Association Of Genetic Polymorphisms In DDAH1 And ALDH2 Genes With Essential Hypertension And Coronary Artery Disease

BackgroundEssential 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.METHODS1. Genotyping the candidate SNPs in cases and controlsBy 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 systemFor 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 HUVECsHUVECs 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). CONCLUSIONDDAH1 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. BackgroundCoronary 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.METHODS1. Genotyping the candidate SNPs in case-control samplesPolymerase 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 experiment3'-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.CONCLUSIONDDAH1 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. BackgroundCoronary 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.METHODS1. Genotyping the candidate SNP in case-control samplesPolymerase 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 HUVECsHUVECs 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. CONCLUSIONALDH2 rs671 polymorphism is associated with an increased risk of CAD in Chinese non-drinkers possibly through influencing HDL-C levels and endothelial ADMA levels.