Supplementary Material for: Analysis of p16CDKN2A Methylation and HPV-16 Infection in Oral Mucosal Dysplasia

Objective: The purpose of this study was to investigate the relationship between p16CDKN2A 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 p16CDKN2A 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 p16CDKN2A methylation status, dmt3b (C46359T) genotyping, and protein levels, respectively. Results: The methylation of p16CDKN2A 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 p16CDKN2A 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 p16CDKN2A methylation. Conclusions: There is an association between the presence of HPV-16 in ED and the occurrence of p16CDKN2A 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.