| Objective To investigate the changes of peripheral blood leukocyte DNA methylation and hydroxymethylation in coronary artery disease(CAD)and the effect of cell subsets.Methods(1)Ultra-high-performance liquid chromatography-mass spectrometry/mass spectrometry(UPLC-MS/MS)was used to detect genomic 5-methlcytosine(5-m C)contents in peripheral blood leukocytes(PBLs)in a case-control study involving265 CAD cases and 270 controls.The correlations between DNA methylation and age,sex,hypertension,hyperlipidemia,type 2 diabetes,PBLs counts and classifications were analyzed by univariate regression and multivariate regression.Associations between global 5-m C contents and CAD were analyzed by multivariate logistic regression.Associations of global 5-m C contents with incident CAD prevalence was analyzed by unconditional logistic regression,progressively adjusted for age,sex,history of hypertension,hyperlipidemia,diabetes,PBLs counts,and PBLs classifications.We characterized the shapes of the associations by calculated ORs using tertiles of global 5-m C contents in all 535 participants.(2)50 CAD patients and 50controls were randomly selected from the 535 samples.Peripheral blood mononuclear cells(PBMCs)were isolated by centrifugation in Ficoll/Hypaque,Monocytes were purified from PBMCs using positive selection in a Magnetic Activated Cell Sorted(MACS)system(Miltenyi Biotech,Bergisch Gladbach,Germany)with CD14microbeads(Miltenyi)according to the manufacturer's instructions.Genomic 5-m C and 5-hydroxymethylcytosine(5-hm C)contents were detected via UPLC-MS/MS.DNMT1 and TETs m RNA expression were detected by reverse-transcription quantitative PCR.Pearson correlation coefficients were used to estimate the within-person correlation by source of DNA.(3)Another group of 67 CAD cases and 71controls were recruited,the distribution of different monocyte subsets was identified using flow cytometry,monocytes were purified via MACS system,genomic 5-m C and5-hm C contents were detected by UPLC-MS/MS.The relationships between monocyte subsets and global 5-m C,5-hm C contents were analyzed.Results(1)The UPLC-MS/MS method has been verified to have high accuracy,reproducibility and reliability when detecting modified DNA nucleosides.The relative error for detection is 4.7-11%,the relative standard deviation is 2.3-3.5%and 2.9-6.7%for intra-day and inter-day respectively.In controls,after forward stepwise multivariate linear regression,only age(?=-0.143,P=0.016),TC(?=-0.158,P=0.010)and PBLs classifications(?=-0.137,P=0.023)were independent factors associated with 5-m C contents,which could partially explain 6.8%individual variation.The mean of 5-m C contents of CAD patients was significantly lower than controls(P=1.10×10-14).After progressive adjustment for various CAD risk factors especially history of hyperlipidemia and PBLs counts and classifications,the association of genomic 5-m C contents with CAD remained significant(OR=0.325,95%CI,0.237?0.445,P=2.62×10-12).In analysis adjusted for various CAD risk factors,the OR for CAD was 5.667in individuals with 5-m C contents in the bottom tertile compared with the top tertile.(2)DNMT1 expression significantly decreased,along with a significant decrease of 5-m C(P=0.017)but a statistical increase of 5-hm C(P=0.005)in CAD in the 100randomly selected subjects.The overall distributions of 5-m C and 5-hm C were not significantly different among all three PBL subtypes,though the highest methylation and hydroxymethylation were in neutrophils while a significantly decreased 5-m C along with elevated 5-hm C in monocytes.In CAD,the 5-m C content of PBL was correlated with lymphocyte(r=0.382,P=0.006)and monocyte(r=0.439,P=0.001),and 5-hm C content was correlated with monocyte(r=0.454,P=0.001)only.(3)Compared with controls,the number of intermediate monocytes in CAD increased significantly(P=0.018),while no significant change was found in classical and non-classical monocytes.In CAD,with the decrease of genomic DNA 5-m C content,the proportion of classical monocytes increased,while intermediate monocytes decreased,no significant change in non-classical monocytes.the change trend of 5-hm C was opposite to that of monocytes.Conclusion(1)Age,TC and PBL classification were independent factors associated with 5-m C contents,which were independent protective factors for CAD.After progressive adjustment for various risk factors,5-m C contents remained significantly associated with CAD prevalence.(2)Global DNA hypomethylation and hyper-hydroxymethylation of blood cells were defined dominantly by the change in monocyte DNA.(3)Intermediate monocytes increased significantly in CAD,the changes of 5-m C and 5-hm C contents in monocytes are correlated with the changes of the proportion of classical and intermediate monocyte subsets. |
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