Epigenetic modification in TNFa-gene

Objective: Periodontitis as a bacterially induced chronic inflammatory disease is triggered by specific host dependent immune response. The expression of the genes involved in inflammatory processes is influenced among others by genetic and epigenetic modifications. Moreover, many risk factors associated with periodontitis, including bacterial occurrence, smoking, or diabetes, are known to induce epigenetic changes. Methods: In order to evaluate the impact of epigenetic modification on regulation of inflammatory genes in periodontitis we established and validated a Combined Bisulfite Restriction Analysis (COBRA) for assessing CpG methylation of TNFa-gene on position c.-668. After bisulfite conversion (EpiTect Bisulfite Kit, Qiagen) specific seminested PCRs were performed. For COBRA restriction enzyme HinfI was used. As positive and negative controls methylated and non-methylated converted DNA (Qiagen) were used. Patients: 24 patients (18 patients with severe chronic or aggressive periodontitis: mean age: 47.5+9.7y, 13males; 6 periodontitisfree healthy controls: mean age: 41.8+6.6y, 4 males) were included in the study. From 12 periodontitis patients the methylation status was determined in gingival tissue. The methylation pattern of 6 controls and 6 periodontitis patients was assessed in venous blood. Results: In gingival tissue there was a significant reduction of CpG methylation in TNFa-promoter compared with venous blood (61% vs. 100%, p0.001) possibly leading to an increase in TNFa-gene expression. However, in venous blood there was no difference in methylation status in dependence periodontal disease (controls: 100%, periodontitis patients: 100% methylation, ns). In gingival tissue a distinct decrease in methylation was obviously in CP-patients compared to AP-patients (56.9% vs. 65%, p=0.085). Conclusion: For the first time a change of epigenetic pattern in TNFa-gene was assessed comparing inflamed gingival tissue and venous blood. However, because of the small cohorts the results obtained could only be regarded as preliminary and should be verified in larger cohorts. Keywords: epigenetics, periodontitis, TNFa