title: Methylation of HPV18, HPV31, and HPV45 genomes and cervical intraepithelial neoplasia grade 3.
abstract: BACKGROUND
  Persistent infections with carcinogenic human papillomavirus (HPV) types are the necessary cause of cervical cancer. We recently demonstrated that the HPV16 genome is strongly methylated in cervical precancer compared with transient infections. However, the extent of methylation in other HPV types and its role in progression to cancer is poorly understood.
  
  
  METHODS
  We analyzed whole-genome methylation patterns of the three next most carcinogenic HPV genotypes: HPV31 (closely related to HPV16), and two other closely related types, HPV18 and HPV45. DNA was extracted from cervical cytology specimens from 92 women with precancer and 96 women infected with HPV31, HPV18, or HPV45, but who had no cytological or histological abnormalities. After bisulfite modification, genome-wide pyrosequencing was performed covering 80-106 sites. We calculated differences in median methylation, odds ratios, areas under the curve, and Spearman rank correlation coefficients for methylation levels between different sites. All statistical tests were two-sided.
  
  
  RESULTS
  For all three HPV types, we observed strongly elevated methylation levels at multiple CpG sites in the E2, L2, and L1 regions among women with cervical intraepithelial neoplasia grade 3 compared with women with transient infections. We observed high correlation of methylation patterns between phylogenetically related types. The highest areas under the curve were 0.81 for HPV31, 0.85 for HPV18, and 0.98 for HPV45. Differential methylation patterns in cervical intraepithelial neoplasia grade 3 patients with multiple infections suggest that methylation can clarify which of the infections is causal.
  
  
  CONCLUSIONS
  Carcinogenic HPV DNA methylation indicates transforming HPV infections. Our findings show that methylation of carcinogenic HPV types is a general phenomenon that warrants development of diagnostic assays.
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, 6120 Executive Blvd, Room 5024, Rockville, MD 20852-7234, USA. wentzenn@mail.nih.gov
authors.forename: Nicolas
authors.initials: N
authors.lastname: Wentzensen
authors.forename: Chang
authors.initials: C
authors.lastname: Sun
authors.forename: Arpita
authors.initials: A
authors.lastname: Ghosh
authors.forename: Walter
authors.initials: W
authors.lastname: Kinney
authors.forename: Lisa
authors.initials: L
authors.lastname: Mirabello
authors.forename: Sholom
authors.initials: S
authors.lastname: Wacholder
authors.forename: Ruth
authors.initials: R
authors.lastname: Shaber
authors.forename: Brandon
authors.initials: B
authors.lastname: LaMere
authors.forename: Megan
authors.initials: M
authors.lastname: Clarke
authors.forename: Attila T
authors.initials: AT
authors.lastname: Lorincz
authors.forename: Philip E
authors.initials: PE
authors.lastname: Castle
authors.forename: Mark
authors.initials: M
authors.lastname: Schiffman
authors.forename: Robert D
authors.initials: RD
authors.lastname: Burk
mesh_terms: D000328:Adult; D052685:Alphapapillomavirus / Q000145:classification / Q000235:genetics*; D016022:Case-Control Studies; D019175:DNA Methylation; D004279:DNA, Viral / Q000378:metabolism*; D005260:Female; D016679:Genome, Viral; D005838:Genotype; D052161:Human papillomavirus 18 / Q000235:genetics*; D058952:Human papillomavirus 31 / Q000235:genetics*; D006801:Humans; D016015:Logistic Models; D008875:Middle Aged; D060787:Neoplasm Grading; D030361:Papillomavirus Infections / Q000150:complications* / Q000821:virology; D011230:Precancerous Conditions / Q000473:pathology / Q000821:virology*; D018401:Sample Size; D002578:Uterine Cervical Dysplasia / Q000473:pathology / Q000821:virology*; D002583:Uterine Cervical Neoplasms / Q000473:pathology / Q000821:virology*; D014626:Vaginal Smears
chemical_list: D004279:DNA, Viral