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Background Dysregulation of the OPG/RANK/RANKL signalling pathway is a key step in the occurrence of steroid-induced osteonecrosis of the femoral head (ONFH). This study aims to understand the degree of methylation of the OPG, RANK, and RANKL genes in steroid-related ONFH. Methods A case-control study was designed, including 50 patients (25 males and 25 females) and 50 matched controls. The European Molecular Biology Open Software Suite (EMBOSS) was used to predict the existence and location of CpG islands in the OPG, RANK, and RANKL genes. The Agena MassARRAY platform was used to detect the methylation status of the above genes in the blood of subjects. The relationship between the methylation level of CpG sites in each gene and steroid-related ONFH was analysed by the chi-square test, logistic regression analysis, and other statistical methods. Results In the CpG islands of the OPG, RANK, and RANKL genes in patients with steroid-related ONFH, several CpG sites with high methylation rates and high methylation levels were found. Some hypermethylated CpG sites increase the risk of steroid-related ONFH. In addition, a few hypermethylated CpG sites have predictive value for the early diagnosis of steroid-related ONFH. Conclusion Methylation of certain sites in the OPG/RANK/RANKL signalling pathway increases the risk of steroid-related ONFH. Some hypermethylated CpG sites may be used as early prediction and diagnostic targets for steroid-related ONFH.

Background and purpose Alcohol-induced osteonecrosis of the femoral head (ONFH) is a complex and heterogeneous disease. Genetic factors and epigenetic modifications are one of the pathogenesis of the disease. However, the influence of epigenetic factors on the disease has not been systematically studied. Our research aims to determine the methylation changes of alcohol-induced ONFH. Methods An analytical cross-sectional study of a Chinese male population (50 alcohol-induced ONFH patients and 50 controls). The EpiTYPER of the Sequenom MassARRAY platform was used to detect the DNA methylation status of 132 cytosine-phosphate-guanine (CpG) sites in the OPG/RANKL/RANK gene promoter region. Results In the whole study group, the chi-square test was used to analyze the methylation rate between the two groups, and six CpG sites were found to be different, among which OPG1_CpG_2, OPG3_CpG_4, RANK1_CpG_6, RANK3_CpG_10, RANKL2_CpG_21, and RANKL2_CpG_46 in the case group were higher than those in the control group, while OPG4_CpG_2 was lower than that in the control group. The results showed that in patients with alcohol-induced ONFH, 146 CpG sites were examined for differences in methylation levels compared with healthy controls, 32 of which were not detected, and 23 of the remaining 114 sites showed differences in methylation levels compared with alcohol-induced ONFH patients. Receiver operator characteristic (ROC) curve analysis demonstrated the methylation levels of OPG/RANKL/RANK could efficiently predict the existence of alcohol-induced ONFH. Conclusion Our study of Chinese men suggests that several CpG sites in the OPG/RANKL/RANK gene in peripheral blood leukocytes of patients with alcohol-induced ONFH are in an abnormal methylation state (hypermethylation tended to be more frequent).