DNA methylation analysis in patients with bisphosphonate-related osteonecrosis of the jaws.

Objectives: Bisphosphonate-related osteonecrosis of the jaws (BRONJ) is a major complication associated with long-term use of bisphosphonates (BP). We performed an epigenome-wide association studies (EWAS) on genomic DNA from peripheral white blood cells (WBC) in a hospital case-control study of 70 Italian patients. To the best of our knowledge, DNA methylation profile has never been investigated in BRONJ patients. Methods: We performed DNA methylation profiling in bisulphite converted DNA from cases and controls using the Illumina Infinium HumanMethylation27 BeadChip, that enables the direct investigation of 27 578 individual cytosines at CpG loci throughout the genome, which are focused on the promoter regions of 14 475 genes. Wilcoxon rank sum test was used to identify differencies in methylation b-values between cases and controls. P-values less than P < 1.8 ˙ 10-6 were considered to be significant at the level of epigenome-wide significance. Results: We observed differential methylation between cases and controls at multiple CpG sites, and one CpG site reached the most stringent level of genome-wide statistical significance (e.g. PRDM2). Other sites of major interest were (in order of relevance): GIPC2, SCTR, DPH5, SNX6, CALCA, APLP2, TMPO, RSHL1. Conclusions: We present preliminary evidence that alterations in peripheral blood cells DNA methylation may be associated with BRONJ development. This high through put platform was able to successfully interrogate the methylation state of individual cytosines and identified nine CpG sites associated with BRONJ in Italian patients. Relevance: Although promising, these differences in DNA methylation are worthy of further follow-up in replication studies using larger cohorts of BRONJ patients.