Association And Functional Characterization Of Single Nucleotide Polymorphism Within GSTM3 Gene's Regulatory Region In Essential Hypertension And Hyperlipidemia

ObjectiveEssential hypertension (EH) and hyperlipidemia are the most commoncardiovascular diseases nowadays that may lead to a series of severe results such asatherosclerosis, stroke, haeat failure and renal failure. More than half of the diseases areattributable to lipid abnormalities. EH and hyperlipidemia are complex diseasesresulting from interaction of cumulative effects of multiple genetic and environmentalfactors. Therefore, associational study and functional identifcation of EH andhyperlipidemia candidate genes relevant to environmental factors could throw light onthe pathogenesis of such diseases.Glutathione S-transferases (GSTs) comprise a supergene family of isoenzymes.GSTM3 is one of the supergene family isoenzymes. GSTs also display glutathioneperoxidase activity and can protect cells from oxidative damage. Their centralimportance against oxidative stress rests on the unique capacity for conjugation of atremendous variety of reactive intermediates such as oxidative species and free radicals.In addition, oxidative stress may also play a critical role in the pathogenesis of coronaryheart disease, atherosclerosis and hypertension. Up to date, there are at least 150hypertension candidate genes, including vasoactiveintestinalpeptide and its acceptor,growth factors, cytokine and their receptor genes, saltivity gene and ion channel, etc.The mechanisms of their pathogenesis are under research. Recently, Aoharu Iwai reported two SNPs, A3207G and C-1305G of GSTM3, tobe significantly associated with hypertension in Japaese women. However, associationof GSTM3/A-63C with hyperlipidemia and EH as well as their functional implicationswere not identified to date. In this work, we focused on SNPs within GSTM3 gene'sregulatory region, and genotyped a relatively isolated population from hypertensionprevalent region in Northeastern China, and undertook case-control association study ofGSTM3 with EH and hyperlipidemia. Furthermore, we analyzed the GSTM3 regulatoryregion by software prediction, constructed reporter vector and pGL3 detection toelucidate the role of GSTM3 in EH and hyperlipidemia pathogenesis.Materials and MethodsMaterials1. Reagents for gene cloning and luciferase assay.2. JM109 competent cell.3. Human hepatoma Bel cell.4. Reagents for electrophoresis mobility shift assay (EMSA).5. All subjects were Han Chinese in origin and from the prevalent region ofhypertension in Liaoning province of northeastern China. 878 subjects were recruitedfor case-control study, including 234 unrelated hypertensives (99 males and 135femles)(systolic pressure of≥140 mm Hg or diastolic pressure≥90 mm Hg or both).316 subjects were with hyperlipidemia (99 males, 217 females) (serum total cholesterolof≥5.70 mmol/L or serum total triglyceride of≥1.70 mmol/L, or both) and 328 controlsubjects (100 males, 228 females) had normal blood pressure (both systolic pressure of＜140mmHg and diastolic pressure＜90 mmHg) and normal blood lipid (both serumtotal cholesterol of＜5.70 mmol/L and serum total triglyceride of＜1.70 mmol/L).Methods 1. Genomic DNA was extracted from white blood cells with a proteinase Kextraction protocol. GSTM3 regulatory region (-427 bp～+40 bp) was amplified bypolymerase chain reaction (PCR). Case-control population was examined usingPCR-restriction fragment length polymorphism (RFLP) analysis. 20 PCR productsfrom case-control population were randomly selected for DNA sequencing.2. Two kinds of luciferase reporter constructs were generated by PCR amplificationand cloned into pGL3-basic. Transient transfection was performed in Bel cells usingliposome, and pGL3 assay was undertaken by luciferase enzyme assay. Alcoholtreatment was performed to estimate its effect on the expression of GSTM3.3. Electrophoretic mobility shift assay (EMSA) was conducted to identifytranscription factor binding element in GSTM3 gene. Nuclear protein was extractedfrom the Bel cells according to the method discribed by Dignam et al. Putative andmutant oligonucleotides according to GSTM3 gene sequence with EFII responsiveelement and TATA box were radiolabeled with [γ-³²p] ATE For competition assay,100-fold molar excess of the unlabeled double stranded oligonucleotides were addedinto reaction mixture. Electrophoresis was performed in a 10% non-denaturingpolyacrylamide gel. To examine whether the activity of the GSTM3 promoter isaffected by alcohol, cells were stimulated with different concentration of alcohol (100mmol/L and 150 mmol/L) for 24 hours.4. Qualitative data and Hardy-Weinberg equilibrium were tested using x² test.Clinical biochemical data were expressed as mean±SD. P＜0.05 was consideredsignificant. Quantitative clinical data were compared between case and controls by theindependent -samples t. Adjusted odds ratios (ORs) with 95% confidence intervalsfrom logistic regression analysis were used to estimate the relative risk factor ofhypertension and hyperlipidemia. Statistical analyses were done with SPSS 12.0.Results1. In the case-control population, body mass index, serum triglyceride, total cholesterol level and low-density lipoprotein cholesterol level were significantly higher in EHgroup than in controls. Genotype distribution of A-63C was in Hardy-Weinbergequilibrium in both study population. In the case-control population, -63C allelefrequency was higher in EH group (20.4 %) than in control (14.8 %) (x²=3.419,p=0.042), and the frequency of -63CC genotype was pronouncedly higher inhypertension group than in control (6% versus 1.8 %, x²=6.92, P=0.011) In females, thefrequency of -63CC genotype was statistically higher in HE group than in control(6.7% versus 0.9%, x²=8.04, p=0.005), Sorting by genotype demonstrated that meanlevel of systolic blood pressure, diastolic blood pressure, total cholesterol, low-densitylipoprotein cholesterol and serum sodium in -63CC individual was statistically higherthan other genotype group (p＜0.05).2. In the case-control population, body mass index, blood pressure, low-densitylipoprotein cholesterol level and serum sodium were significantly higher inhyperlipidemia group than in control (p＜0.05). In females, the frequency of -63CCgenotype was statistically higher in hyperlipidemia group than in control (4.6 % versus0.4 %, x²=5.899, p=0.018,OR=5.459, 95%CI: 1.182～8.206). Logistic regressionanalysis revealed that homozygous -63CC genotype was one of independent riskfactors for hyperlipidemia (adjusted OR=2.78, 95%CI:1～7.69, p=0.045).Meanwhile T-chol, triglyceride, LDL-C and serum calcium were significantly higher insubjects with CC genotype than with AA+AC genotype (p＜0.05).3. 970 bp and 636bp of the GSTM3 promoter were amplified and cloned into theupstream of luciferase reporter of pGL3-basic vector. Relative promoter activities ofA-63C alleles were assessed by measuring luciferase activity. Higher luciferaseexpression was observed for the -63C/-63A vectors compared with the emptypGL3-basic vector. Luciferase assay showed that the transcriptional activities of thepGL3-636C was 9.9-fold lower than that of the pGL3-636A(p＜0.05), and thepGL3-970C had 5.7-fold lower activities relative to the pGL3-970A (p＜0.05).Meanwhile, there was an increase in transcriptional activity of the p GL3-636A in the presence of alcohol compared with absence of alcohol, and slightly increasedtranscriptional activity was observed for the pGL3-636C with increasing concentrationof alcohol, but these differences did not reach statistical significances.4. Putative TATA box in GSTM3 around A-63C was identified by EMSA,suggesting that TATA-a and TATA-c probes could form protein-DNA complex withnuclear protein, and the -63A probe binding band with nuclear protein was strongerthan the -63C probe, which showed A-63C polymorphisms could change the bindingactivity of nuclear protein with TATA box, and GSTM3 -63A locus could enhance thebinding activity. There was a marked increase in binding of nuclear protein withTATA-a radiolabeled probe with increasing concentration of alcohol.Conclusions1. A-63C polymorphism of GSTM3 gene regulatory region is associated with EH.And in females, A-63C polymorphism is associated with hyperlipidemia, and -63CCgenotype was one of independent risk factors for hyperlipidemia.2. A-63C polymorphisms may change the binding affinity to nuclear protein withTATA box. As a result, transcriptional activity of 636A promoter was much higher thanthat of 636C promoter.3. Alcohol can stimulate the transcriptional activity of 636A promoter, but canhardly influence that of 636C promoter.4. GSTM3 maybe a EH and hyperlipidemia causative gene. Different susceptibilityfor EH and hyperlipidemia may be determined by variant of GSTM3 gene regulatoryregion through TATA box pathway.