title: HPV16 CpG methyl-haplotypes are associated with cervix precancer and cancer in the Guanacaste natural history study.
abstract: OBJECTIVE
  To evaluate HPV16 CpG methylation and methyl-haplotypes and their association with cervix precancer and cancer utilizing massively parallel single molecule next-generation sequencing (NGS).
  
  
  METHODS
  A nested case-control study of HPV16 positive women was performed in a prospective cohort from Guanacaste, Costa Rica designed to study the natural history of HPV and cervical neoplasia. Controls encompassed 31 women with transient infections; there were 44 cases, including 31 women with CIN3 and 13 with cervical cancer. DNA samples from exfoliated cervical cells were treated with bisulfite and four regions (E6, E2, L2 and L1) were amplified with barcoded primers and tested by NGS. CpG methylation was quantified using a bioinformatics pipeline.
  
  
  RESULTS
  Median methylation levels were significantly different between the CIN3+ cases versus controls in the E2, L2, and L1 regions. Methyl-haplotypes, specifically in 5 CpG sites included in the targeted L2 region, with the pattern "--+-+" had the highest Area Under the Curve value (AUC=88.40%) observed for CIN3 vs.
  
  
  METHODS
  The most significant CpG site, L2 4277, determined by bisulfite NGS had an AUC=78.62%.
  
  
  CONCLUSIONS
  This study demonstrates that NGS of bisulfite treated HPV DNA is a useful and efficient technique to survey methylation patterns in HPV16. This procedure provides quantitative information on both individual CpG sites and methyl-haplotypes that identify women with elevated present or subsequent risk for HPV16 CIN3 and cancer.
authors.affiliation: Division of Gynecologic Oncology, Department of Obstetrics & Gynecology and Women's Health, Albert Einstein College of Medicine/Montefiore Medical Center, United States. Electronic address: mfrimer@montefiore.org.
authors.forename: Marina
authors.initials: M
authors.lastname: Frimer
authors.affiliation: Department of Pediatrics, Albert Einstein College of Medicine, United States.
authors.forename: Chang
authors.initials: C
authors.lastname: Sun
authors.affiliation: Department of Obstetrics & Gynecology and Women's Health, Albert Einstein College of Medicine, United States.
authors.forename: Thomas
authors.initials: T
authors.lastname: McAndrew
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, United States.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Smith
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, United States.
authors.forename: Ariana
authors.initials: A
authors.lastname: Harari
authors.affiliation: Department of Pediatrics, Albert Einstein College of Medicine, United States.
authors.forename: Zigui
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, United States.
authors.forename: Lisa
authors.initials: L
authors.lastname: Mirabello
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, United States.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Wentzensen
authors.affiliation: Division of Gynecologic Oncology, Department of Obstetrics & Gynecology and Women's Health, Albert Einstein College of Medicine/Montefiore Medical Center, United States.
authors.forename: Gary L
authors.initials: GL
authors.lastname: Goldberg
authors.affiliation: Proyecto Epidemiologico Guanacaste, Fundacion INCIENSA, San Jose, Costa Rica.
authors.forename: Ana C
authors.initials: AC
authors.lastname: Rodriguez
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, United States.
authors.forename: Mark
authors.initials: M
authors.lastname: Schiffman
authors.affiliation: Department of Pediatrics, Albert Einstein College of Medicine, United States; Department of Obstetrics & Gynecology and Women's Health, Albert Einstein College of Medicine, United States; Department of Microbiology and Immunology, Albert Einstein College of Medicine, United States.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Burk
mesh_terms: D000328:Adult; D016022:Case-Control Studies; D003364:Costa Rica; D018899:CpG Islands; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D005260:Female; D006239:Haplotypes; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D008137:Longitudinal Studies; D030361:Papillomavirus Infections / Q000473:pathology / Q000821:virology*; D017422:Sequence Analysis, DNA / Q000379:methods; D013447:Sulfites / Q000737:chemistry; D002583:Uterine Cervical Neoplasms / Q000821:virology*; D055815:Young Adult; D002578:Uterine Cervical Dysplasia / Q000821:virology*
chemical_list: D004273:DNA, Neoplasm; D004279:DNA, Viral; D013447:Sulfites; C042345:hydrogen sulfite
keywords: Cervix cancer; DNA methylation; HPV16; Next generation sequencing