A Study On Relationship Between ESR1 Gene Methylation And Bone Mineral Density In Postmenopausal Women

Objective: 1.To investigate the relationship of peripheral blood ESR1 gene promoter methylation level with bone mineral density(BMD)and osteoporosis(OP)in postmenopausal women;2.To investigate the association betweenperipheral blood ESR1 gene promoter methylation level and bone turnover markers.Methods:1.301 postmenopausal Chinese Han women aged from 49 to 65 were recruited according to inclusion and exclusion criteria.Demographic characteristics,disease and medication history as well asother information of the subjects were collected,and blood samples were obtained;2.Whole-body BMD scan was performed with dual-energy X-ray absorptiometry(DXA).Bone measurements included total body BMD(T-BMD),total hip BMD(H-BMD),total spine BMD(S-BMD),lumbar spine 1-4 BMD(L1-4 BMD),and femoral neck BMD(FN-BMD).Serum estradiol(E2),bone specific alkaline phosphatase(BALP)and human tartrate resistant acid phosphatase 5b(TRACP-5b)were determined by ELISA.Electrochemical luminescence was used to detect the serum sex hormone binding globulin(SHBG)and N-terminal propeptide of type Iprocollagen(PINP).Free estradiol(FE2)was calculated;3.Whole blood DNA was extracted and Sanger sequencing was performed after bisulfite transformation to detect the methylation levels at each 6 Cp G sites of ESR1 gene promoter.The expression level of ESR1 m RNA in peripheral blood was detected by quantitative real-time PCR.Results:1.The methylation levels of Cp G1,Cp G3,Cp G4 and Cp G5 sites in ESR1 gene promoter region in the osteopenia(ON)and OP groups were significantly higher than those in the Control group(all P < 0.05).There was no significant difference in methylation level between ON group and OP group(all P > 0.05).2.Methylation levels of Cp G1,Cp G3,Cp G4,Cp G5 and Cp G6 sites in ESR1 gene promoter region showed significant negative correlation with L1-4 BMD and/or FN-BMD(r =-0.117--0.198,all P < 0.05).3.The correlation of mean methylation level of ESR1 gene promoter(A-Cp G)with BMD at all body sites(r =-0.261--0.227,all P < 0.05)was stronger than independent Cp G methylation level,and showed the best diagnostic value for OP risk with an AUC0.688(0.613,0.764).4.A-Cp G was associated negatively with peripheral blood ESR1 m RNA expression(r =-0.163,P = 0.006),negatively with bone formation marker PINP(r =-0.151,P =0.008),and positively with bone resorption marker TRACP-5b(r = 0.117,P = 0.045).5.Multiple linear stepwise regression analysis showed that after adjusting for estradiol level,age,and BMI,A-Cp G was still negatively correlated with BMD at all sites.For each 1SD increase of A-Cp G,L1-4 BMD,FN-BMD,H-BMD,S-BMD,and T-BMD decreased by 0.131,0.192,0.172,0.137,and 0.143 SD,respectively.A-Cp G independently explained 1.7%,4.1%,3.4%,1.8% and 2.0% of the variance of L1-4 BMD,FN-BMD,H-BMD,S-BMD and T-BMD,respectively.Conclusion: The methylation of ESR1 gene promoter in peripheral blood of postmenopausal women is related to BMD,suggesting that the regulation of ESR1 gene methylation plays an important role in the etiology and development of OP.A-Cp G,an indicator of the mean methylation level of ESR1 gene in peripheral blood,is of good diagnostic value for OP,and the monitoring of ESR1 A-Cp G in peripheral blood may be helpful to predict bone loss and the severity of OP in postmenopausal women.