The Association Study Of OPG,RANK,and RANKL Gene Methylation With Steroid-induced Osteonecrosis Of The Femoral Head

Objective: Steroid-induced osteonecrosis of the femoral head(SONFH)is a disease in which the blood supply to the femoral head is reduced or interrupted mostly by the massive use of hormones.It is a complex disease affected by the interaction of genetics and environment,which can cause avascular necrosis of bone cells,the irreversible collapse of the femoral head,and ultimately lead to dysfunction of the hip joint.So far,the cause of SONFH is still unclear.However,among the widely accepted theories,abnormal bone metabolism is undoubtedly one of the important risk factors for its pathogenesis,and the OPG/RANK/RANKL signaling pathway is involved in this process.This signaling pathway mainly regulates the activity of osteoclasts to maintain the bone metabolism balance of the femoral head during the reconstruction process.Existing research indicates that the polymorphisms of OPG,RANK and RANKL genes are closely related to the pathogenesis of SONFH,but the effect of the methylation of genes in the OPG/RANK/RANKL signaling pathway on SONFH is still unclear.Therefore,this study aims to understand the degree of methylation of OPG,RANK,and RANKL genes in this pathway and their possible association with SONFH.Methods: A case-control study was designed,including 50 patients(25 male and 25female)and 50 matched controls.EMBOSS(The European Molecular Biology Open Software Suite)was used to predict the existence and location of CpG islands in OPG,RANK,and RANKL genes.Epi Typer Mass Array was used for quantitative methylation analysis of CpG islands of the above genes.Chi-square test,logistic regression analysis,and other statistical methods were used to analyze the methylation degree of CpG sites of each gene and their correlation with SONFH.Results:(1)One CpG island(chr8:118952332～118952550)was found in the OPG gene,and the methylation level of 4 CpG sites in the case group was significantly higher than that in the control group,with statistically significant differences(p <0.05).There were two CpG islands(chr18:62325388～62326216 and 62326241～62326536)in the RANK gene,and the methylation level of 15 CpG sites in the case group was significantly increased(p < 0.05).A CpG island(chr13:42574105～42574458)was found in the RANKL gene,and methylation levels at 10 CpG sites were significantly lower in the control group(p < 0.05).(2)The methylation rates of 3CpG sites in the OPG gene(such as OPG2-CPG1),10 CpG sites in the RANK gene(such as RANK1-CPG20)and 6 CpG sites in the RANKL gene(such as RANKL2-CPG2)in the SONFH group were significantly higher than those in the control group(p < 0.05).(3)Two highly methylated CpG sites(OPG2-CpG1 and OPG2-CpG5)in the OPG gene increased the risk of SONFH(p < 0.05).In the RANK gene,the elevated methylation level of 12 CpG sites(such as RANK1-CpG15)was a risk factor for SONFH(p < 0.05).In the RANKL gene,10 CpG sites with high methylated levels were positively correlated with disease risk(p < 0.05).(4)In the OPG gene,the Area Under Curve(AUC)of the 3 CpG sites was greater than 0.5,and OPG2-CpG1(AUC: 0.679 95%CI: 0.57-0.79 p = 0.002)had a higher diagnostic value.In the RANK gene,15 CpG sites had AUC values greater than 0.5,and RANK3-CpG8(AUC: 0.778 95%CI: 0.69-0.87 p < 0.001)was the most diagnostic site.In the RANKL gene,10 CpG sites had AUC values greater than 0.5,and RANKL1-CpG1(AUC: 0.764 95%CI: 0.66-0.87 p < 0.001)was more meaningful for the diagnosis of SONFH.Conclusion: A large number of hypermethylated CpG sites were found in the CpG islands of OPG,RANK,and RANKL genes in patients with SONFH.Many hypermethylated CpG sites increased the risk of disease.And,some hypermethylated CpG sites had predictive value for the early diagnosis of SONFH.In short,in SONFH,OPG/RANK/RANKL signaling pathway does have DNA methylation changes,and part of hypermethylated CpG sites may serve as targets for early prediction and diagnosis of SONFH.