Methylation at 3′LCR of HPV16 can be affected by patient age and disruption of E1 or E2 genes

•The effect of HPV DNA methylation in regulation of viral transcription is unclear.•Higher methylation of E2-binding-sites at 3′LCR up regulates E6 and E7 transcription.•E2 are frequently lost due to integration of the HPV genome in the cellular genome.•We found that presence of intact E2 ORF was associated with HPV DNA methylation.•Patient age at diagnosis was independently associates with HPV DNA methylation.

CpG methylation at early promoter of HPV16 DNA, in the 3′ end of the Long Control Region (3′LCR), has been associated to the presence of episomal forms of viral genome and, consequently, intact E1 and E2 ORFs. The DNA methylation would block the access of E2 viral protein to the E2 binding sites at early-promoter. However, is still unclear if methylation at 3′LCR of HPV16 DNA can also vary depending of other tumor characteristics in addition to viral DNA physical state. In this study, we evaluate whether the methylation level at the five CpG located at 3′LCR of HPV16 is associated to patient age and E1 and/or E2 ORFs integrity. DNA pyrosequencing was used to measure the methylation level in 69 invasive cervical cancer samples obtained from biopsies of patients attended at Brazilian National Institute of Cancer (INCA). PCR amplifications were performed to assess disruption status of E1 and E2 genes of HPV16. The methylation average per sample ranged widely, from <1 to 88.00%. Presence of intact E1/E2 genes and patient age were positively associated with average methylation in both bivariate analyses (p = 0.003 and p = 0.006, respectively), and multivariate analysis (p = 0.002 and p = 0.021, respectively), adjusted for tumor type (squamous cell carcinomas or adenocarcinomas) and HPV16 lineage. These findings showed that presence of intact E1/E2 open reading frames was associated with high levels of DNA methylation, and older patients showed higher levels of methylation than younger ones independently of viral genome disruption.