| Objective:This case-control study was tried to explore the association of inflammatory gene promoter methylation with the risk of essential hypertension(EH).We also tried to investigate if the inflammatory gene promoter methylation could be changed by the environmental factors(for example: gender,smoking and drinking et al).In addition,we tried to study the diagnostic value to predict the onset of EH between controls and EH with gene methylation preliminarily.Methods : 1.In the current case-control study,a total of 192 individuals including 96 controls and 96 gender-and age-matched incidence essential hypertension patients(In-EH)were recruited from Han Chinese families in Ningbo China by multistage sampling method.The baseline data,serum biochemical indexes and blood samples of participants were obtained through questionnaire,conventional check-up and laboratory detection.2.Genomic DNA was extracted from peripheral blood samples and DNA sequences were reacted with sodium bisulfite to convert unmethylated cytosine residues to thymine.Methylation levels of CpG sites in inflammatory gene promoter were measured using bisulfite pyrosequencing.With database selection,software prediction and the results of preliminary experiment,TLR2?IL6and IFN-? genes were selected and tested.3.The differences of three genes promoter CpG site were analyzed between controls and incidence essential hypertension patients.Logistic regression was used to adjust for confounding factors,and find the CpG sites which were sensitive to essential and environmental factors.Results: 1.Logistic regression analysis showed the following results:(1)TLR2 gene:Incidence essential hypertension patients(In-EH)compared with controls,methylation level of CpG6 was significantly lower in In-EH than in controls(Controls vs.In-EH(%): CpG6: 8.30 ±4.13 VS 3.58 ± 3.64,odd ratio(OR(95%CI))= 0.91(0.861~0.979),adjusted P = 0.009).(2)IL6gene: Incidence essential hypertension patients(In-EH)compared with controls methylation of CpG3 was significantly lower in In-EH than in controls(Controls vs.In-EH(%): Cp G3: 57.45 ±8.29 VS 51.52 ± 6.18,odd ratio(OR(95%CI))= 0.90(0.838~0.965),adjusted P =0.004).(3)IFN-? gene: Incidence essential hypertension patients(In-EH)compared with controls methylation of CpG2 was significantly lower in In-EH than in controls(Controls vs.In-EH(%):Cp G2: 10.50 ± 0.92 VS 9.23±0.83,odd ratio(OR(95%CI))= 0.64(0.480~0.856),adjusted P = 0.003).2.Gender compared of three genes methylation levels in controls:(1)TLR2 gene:Methylation levels of CpG6 was significantly higher in females than males(males vs.females(%):Cp G6:7.31 ± 1.49 VS 8.94 ± 5.09,adjusted P = 0.013).(2)IL6 gene: Methylation levels of CpG2 was significantly lower in females than males(males vs.females(%): Cp G2: 65.53 ±7.31 VS 60.26 ± 10.46,adjusted P = 0.046).(3)IFN-? gene: Methylation levels of CpG1 was significantly lower in females than males(males vs.females(%): Cp G1: 37.58±1.31 VS36.52±1.64,adjusted P = 0.020).3.Methylation levels of inflammatory genes in drinkers and smokers in males:(1)IL6 gene:Smokers compared no-smokers,methylation levels of CpG2 and CpG3 were significantly lower in smokers than no-smokers(no-smokers vs.smokers(%): Cp G2: 64.28 ± 6.36 VS 60.41 ±7.74,adjusted P = 0.023;Cp G3: 57.78 ± 7.87 VS 53.70 ± 8.62,adjusted P = 0.038).Drinkers compared with no-drinkers,methylation levels of CpG2 and CpG3 were significantly lower in drinkers than no-drinkers(no-drinkers vs.drinkers(%):Cp G2: 64.70 ± 7.03 VS 60.89± 7.32,adjusted P = 0.038;Cp G3: 60.48 ± 7.58 VS 53.23 ± 7.99,adjusted P =4.24×10-4).(2)TLR2 and IFN-? gene: No significances were found.4.Correlation analysis:(1)TLR2 gene: Pearson correlation analysis found that CpG6 of TLR2 gene promoter was negative correlated with blood pressure(Cp G6 and SBP: r =-0.329,adjusted P = 3.95×10-5;Cp G6 and DBP: r =-0.304;adjusted P = 1.80×10-4).(2)IL6gene: Pearson correlation analysis found that CpG2-3 of IL6 gene promoter was negative correlated with blood pressure(Cp G2 and SBP: r =-0.274,adjusted P = 0.009;Cp G2 and DBP: r =-0.183,adjusted P = 0.011;Cp G3 and SBP: r =-0.321,adjusted P =5.60×10-6;Cp G3 and DBP: r =-0.274,adjusted P =1.17×10-4).(3)IFN-? gene: Spearman rank correlation analysis found that CpG2 of IFN-? gene promoter was negative correlated with blood pressure(Cp G2 and SBP: rs =-0.546,adjusted P = 2.60×10-16;Cp G2 and DBP: rs=-0.539,adjusted P =7.60×10-16).5.Receiver operating characteristic curve(ROC)analysis:(1)TLR2 gene:Analysis found that Cp G1 and Cp G6 of TLR2 gene promoter had a great diagnostic value to predict the onset of EH(Cp G1: AUC(area under curve)= 0.612,P =0.007;Cp G6: AUC(area under curve)= 0.834,P = 1.31×10-15).(2)IL6 gene: Analysis found that Cp G2-3of IL6 gene promoter has a great diagnostic value to predict the onset of EH(Cp G2: AUC(area under curve)= 0.638,P = 0.001;Cp G3: AUC(area under curve)= 0.704,P=8.12×10-7).(3)IFN-? gene: Analysis found that Cp G2 of IFN-? gene promoter has a great diagnostic value to predict the onset of EH(Cp G2: AUC(area under curve)= 0.834,P =1.40×10-15).Conclusions:1.Hypomethylation of CpG6 in TLR2 gene,hypomethylation of CpG3 in IL6 gene and hypomethylation of CpG2 in IFN-? gene are risk factors for essential hypertension.2.CpG6 methylation levels of TLR2 gene,CpG2 methylation levels of IL6 gene and CpG1 methylation levels of IFN-? gene are significant difference between male and female in controls.In males,CpG2 and CpG3 methylation levels of IL6 gene are influenced by smoking and drinking.Smoking and drinking played a great role in the pathophysiology of essential hypertension as important risk factors by the DNA methylation molecular mechanisms.3.Correlation analysis found that CpG6 of of TLR2 gene promoter,CpG2 and CpG3 of IL6 gene promoter had a negative correlation with blood pressure.CpG2 of IFN-? gene promoter had a negative correlation with blood pressure.4.CpG1 and CpG6 of TLR2 gene promoter,CpG2 and CpG3 of IL6 gene promoter and CpG2 of IFN-? gene promoter had a great diagnostic value to predict the onset of essential hypertension gene methylation preliminarily by ROC analysis. |
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