title: Methylation at 3'LCR of HPV16 can be affected by patient age and disruption of E1 or E2 genes.
abstract: 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.
authors.affiliation: Genetics Program - Instituto Nacional de Câncer (INCA), Rio de Janeiro, Brazil.
authors.forename: Sérgio Menezes Amaro
authors.initials: SMA
authors.lastname: Filho
authors.affiliation: Population Research Department - Instituto Nacional de Câncer (INCA), Rio de Janeiro, Brazil.
authors.forename: Neilane
authors.initials: N
authors.lastname: Bertoni
authors.affiliation: Genetics Program - Instituto Nacional de Câncer (INCA), Rio de Janeiro, Brazil; Genetics Department - Universidade Federal do Rio de Janeiro, Brazil.
authors.forename: Ayslan Castro
authors.initials: AC
authors.lastname: Brant
authors.affiliation: Genetics Program - Instituto Nacional de Câncer (INCA), Rio de Janeiro, Brazil.
authors.forename: João Paulo Castello Branco
authors.initials: JPCB
authors.lastname: Vidal
authors.affiliation: Genetics Program - Instituto Nacional de Câncer (INCA), Rio de Janeiro, Brazil.
authors.forename: Shayany Pinto
authors.initials: SP
authors.lastname: Felix
authors.affiliation: Department of Microbiology and Parasitology, Universidade Federal Fluminense, Niterói, Brazil.
authors.forename: Silvia Maria Baeta
authors.initials: SMB
authors.lastname: Cavalcanti
authors.affiliation: Department of Microbiology and Parasitology, Universidade Federal Fluminense, Niterói, Brazil.
authors.forename: Fernanda N
authors.initials: FN
authors.lastname: Carestiato
authors.affiliation: Population Research Department - Instituto Nacional de Câncer (INCA), Rio de Janeiro, Brazil.
authors.forename: Luís Felipe Leite
authors.initials: LFL
authors.lastname: Martins
authors.affiliation: Population Research Department - Instituto Nacional de Câncer (INCA), Rio de Janeiro, Brazil.
authors.forename: Liz Maria de
authors.initials: LM
authors.lastname: Almeida
authors.affiliation: Genetics Program - Instituto Nacional de Câncer (INCA), Rio de Janeiro, Brazil. Electronic address: miguelm@inca.gov.
authors.forename: Miguel Angelo Martins
authors.initials: MAM
authors.lastname: Moreira
mesh_terms: D000230:Adenocarcinoma / Q000473:pathology / Q000821:virology; D000328:Adult; D000367:Age Factors; D001665:Binding Sites; D002294:Carcinoma, Squamous Cell / Q000473:pathology / Q000821:virology; D018899:CpG Islands; D019175:DNA Methylation; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D017353:Gene Deletion; D054884:Host-Pathogen Interactions; D052162:Human papillomavirus 16 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008875:Middle Aged; D009856:Oncogene Proteins, Viral / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames; D030361:Papillomavirus Infections / Q000473:pathology / Q000821:virology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D002583:Uterine Cervical Neoplasms / Q000473:pathology / Q000821:virology*
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; C093478:E1 protein, Human papillomavirus 16; C054855:E2 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral
keywords: Episomal HPV genome; HPV integration; HPV16; Human papillomavirus; Invasive cervical cancer; Methylation