title: Increased methylation of Human Papillomavirus type 16 DNA correlates with viral integration in Vulval Intraepithelial Neoplasia.
abstract: BACKGROUND
  Methylation of HPV16 DNA is a promising biomarker for triage of HPV positive cervical screening samples but the biological basis for the association between HPV-associated neoplasia and increased methylation is unclear.
  
  
  OBJECTIVE
  To determine whether HPV16 DNA methylation was associated with viral integration, and investigate the relationships between viral DNA methylation, integration and gene expression.
  
  
  METHODS
  HPV16 DNA methylation, integration and gene expression were assessed using pyrosequencing, ligation-mediated PCR and QPCR, in biopsies from 25 patients attending a specialist vulval neoplasia clinic and in short-term clonal cell lines derived from vulval and vaginal neoplasia.
  
  
  RESULTS
  Increased methylation of the HPV16 L1/L2 and E2 regions was associated with integration of viral DNA into the host genome. This relationship was observed both in vivo and in vitro. Increased methylation of E2 binding sites did not appear to be associated with greater expression of viral early genes. Expression of HPV E6 and E7 did not correlate with either integration state or increased L1/L2 methylation.
  
  
  CONCLUSIONS
  The data suggest that increased HPV DNA methylation may be partly attributable to viral integration, and provide a biological rationale for quantification of L1/L2 methylation in triage of HPV positive cervical screening samples.
authors.affiliation: HPV Research Group, Institute of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. Electronic address: dean.bryant@gmail.com.
authors.forename: Dean
authors.initials: D
authors.lastname: Bryant
authors.affiliation: HPV Research Group, Institute of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. Electronic address: onionst@cf.ac.uk.
authors.forename: Tiffany
authors.initials: T
authors.lastname: Onions
authors.affiliation: HPV Research Group, Institute of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. Electronic address: raybouldre1@cf.ac.uk.
authors.forename: Rachel
authors.initials: R
authors.lastname: Raybould
authors.affiliation: HPV Research Group, Institute of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. Electronic address: jonessadie@me.com.
authors.forename: Sadie
authors.initials: S
authors.lastname: Jones
authors.affiliation: HPV Research Group, Institute of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. Electronic address: tristramaj@cf.ac.uk.
authors.forename: Amanda
authors.initials: A
authors.lastname: Tristram
authors.affiliation: HPV Research Group, Institute of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. Electronic address: hibbittssj@cf.ac.uk.
authors.forename: Samantha
authors.initials: S
authors.lastname: Hibbitts
authors.affiliation: HPV Research Group, Institute of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. Electronic address: fianderan@cf.ac.uk.
authors.forename: Alison
authors.initials: A
authors.lastname: Fiander
authors.affiliation: HPV Research Group, Institute of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. Electronic address: powellng@cf.ac.uk.
authors.forename: Ned
authors.initials: N
authors.lastname: Powell
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015415:Biomarkers / Q000032:analysis; D002278:Carcinoma in Situ / Q000473:pathology / Q000821:virology*; D019175:DNA Methylation; D004279:DNA, Viral / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D052162:Human papillomavirus 16 / Q000502:physiology*; D006801:Humans; D008875:Middle Aged; D016662:Virus Integration; D014846:Vulvar Neoplasms / Q000473:pathology / Q000821:virology*; D055815:Young Adult
chemical_list: D015415:Biomarkers; D004279:DNA, Viral
keywords: HPV; Human papillomavirus; Integration; Methylation; Vulval Intraepithelial Neoplasia