基因型分布存在显著的差异(P值分别为0.003、0.003和0.042),且rs3087894 C/G和rs233113 A/T多态性呈完全连锁不平衡(D'=1,r2=1)；病例和对照人群中rs3813600 C/T多态位点基因型分布差异不明显(p=0.073)。
(3)通过对高血压的风险因素进行校正,Logistic回归分析结果显示,携带rs233113 T(或rs3087894 G)等位的个体患原发性高血压病的风险显著降低(比值比=0.747,95%可信区间：0.618-0.904,P=0.005); rs233112GG基因型也可降低高血压病的发病风险(比值比=0.772；95%可信区间：0.607-0.983,p=0.035)；而rs3813600多态位点与高血压病的发病风险不相关(p>0.05)。
(4)病例组人群血浆ADMA的浓度显著高于对照人群组(p<0.001)。在对照人群中,携带rs233113 T等位的个体血浆中ADMA浓度显著高于基因型为AA的个体(p=0.016),而在病例人群中,rs233113 TT基因型个体血浆ADMA浓度与rs233113 AA基因型个体相比有降低的趋势(p=0.089)；对照组中携带rs3813600 T等位的个体血浆ADMA的浓度显著低于该位点基因型为CC的个体(p=0.033),rs233112GG基因型个体血浆ADMA的浓度显著高于该位点为AA基因型的个体(p=0.007)；而在病例组中,rs3813600位点携带T等位的个体血浆ADMA的浓度显著高于该位点为CC基因型的个体(p=0.048); rs233112GG基因型个体血浆ADMA的浓度显著低于该位点为AA基因型的个体(p=0.036)。
(5)携带rs233113 T等位的pGL3质粒荧光素酶活性显著高于携带rs233113 A等位的质粒；与rs233113 (A/T)多态性呈完全连锁不平衡的SNP rs3087894 (C/G)/rs233115 (C/T)/rs233114 (A/C)构成的两种单倍型(rs3087894C-rs233115C-rs233114A和rs3087894G-rs233115T-rs233114C)间报告基因的活性无显著差别。
(6)携带rs233113 T等位的HUVECs (n=4)细胞内DDAH的活性显著高于基因型为rs233113 AA的HUVECs (n=5, p=0.039),而细胞内ADMA的浓度显著低于rs233113 AA基因型HUVECs(p=0.037)。放线菌酮处理前及处理后4小时和8小时,携带rs233113T等位的HUVECs (n=4)内DDAH1 mRNA表达水平显著高于基因型为rs233113 AA的HUVECs (n=5,p<0.05)。与基因型为rs233113AA的HUVECs (n=5)相比,携带rs233113 T等位的HUVECs (n=4)内DDAH1 mRNA消除半衰期有缩短的趋势(t1/2分别为17.3±3.4h和51.0±31.5h,p=0.073)。
结论
DDAH1基因rs233113A/T和rs233112A/G多态性与中国长沙地区汉族人群原发性高血压的遗传易感性相关,rs233113A/T多态性可通过增加报告基因的活性、DDAH1 mRNA表达和活性,从而降低体内ADMA的水平,降低原发性高血压的易感性。
研究背景
冠心病是人类死亡的主要原因并已成为世界主要卫生问题之一。流行病学调查资料表明,冠心病是由环境因素和遗传因素相互作用所导致的多基因疾病。
血管内皮在维持心血管系统的稳态中起极其重要的作用,内皮功能受损是包括冠心病在内的多种心血管疾病的早期表现。NO能够调控内皮功能且能反映内皮功能的健康状况,其生成减少或活性降低在动脉粥样硬化(Atherosclerosis, AS)和冠心病的发生发展中起到关键作用。
非对称二甲基精氨酸(ADMA)是一种内源性NOS抑制剂,能竞争性的抑制NO的生成。研究表明,血浆ADMA水平和冠心病病变程度间呈强关联。此外,冠心病患者体内活性氧(Reactive oxygen species, ROS)水平显著增加,氧化应激在AS和冠心病的发生发展中起重要作用。研究表明,病理生理浓度的ADMA也可通过增加培养的人脐静脉内皮细胞和脂肪细胞细胞内ROS的生成,从而参与AS的发生发展。
人体血浆中的ADMA主要经二甲基精氨酸二甲胺水解酶1和2(Dimethylarginine Dimethylaminohydrolases, DDAH1和DDAH2)水解代谢而失活。研究表明,遗传因素可能是影响体内DDAH酶活性的因素之一。因此,由遗传因素所导致的个体间DDAH酶活性的差异可能通过影响ADMA的体内浓度以及NO的水平,从而影响机体对冠心病的易感性。本文通过病例-对照研究,探讨DDAH1基因多态性与中国汉族人群冠心病遗传易感性的关系。
方法
1.病例对照标本候选SNP位点基因分型
应用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)的分析方法,在544例冠心病患者和992例健康对照人群中对DDAH1基因的标签SNP进行基因分型。
2.荧光素酶报告基因实验对SNP的功能进行验证
PCR扩增含不同等位基因的3’-UTR区段并克隆入pGL3-Control质粒中,以pRL-SV40载体为内参,两种质粒以及microRNA 218(miR-218)模拟物共同转染人脐静脉内皮细胞株HUVECs。双荧光素酶报告实验测定荧光素酶活性,分析和比较含不同等位的3’-UTR序列在不同浓度的miR-218模拟物处理下的报告基因活性。
结果
(1)冠心病病例组和对照组中rs233113位点AA、AT和TT基因型频率分别为53.5%、37.1%、9.4%和47.3%、43.3%、9.4%,病例组rs233113 AA基因型频率显著高于对照组(P=0.020)。病例组和对照组中rs233112位点AA、AG和GG基因型频率分别为32.8%、50.6%、16.6%和28.9%、49.9%、21.2%,病例组rs233112GG基因型频率显著低于对照组(P=0.036)。
(2)通过对冠心病的风险因素进行校正,Logistic回归分析结果显示,携带rs233113 T等位的个体患冠心病的风险显著降低(OR=0.503,95%CI:0.316-0.801, P=0.004),而携带rs233112 A等位的个体患冠心病的风险显著增加(OR=1.969,95%CI:1.040-3.728,P=0.037)。
(3)冠心病病例组人群血浆ADMA的浓度显著高于对照人群组(p<0.05)；病例组携带rs233113 T等位的个体血浆中ADMA浓度略低于AA基因型个体；携带rs233112 G等位的个体血浆中ADMA浓度显著低于基因型为AA的个体(p=0.024)。
(4)携带rs233112 A等位的质粒的荧光素酶活性显著高于携带rs233112 G等位的质粒(p<0.05), miR-218 (2.5nM-20nM)可显著降低含rs233112 A质粒的荧光素酶活性,而对携带rs233112 G等位的质粒荧光素酶活性无影响。
结论
DDAH1基因rs233113A/T多态性和rs233112A/G多态性可分别降低和增加中国长沙地区汉族人群冠心病的发病风险,rs233112多态位点可影响报告基因活性和miR-218的结合,可能是影响冠心病易感性的功能性SNP位点。
研究背景
冠心病是一种由环境因素和遗传因素相互作用所导致的复杂性多因素疾病。大量的研究结果表明,氧化应激和活性氧(ROS)在动脉粥样硬化(AS)及其并发症的发生发展中起到重要作用。ROS可导致多种活性醛如4-羟基壬烯醛(4-HNE)等生成增加,而活性醛类可进一步加重氧化应激,引起血管内皮功能障碍,促进AS的病理生理过程。
人类线粒体醛脱氢酶(ALDH2)参与多种活性醛类的代谢灭活,其中包括4-HNE。ALDH2基因的第12号外显子上存在功能性单核苷酸序列多态性rs671多态,该SNP使ALDH2多肽第504位的谷氨酸被赖氨酸取代,从而导致ALDH2酶活性降低,代谢乙醛等活性醛的能力下降。
非对称二甲基精氨酸(ADMA)是一种内源性的一氧化氮合酶(NOS)抑制剂,它能够降低NO的生物利用度而导致内皮功能障碍,因此ADMA被视为一种新的冠心病和AS的危险因子。ADMA在体内主要是由二甲基精氨酸二甲胺水解酶(DDAH1和DDAH2)代谢灭活。在培养的内皮细胞中,4-HNE能通过与DDAH1形成Michael加合物而剂量依赖性地抑制]DDAH1活性,进而增加ADMA产生并降低NO生成。
本实验在中国人群中研究了ALDH2 rs671多态性是否与冠心病发病风险相关,并研究该多态是否可影响DDAH1的mRNA表达和ADMA的浓度,从DDAH/ADMA系统探讨其影响冠心病易感性的可能机制。
方法
1.病例对照标本候选SNP位点基因分型
收集冠心病和性别年龄相匹配的健康对照人群血液标本。应用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)的分析方法,对所有病例和对照人群ALDH2基因rs671多态性进行基因分型。
2. ALDH2 rs671多态性对原代人脐静脉内皮细胞血管紧张素Ⅱ处理前后DDAH1/ADMA系统的影响
取11根人脐带进行ALDH2基因分型,分离培养人脐静脉内皮细胞HUVECs。第4代细胞用不同浓度(10-7至10-5mol/L)的血管紧张素Ⅱ处理24小时。应用实时定量PCR法检测细胞内DDAH1 mRNA表达水平。应用高效液相色谱-质谱联用法检测细胞内及培养基中ADMA的水平。
结果
(1)冠心病病例组和对照组中rs671位点GG、GA和AA基因型频率分别为52.5%、41.0%、6.5%和65.0%、30.1%、4.9%,病例组rs671 GA+AA基因型频率显著高于对照组(χ2=13.807,P=0.0002)。通过对冠心病风险因素进行校正,Logistic回归分析结果显示,在总人群中,携带rs671 A等位的个体患冠心病的风险显著增高(OR=1.85,95%CI：1.38-2.49,P=0.00005)。
(2)在病例和对照人群中,不饮酒人群携带rs671 A等位个体的频率均显著高于饮酒人群(病例组中分别为53.7%和28.8%,P=1.1×10-5；对照组中分别为38.8%和22.5%,P=0.003)；不饮酒人群中病例组携带rs671 A等位个体的频率(53.7%)显著高于对照组人群(38.8%,P=0.00012)。通过对冠心病的风险因素进行校正,Logistic回归分析结果显示,携带rs671 A等位可显著增加不饮酒人群冠心病的发病风险(OR=1.95,95%CI:1.40-2.70, P=0.00007),但不影响饮酒人群冠心病的发病风险(OR= 1.56,95%CI:0.77-3.18, P=0.22)。
(3)在不饮酒健康人群,rs671 AA纯合子个体血清高密度脂蛋白(HDL-C)浓度(1.20±0.22 mmol/L, n=22)显著低于GG纯合子个体(1.39±0.36 mmol/L, n=212, P=0.015)。
(4)基因型为rs671 GG的HUVECs (n=6) DDAH1 mRNA表达水平显著高于基因型为rs671 GA的HUVECs (n=5) (p＜0.05),而细胞内ADMA水平显著低于基因型为GA的HUVECs (p＜0.05)。高浓度的血管紧张素Ⅱ(10-5mol/L)可显著降低基因型为rs671 GG的HUVECs中DDAH1 mRNA表达,升高该基因型HUVECs细胞内ADMA浓度,但对基因型为GA的HUVECs DDAH1 mRNA表达和ADMA浓度影响不明显(p>0.05)。
结论
ALDH2基因rs671多态性可能通过降低HDL-C水平和HUVECs内DDAH1的mRNA表达,升高HUVECs内ADMA的水平,从而增加不饮酒的中国长沙地区汉族人群冠心病的发病风险。
Background
Essential hypertension (EH) is one of the most important global health problems beacause of its high prevalence and high mortality. EH is a multigenic disease with components of environmental and genetic factors, genetic factors may account for about 30%-60% of blood pressure (BP) variance.
The vascular endothelium plays an important role in the maintainance of the homeostasis of cardiovascular system. Endothelial dysfunction is the early characterization of cardiovascular diseases including EH. Nitric oxide (NO) is one of the most important mediators that are involved in the maintainance of vascular endothelium function. NO is an endogeneous vasodilator, and is synthesized from L-arginine catalyzed by NO synthase (NOS).
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NOS which can competitively inhibit NO production. About 80% of ADMA in plasma is metabolized and inactivated by dimethylarginine dimethylaminohydrolases (DDAH1 and DDAH2). Therefore, variation in the DDAH activity may influence NO level through affecting plasma ADMA level and NOS activity, and consequently influence EH predisposition. This study was designed to evaluate whether genetic polymorphisms in DDAH1 is associated with hypertension susceptibility by a case-control study.
METHODS
1. Genotyping the candidate SNPs in cases and controls
By using the public data deposited in the NCBI database, single nucleotide polymorphisms (SNPs) at DDAH1 locus in Beijing Chinese Han were recorded. After analysis of linkage disequilibrium (LD) and establishment of haplotypes, haplotype tag SNPs (htSNPs) were selected for further study. DNA discovery was also carried out by resequencing all exons, exon-intron boundaries, and 2kb region upstream the transcriptional start site in the DDAH1 promoter in 20 randomly selected EH patients. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to genotype the candidate SNPs. A case-control study of 1318 newly diagonised EH patients and 1005 normotensive controls of Han nationality recruited from Hunan province was carried out.
2. Functional analysis of DDAH1 SNPs by luciferase reporter gene assay system
For the positively associated SNPs in 3'-untranslated region (3'-UTR) of DDAH1,3'-UTR encompassing the SNPs were cloned to downstream of pGL3-Control vectors. The vectors were then transfected into human umbilical vein endothelial cells (HUVECs) by using pRL-SV40 vector as an internal standard. Dual-luciferase reporter assay system was used to determine the luciferase activity.
3. Verifying the function of SNPs in HUVECs
HUVECs were isolated from 9 human umbilical cords, DDAH1 SNPs were genotyped, and the cells were cultured in EGM-2 medium. Cells of the fourth passage were cultured for 24 hours, and then cycloheximide (CHX) were added. ADMA in lysate and cell medium and DDAH1 activity were determined by ELISA. RNA were extracted just before and at 4h,8h,12h,16h,24h, and 36h, respectively after CHX treatment. DDAH1 mRNA expression were determined by real-time quatitative PCR.
RESULTS
(1) A total of 46 SNPs were idenfied at the DDAH1 locus in 20 Chinese EH patients,10 of the SNPs were newly discovered. None of the SNPs lead to change in the amino acid sequence of DDAH1, and 12 of the SNPs were located in 3'-UTR.
(2) Significant difference in genotype distribution for rs233113 A/T, rs3087894 C/G and rs233112 A/G polymorphisms were observed between EH cases and controls (p values were 0.003,0.003, and 0.042, respectively). The rs233113 A/T and rs3087894 C/G polymorphisms were in complete linkage disequilibrium (D'=1, r2=1). No difference in genotype distribution of the rs3813600 C/T polymorphism was observed between cases and controls (p=0.073).
(3) When adjusted by EH risk factors, results of unconditional logistic regression analysis showed that carriers of the rs233113 T (or rs3087894 G) allele showed significantly decreased risk for EH (odds ratio [OR]=0.747,95% confidence interval [CI]:0.618-0.904, p=0.005). The rs233112 GG was also associated with decreased risk for EH (OR=0.772, 95%CI:0.607-0.983,p=0.035). No association between rs3813600 polymorphism and EH risk was observed.
(4) EH cases showed significantly higher plasma ADMA level than controls (p<0.001). In controls, carriers of the rs233113 T allele showed significantly higher mean plasma ADMA level as compared with individuals with the rs233113 AA genotype, carriers of the rs3813600 T allele showed significantly lower mean plasma ADMA level as compared with individuals with the rs3813600 CC genotype, carriers of the rs233112 G allele showed significantly higher mean plasma ADMA level as compared with individuals with the rs233112 AA genotype. While in EH cases, carriers of the rs3813600 T allele showed significantly higher mean plasma ADMA level as compared with individuals with the rs3813600 CC genotype, individuals genotyped as rs233112 GG showed significantly lower mean plasma ADMA level as compared with individuals with the rs233112 AA genotype.
(5)pGL3 vectors bearing the rs233113 T allele showed significantly higher luciferase activity than those bearing the rs233113 A allele. No difference in luciferase activity was observed between bearing the rs3087894 C-rs233115 C-rs233114 A and rs3087894 G-rs233115 T-rs233114Calleles.
(6) HUVECs carrying the rs233113 T allele showed significantly higher DDAH1 activity and lower intracellular ADMA levels as compared those with the rs233113 AA genotype. HUVECs carrying the rs233113 T allele showed significantly higher DDAH1 mRNA expression levels as compared those with the rs233113 AA genotype before (0 hour) and at 4 hour and 8 hour after CHX treatment (p<0.05, respectively). As compared HUVECs with the rs233113 AA genotype, half-life time of DDAH1 mRNA in HUVECs bearing the rs233113 T allele trended to be decreased (17.3±3.4 h vs 51.0±31.5 h,p=0.073).
CONCLUSION
DDAH1 rs233113 A/T and rs233112 A/G genetic polymorphisms are associated with EH susceptibility in Chinese. The rs233113 A/T polymorphism can increase the activity of reporter gene and increase DDAHl mRNA expression.
Background
Coronary artery disease (CAD) has become a leading cause of death and a major health problem worldwide. Epidemiological studies have indicated that CAD is a complex, multifactorial disease with both genetic and environmental components.
The vascular endothelium plays an important role in maintaining the homeostasis of cardiovascular system. Endothelial dysfunction is the early characterization of many cardiovascular diseases including CAD. Nitric oxide (NO) plays important roles in the regulation of endothelium function. Decrease in NO bioavailability can lead to endothelial dysfunction, and thus contribute to the initiation and progression of atherosclerosis (AS) and CAD.
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NOS which can competitively inhibit NO production. A strong relationship between ADMA plasma levels and CAD severity was reported. Oxidative stress and ROS also play important roles in the pathogenesis of AS. Pathophysiological concentration of ADMA can increase production of intracellular reactive oxygen species (ROS) in cultured HUVECs and adipocytes.
About 80% of ADMA in plasma is metabolized and inactivated by dimethylarginine dimethylaminohydrolases (DDAH1 and DDAH2). Therefore, variation in the DDAH activity may influence plasma ADMA level and NO level, and consequently influence CAD predisposition. This study was designed to evaluate whether genetic polymorphisms in DDAH1 is associated with CAD susceptibility by a case-control study.
METHODS
1. Genotyping the candidate SNPs in case-control samples
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to genotype the candidate SNPs. A case-control study of 544 newly diagonised CAD patients and 992 healthy controls of Han nationality recruited from Hunan province was carried out.
2. Verifying the function of SNPs by luciferase reporter gene experiment
3'-UTR encompassing the SNPs of interesting were cloned to downstream of pGL3-Control vectors. The vectors were then transfected into human umbilical vein endothelial cells (HUVECs) by using pRL-SV40 vector as an internal standard. MicroRNA 218 (miR-218) mimic were also co-transfected in combination with the vectors. Dual-luciferase reporter assay system was used to determine the luciferase activity.
RESULTS
(1) Genotype frequencies for DDAH1 rs233113 AA、AT and TT genotypes were 53.5%、37.1% and 9.4%, respectively, in cases, and 47.3%、43.3% and 9.4%, respectively, in controls. Significant difference in genotype distribution of rs233113 polymorphism was observed (P=0.020) between cases and controls. Genotype frequencies for DDAH1 rs233112 AA、AG and GG genotypes were 32.8%、50.6% and 16.6%, respectively, in cases, and 28.9%, 49.9% and 21.2%, respectively, in controls. Significant difference in genotype distribution of rs233112 polymorphism was observed (P=0.036) between cases and controls.
(2) When adjusted by CAD risk factors, results of unconditional logistic regression analysis showed that carriers of the rs233113 T allele showed significantly decreased CAD risk (OR=0.503,95%CI: 0.316-0.801, P=0.004), while carriers of the rs233112 A allele showed significantly increased risk for CAD (OR=1.969,95%CI:1.040-3.728, P=0.037).
(3) CAD cases showed significantly higher plasma ADMA level than controls (p<0.05). Carriers of the rs233113 T allele showed lower mean plasma ADMA level as compared with individuals with the rs233113 AA genotype, carriers of the rs233112 G allele showed significantly lower mean plasma ADMA level as compared with individuals with the rs233112 AA genotype (P=0.024).
(4) The pGL3 vectors bearing the rs233112 A allele showed significantly higher luciferase activity than those bearing the rs233112 G allele. When treated with microRNA 218 mimic, luciferase activity decreased significantly in vectors bearing the rs233112 A allele but not in vectors bearing the rs233112 G allele.
CONCLUSION
DDAH1 rs233113 A/T and rs233112 A/G genetic polymorphisms are associated with decreased and increased risk for CAD, respectively, in Chinese population. The rs233112 A/G genetic polymorphism may act through affecting miR-218 binding.
Background
Coronary artery disease (CAD) is a complex, multifactorial disease with both genetic and environmental components. A growing body of evidence suggests that oxidative stress and reactive oxygen species (ROS) play an important role in the pathogenesis of atherosclerosis (AS) and its complications. ROS can increase the production of active aldehydes, such as 4-hydroxy-2-nonenal (4-HNE).4-HNE is also implicated in atherosclerosis through induction of ROS generation and endothelial barrier dysfunction.
Decrease in NO bioavailability can lead to endothelial dysfunction, and thus contribute to the initiation and progression of atherosclerosis. An increase in plasma concentration of the endogenous inhibitor of nitric oxide synthase, asymmetric dimethylarginine (ADMA), is regarded as a novel independent cardiovascular risk factor. Dimethylarginine dimethylaminohydrolases 1 and 2 (DDAH1 and DDAH2) are two enzymes responsible for the metabolism of ADMA. In cultured vascular endothelial cells,4-HNE can form Michael adducts with DDAH1, decrease DDAH1 activity, increase ADMA formation, and decrease NO generation.
Mitochondrial aldehyde dehydrogenase (ALDH2) is an enzyme responsible for the detoxification of aldehydes such as 4-HNE. A common Glu504Lys polymorphism (rs671) of ALDH2, which accounts for decreased ALDH2 activity, is common in Asian population. In the present study, we investigated the association of ALDH2 rs671 polymorphism with CAD susceptibility and the possible mechanism in Chinese population.
METHODS
1. Genotyping the candidate SNP in case-control samples
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to genotype the candidate SNP. A case-control study of 417 newly diagonised CAD patients and 448 healthy controls of Han nationality recruited from Hunan province was carried out.
2. Effect of ALDH2 rs671 on DDAH1/ADMA system in HUVECs
HUVECs from 11 human umbilical cords were genotyped, cultured and treated with angiotensin II (Ang II,10-7-10-5 mol/L). DDAH1 mRNA expression was detected by real-time PCR. ADMA in lysate and cell medium was determined by high performance liquid chromatography-mass spectrometry (HPLC-MS).
RESULTS
(1) Genotype frequencies for ALDH2 rs671 GG、GA and AA genotypes were 52.5%、41.0% and 6.5%, respectively, in cases, and 65.0%、30.1% and 4.9%, respectively, in controls. Significant difference in genotype distribution of rs671 polymorphism was observed (P=0.0002) between cases and controls. When adjusted by CAD risk factors, results of unconditional logistic regression analysis showed that carriers of the rs671 A allele were associated significantly with increased CAD susceptibility in overall subjects (P=0.00005).
(2) In both cases and controls, carriers of the rs671 A allele were significantly overrepresented in non-drinkers than in drinkers (53.7% vs 28.8%, non-drinkers vs drinkers, p=1.1×10-5 for cases; 38.8% vs 22.5%, non-drinkers vs drinkers,p=0.003 for controls). Carriers of the rs671 A allele were also significantly overrepresented in non-drinking patients with CAD (53.7%) than in non-drinking controls (38.8%) (p=0.00012). After adjustment for CAD risk factors, carriers of the rs671 A allele were at an increased risk of CAD among non-drinkers (OR=1.95,95% CI: 1.40-2.70,p=0.00007) but not among drinkers (OR=1.56,95%CI: 0.77-3.18,p=0.22).
(3) In non-drinking controls, rs671 AA homozygotes showed significantly lower serum concentration of high density lipoprotein cholesterol (HDL-C) than GG homozygotes.
(4) HUVECs from rs671 GG homozygotes showed significantly higher DDAH1 mRNA expression and lower intracellular ADMA levels than those from GA heterozygotes. High concentration of Ang II (10-5 mol/L) decreased DDAH1 mRNA expression and increased intracellular ADMA concentration in HUVECs with the rs671 GG genotype more obviously. CONCLUSION
ALDH2 rs671 polymorphism is associated with an increased risk of CAD in Chinese non-drinkers possibly through influencing HDL-C levels and endothelial ADMA levels.
引文
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