Human Papillomavirus Type 16 L2 DNA Methylation in Exfoliated Cervical Cells From College-Age Women

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Abstract

Objectives

The Carolina Women’s Care Study (CWCS) at the University of South Carolina followed 467 young women with the goal of identifying biomarkers of human papillomavirus (HPV) persistence. In this study, we analyzed the methylation of HPV16 DNA.

Methods

The aims of this study were to determine the methylation status of the HPV16 L2 gene in DNA isolated from exfoliated cervical cells collected longitudinally as part of the CWCS and to determine the prevalence of polymorphisms (single nucleotide polymorphisms [SNPs]) in folate metabolizing enzymes and DNA repair enzymes known to affect DNA methylation in blood-derived genomic DNA from CWCS participants. For methylation studies, DNA samples were bisulfite converted and amplified with the EpiTect Whole Bisulfitome kit. Polymerase chain reaction was performed for amplicons containing 5 CpG sites in L2. Pyrosequencing was carried out using EpigenDx and analyzed with PyroMark Software. Taqman genotyping assays were performed to determine selected SNP alleles in the CWCS cohort.

Results and Conclusions

Methylation data were obtained for 82 samples from 27 participants. Of these, 22 participants were positive for HPV16 for 3 or more visits (≥12 months). Methylation in L2 was detectable, but methylation levels varied and were not associated with HPV16 persistence. No linearity of methylation levels over time was observed in participants for whom longitudinal data could be analyzed. Analysis of 9 selected SNPs did not reveal an association with persistence. We conclude that at early stages of infection methylation of HPV16 L2 DNA in Pap test samples is not a predictive biomarker of HPV persistence.

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