Assessment of viral methylation levels for high risk HPV types by newly designed consensus primers PCR and pyrosequencing

A. Gillio-Tos, V. Fiano, C. Grasso, M. Trevisan, S. Gori, A. Mongia, L. De Marco, G. Ronco, N. Segnan, R. Rizzolo, P. Giubilato, B. Ghiringhello, G. Maina, L. Pasero, A. Sapino, M.G. Accinelli, S. Girlando, M. Barbareschi, A. Del Mistro, L. BabociH. Frayle, R. Trevisan, M. Zorzi, C. Fedato, S. Baracco, E. Insacco, D. Minucci, M. Matteucci, G.L. Onnis, M. Manfredi, G.P. Casadei, G. Collina, P. Schincaglia, M. Serafini, B. Vitali, M. Aldi, S. Folicaldi, R. Nannini, G. Galanti, M. De Lillo, P. Giorgi Rossi, C. Naldoni, F. Carozzi, M. Confortini, A. Iossa, C. Sani, M. Zappa, G.L. Taddei, S. Brezzi, P. Raggi, E. Gomes, A. Pellegrini, M.L. Schiboni, P. Borgia, F. Chini, New Technologies for Cervical Cancer Screening (NTCC) Working Group

Research output: Contribution to journalArticlepeer-review

Abstract

Background: Measuring viral DNA methylation in human papillomavirus (HPV) infected women showed promise for accurate detection of high-grade cervical lesions and cancer. Methylation status has been widely investigated for HPV16, sporadically for other HPV types. Methods: Objective of this methodological study was to set up molecular methods to test the methylation levels in the twelve oncogenic HPV types by pyrosequencing, minimizing the number of HPV type-specific PCR protocols. Target CpGs were selected on the HPV L1 (two regions, L1 I and L1 II) and L2 genes. Study samples included DNA stored at Turin, Italy, purified by cervical cells collected in Standard Transport Medium or PreservCyt from women who participated in two studies (N = 126 and 140) nested within the regional organized screening programme. PCR consensus primers were designed by PyroMark Assay Design software to be suitable for amplification of many different oncogenic HPV types. Results: Generation of consensus primers was successful for L1 I and II regions, unsuccessful for L2 region, for which HPV type-specific primers remained necessary. The difference between replicated tests on the same sample was ≤4% in 88%, 77% and 91% of cases when targeting the L1 I, L1 II and L2 regions, respectively. The corresponding intra-class correlation coefficients (ICC) were 0.94, 0.87 and 0.97 respectively. When comparing methylation measures based on consensus and type-specific primers, ICC was 0.97 for the L1 I region and 0.99 the for L1 II region. Conclusions: The proposed protocols, applying consensus primers suitable to amplify the oncogenic HPV types and minimize the number of PCR reactions, represent a promising tool to quantify viral methylation in women positive for any high risk HPV type. Impact: Potential application of these methylation protocols in screening settings can be explored to identify women with high probability of progression to high grade lesions. © 2018 Gillio-Tos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Original languageEnglish
Article number0194619
JournalPLoS One
Volume13
Issue number3
DOIs
Publication statusPublished - Mar 2018

Keywords

  • capsid protein
  • primer DNA
  • virus DNA, CpG island
  • DNA methylation
  • DNA sequence
  • female
  • genetics
  • human
  • isolation and purification
  • metabolism
  • Papillomaviridae
  • papillomavirus infection
  • polymerase chain reaction
  • procedures
  • reproducibility
  • risk
  • software
  • virology, Capsid Proteins
  • CpG Islands
  • DNA Methylation
  • DNA Primers
  • DNA, Viral
  • Female
  • Humans
  • Papillomavirus Infections
  • Polymerase Chain Reaction
  • Reproducibility of Results
  • Risk
  • Sequence Analysis, DNA
  • Software

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