CpG island methylation of COX-2 and IL1B genes in periodontitis

Objective: This pilot study aimed to analyze DNA methylation status of the promoter regions of the Cox-2 and interleukin-1 beta (IL-1b) genes in human gingival tissue samples from patients with periodontal disease in comparison to healthy gingival tissue.

Methods: Genomic DNA was extracted from 7 surgically removed gingival tissues with chronic periodontitis and 3 healthy gingival biopsy samples. After bisulfite conversion, a promoter region of Cox-2 gene (position -531bp to -232) was amplified by PCR. The PCR products were cloned into the pGEM-T Easy vector (Promega A1380), and five clones from each biopsy sample were then sequenced by using the Sp6 primer. The bisulfite converted DNA template samples were also analyzed by Pyrosequencing technology (Biotage) checking for potential methylation sites in the IL-1 b promoter region (-3757 to -2508) and PCR products were then directly sequenced. The methylation level of individual potential methylation site for both genes was compared between diseased and healthy samples.

Results: For Cox-2 gene, there was in general a low level of hypermethylation around all the 23 CpG sites within the targeted promoter region in the diseased tissue samples, except in one site that showed a distinct higher level of hypermethylation (29%) whereas for the same CG site increased methylation in the tissue free of periodontal diseases was found in 13% of the clones. For IL-1b, there was a general trend of hypomethylation in diseased sites in all the sequenced CpG sites as compared to samples free of periodontal diseases.

Conclusion: This preliminary data indicates differential DNA methylation patterns of CpG regions in the promoter of both Cox-2 and IL-1b genes in gingival tissues with periodontal disease. The relationship between these specific differentially methylated sites in the promoter and gene expression remains to be determined.