Supplementary Material for: Analysis of p16_CDKN2A Methylation and HPV-16 Infection in Oral Mucosal Dysplasia

Objective: The purpose of this study was to investigate the relationship between p16_CDKN2A methylation and epithelial dysplasia (ED). We also evaluated the expressions of proteins related to methylation (DNMT3B and DNMT1). Finally, we tested whether HPV-16/18 or the dmt3b (C46359T) polymorphism is associated with p16_CDKN2A methylation status. Methods: To test the hypothesis, a case-control study with 72 (control, n = 24; ED, n = 48) tissue samples from subjects was performed. Methylation-specific PCR, RFLP, and immunohistochemical analyses were performed to evaluate p16_CDKN2A methylation status, dmt3b (C46359T) genotyping, and protein levels, respectively. Results: The methylation of p16_CDKN2A and HPV-16 was associated with ED gradation (p = 0.001 and 0.002, respectively). In addition, most HPV-16-positive samples (77.8%) exhibited p16_CDKN2A methylation; however, changes in DNMT3B and DNMT1 protein levels were not observed in HPV-positive samples. Neither HPV-18 nor the dmt3b polymorphism was associated with p16_CDKN2A methylation. Conclusions: There is an association between the presence of HPV-16 in ED and the occurrence of p16_CDKN2A methylation. Both variables are also associated with ED development, but further studies are necessary to clarify if they operate independently and if they have any impact on OD malignization.