title: Case-control study of HLA-G promoter methylation status, HPV infection and cervical neoplasia in Curitiba, Brazil: a pilot analysis.
abstract: BACKGROUND
  The causal association between persistent human papillomavirus (HPV) infection and cervical cancer has been established, but the mechanisms that favor HPV persistence in cervical cells are still unknown. The diminished capability of the immune system to control and resolve HPV infection is one of several hypotheses. The tolerogenic protein HLA-G has shown aberrant expression in a variety of cancers, which has been suggested as a mechanism for tumor escape from immunosurveillance. In the present study we evaluate the role of epigenetic modification (promoter de-methylation) of the HLA-G gene on susceptibility to HPV infection and development of high-grade cervical lesions.
  
  
  METHODS
  A case-control study was carried out in Curitiba, Brazil, between February and June 2010. A total of 789 women aged 15-47 years were recruited: 510 controls with normal cervical cytology, and 279 cases with histologically confirmed cervical intraepithelial neoplasia grade 2 (CIN2, N = 150) or grade 3 (CIN3, N = 129). All women were administered a questionnaire by interview, which collected information on demographic and lifestyle factors, and a cervical sample was collected. HPV DNA detection was performed by GP5+/GP6+ primer-mediated PCR. HPV-positive samples were genotyped by multiplex PCR. A pilot analysis of HLA-G promoter methylation was carried out in a subset of the study population (96 cases and 76 controls) by pyrosequencing. HLA-G methylation and HPV infection status of cases and controls were compared, and confounding factors were computed by t Student and non-parametric Wilcoxon tests. Comparison of HLA-G methylation between cases and controls was assessed by the Bonferroni correction. The association of HLA-G methylation with CIN2/3 was evaluated by logistic regression.
  
  
  RESULTS
  HPV prevalence was 19.6% in controls and 94.3% in CIN2/3 cases. HPV16, 31, 33, 35 and 18 were the most prevalent types. Methylation analysis of seven CpGs in the HLA-G promoter did not reveal any spontaneous de-methylation events in CIN2/3 cases (mean proportion of methylation: 75.8%) with respect to controls (mean 73.7%; odds ratio 1.01, 95% confidence interval 0.96, 1.07).
  
  
  CONCLUSIONS
  This study did not support the hypothesis that spontaneous de-methylation events in the HLA-G promoter play a primary role in promoting escape from immunosurveillance in the development of precancerous cervical lesions.
authors.affiliation: Department of Medical Sciences, Unit of Cancer Epidemiology - C,E,R,M,S, University of Turin, Turin, Italy. gilliotos.demarco@cpo.it
authors.forename: Anna
authors.initials: A
authors.lastname: Gillio-Tos
authors.forename: Maria da Graça
authors.initials: Mda G
authors.lastname: Bicalho
authors.forename: Valentina
authors.initials: V
authors.lastname: Fiano
authors.forename: Chiara
authors.initials: C
authors.lastname: Grasso
authors.forename: Valentina
authors.initials: V
authors.lastname: Tarallo
authors.forename: Laura
authors.initials: L
authors.lastname: De Marco
authors.forename: Morena
authors.initials: M
authors.lastname: Trevisan
authors.forename: Marinabarbaradesousa
authors.initials: M
authors.lastname: Xavier
authors.forename: Renata
authors.initials: R
authors.lastname: Slowik
authors.forename: Newton S
authors.initials: NS
authors.lastname: Carvalho
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Maestri
authors.forename: Hadriano M
authors.initials: HM
authors.lastname: Lacerda
authors.forename: Daniela
authors.initials: D
authors.lastname: Zugna
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Richiardi
authors.forename: Franco
authors.initials: F
authors.lastname: Merletti
mesh_terms: D000293:Adolescent; D000328:Adult; D001938:Brazil; D016022:Case-Control Studies; D019175:DNA Methylation; D004279:DNA, Viral / Q000032:analysis; D005260:Female; D020022:Genetic Predisposition to Disease; D059951:HLA-G Antigens / Q000235:genetics*; D006801:Humans; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction; D027383:Papillomaviridae / Q000235:genetics; D030361:Papillomavirus Infections / Q000453:epidemiology* / Q000235:genetics*; D010865:Pilot Projects; D015995:Prevalence; D011401:Promoter Regions, Genetic / Q000235:genetics; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000821:virology; D055815:Young Adult; D002578:Uterine Cervical Dysplasia / Q000235:genetics* / Q000821:virology
chemical_list: D004279:DNA, Viral; D059951:HLA-G Antigens