HPV16 Lineages Research Articles

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

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.

Genetic diversity of HPV16 and HPV18 in Brazilian patients with invasive cervical cancer.

Cervical cancer is the fourth most common cancer among women, and ∼70-80% of these cancers are associated with two human papillomavirus types: HPV16 and HPV18. Several studies have reported that intra-type diversity is associated with the progression of infection to invasive cancer. Herein, we report the genetic diversity of HPV16 and HPV18 in a cohort of 594 Brazilian women with invasive cervical cancer and describe the prevalence of lineages and intra-type diversity prior to the implementation of the public immunization program in Brazil. HPV detection and genotyping were performed using PCR, PGMY/GP primers, and DNA extracted from fresh tumors. The HPV16 (378 women) and HPV18 (80 women) lineages were identified by PCR and sequencing of the LCR and E6 fragments, followed by SNV comparison and phylogenetic analysis. In our cohort, was found a higher frequency of the lineage A (in 217 women), followed by lineage D (in 97 women) and lineages B and C (in 10 women each) for HPV16; and a higher frequency of lineage A (in 56 women) followed by lineage B (in 15 women) in HPV18. The genetic diversity of HPV16 indicated a recent expansion of specific variants or a selective advantage that is associated with invasive cancer; this pattern was not observed for HPV18.