A comparison of methylation levels in HPV18, HPV31 and HPV33 genomes reveals similar associations with cervical precancers()

BACKGROUND: High risk human papillomavirus (HR-HPV) infection is common and only a small minority of infections become persistent and lead to cervical cancers. Women positive for HR-HPV usually require a second test to avoid unnecessary colposcopies and over treatment. Elevated DNA methylation of HR-HPV L1 and L2 genes in high grade disease has emerged as a promising molecular triage tool. OBJECTIVES: Our aim was to accurately measure methylation levels at selected CpG positions in the HPV18, HPV31 and HPV33 genomes. We focused on the L2, L1, URR and E6 regions because these were previously shown to be interesting areas for study. STUDY DESIGN: Pyrosequencing was used to measure methylation in 208 HPV18, 207 HPV31, and 126 HPV33 positive women selected from a London colposcopy referral population. RESULTS: After adjustment for multiple testing, at FDR 5%, elevated methylation was significantly associated with cervical intraepithelial neoplasia grades 2 or worse (CIN2+) in all investigated CpGs in HPV18 L2 and L1. Two of 6 L2 and 12 of 15 L1 sites in HPV31 and 6 of 8 L2 and 3 of 13 L1 sites in HPV33 showed significantly elevated methylation in CIN2+. Methylation of CpG sites in the URR and E6 region of the HPV types was low and most differences were not significant. CONCLUSION: Elevated methylation of CpG sites in the L1 and L2 regions of HPV18, HPV31 and HPV33 is associated with CIN2+ and a panel test may be useful for triage of women with HR-HPV infections.