Supplementary Material for: Analysis of p16<sub><i>CDKN2A</i></sub> Methylation and HPV-16 Infection in Oral Mucosal Dysplasia

<i>Objective:</i> The purpose of this study was to investigate the relationship between <i>p16</i><sub><i>CDKN2A</i></sub> 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 <i>dmt3b</i> (C46359T) polymorphism is associated with <i>p16</i><sub><i>CDKN2A</i></sub> methylation status. <i>Methods:</i> 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 <i>p16</i><sub><i>CDKN2A</i></sub> methylation status, <i>dmt3b</i> (C46359T) genotyping, and protein levels, respectively. <i>Results:</i> The methylation of <i>p16</i><sub><i>CDKN2A</i></sub> 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 <i>p16</i><sub><i>CDKN2A</i></sub> methylation; however, changes in DNMT3B and DNMT1 protein levels were not observed in HPV-positive samples. Neither HPV-18 nor the <i>dmt3b</i> polymorphism was associated with <i>p16</i><sub><i>CDKN2A</i></sub> methylation. <i>Conclusions:</i> There is an association between the presence of HPV-16 in ED and the occurrence of <i>p16</i><sub><i>CDKN2A</i></sub> 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.