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 journalArticle

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

Fingerprint

Methylation
Papillomaviridae
methylation
Polymerase Chain Reaction
Network protocols
Screening
Viral DNA
lesions (animal)
Software design
Amplification
Assays
Genes
Software Design
screening
DNA methylation
DNA Methylation
Licensure
DNA
Uterine Cervical Neoplasms
Italy

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

Cite this

Gillio-Tos, A., Fiano, V., Grasso, C., Trevisan, M., Gori, S., Mongia, A., ... Group, N. T. F. C. C. S. NTCC. W. (2018). Assessment of viral methylation levels for high risk HPV types by newly designed consensus primers PCR and pyrosequencing. PLoS One, 13(3), [0194619]. https://doi.org/10.1371/journal.pone.0194619

Assessment of viral methylation levels for high risk HPV types by newly designed consensus primers PCR and pyrosequencing. / Gillio-Tos, A.; Fiano, V.; Grasso, C.; Trevisan, M.; Gori, S.; Mongia, A.; De Marco, L.; Ronco, G.; Segnan, N.; Rizzolo, R.; Giubilato, P.; Ghiringhello, B.; Maina, G.; Pasero, L.; Sapino, A.; Accinelli, M.G.; Girlando, S.; Barbareschi, M.; Del Mistro, A.; Baboci, L.; Frayle, H.; Trevisan, R.; Zorzi, M.; Fedato, C.; Baracco, S.; Insacco, E.; Minucci, D.; Matteucci, M.; Onnis, G.L.; Manfredi, M.; Casadei, G.P.; Collina, G.; Schincaglia, P.; Serafini, M.; Vitali, B.; Aldi, M.; Folicaldi, S.; Nannini, R.; Galanti, G.; De Lillo, M.; Giorgi Rossi, P.; Naldoni, C.; Carozzi, F.; Confortini, M.; Iossa, A.; Sani, C.; Zappa, M.; Taddei, G.L.; Brezzi, S.; Raggi, P.; Gomes, E.; Pellegrini, A.; Schiboni, M.L.; Borgia, P.; Chini, F.; Group, New Technologies for Cervical Cancer Screening (NTCC) Working.

In: PLoS One, Vol. 13, No. 3, 0194619, 03.2018.

Research output: Contribution to journalArticle

Gillio-Tos, A, Fiano, V, Grasso, C, Trevisan, M, Gori, S, Mongia, A, De Marco, L, Ronco, G, Segnan, N, Rizzolo, R, Giubilato, P, Ghiringhello, B, Maina, G, Pasero, L, Sapino, A, Accinelli, MG, Girlando, S, Barbareschi, M, Del Mistro, A, Baboci, L, Frayle, H, Trevisan, R, Zorzi, M, Fedato, C, Baracco, S, Insacco, E, Minucci, D, Matteucci, M, Onnis, GL, Manfredi, M, Casadei, GP, Collina, G, Schincaglia, P, Serafini, M, Vitali, B, Aldi, M, Folicaldi, S, Nannini, R, Galanti, G, De Lillo, M, Giorgi Rossi, P, Naldoni, C, Carozzi, F, Confortini, M, Iossa, A, Sani, C, Zappa, M, Taddei, GL, Brezzi, S, Raggi, P, Gomes, E, Pellegrini, A, Schiboni, ML, Borgia, P, Chini, F & Group, NTFCCSNTCCW 2018, 'Assessment of viral methylation levels for high risk HPV types by newly designed consensus primers PCR and pyrosequencing', PLoS One, vol. 13, no. 3, 0194619. https://doi.org/10.1371/journal.pone.0194619
Gillio-Tos, A. ; Fiano, V. ; Grasso, C. ; Trevisan, M. ; Gori, S. ; Mongia, A. ; De Marco, L. ; Ronco, G. ; Segnan, N. ; Rizzolo, R. ; Giubilato, P. ; Ghiringhello, B. ; Maina, G. ; Pasero, L. ; Sapino, A. ; Accinelli, M.G. ; Girlando, S. ; Barbareschi, M. ; Del Mistro, A. ; Baboci, L. ; Frayle, H. ; Trevisan, R. ; Zorzi, M. ; Fedato, C. ; Baracco, S. ; Insacco, E. ; Minucci, D. ; Matteucci, M. ; Onnis, G.L. ; Manfredi, M. ; Casadei, G.P. ; Collina, G. ; Schincaglia, P. ; Serafini, M. ; Vitali, B. ; Aldi, M. ; Folicaldi, S. ; Nannini, R. ; Galanti, G. ; De Lillo, M. ; Giorgi Rossi, P. ; Naldoni, C. ; Carozzi, F. ; Confortini, M. ; Iossa, A. ; Sani, C. ; Zappa, M. ; Taddei, G.L. ; Brezzi, S. ; Raggi, P. ; Gomes, E. ; Pellegrini, A. ; Schiboni, M.L. ; Borgia, P. ; Chini, F. ; Group, New Technologies for Cervical Cancer Screening (NTCC) Working. / Assessment of viral methylation levels for high risk HPV types by newly designed consensus primers PCR and pyrosequencing. In: PLoS One. 2018 ; Vol. 13, No. 3.
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title = "Assessment of viral methylation levels for high risk HPV types by newly designed consensus primers PCR and pyrosequencing",
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. {\circledC} 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.",
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author = "A. Gillio-Tos and V. Fiano and C. Grasso and M. Trevisan and S. Gori and A. Mongia and {De Marco}, L. and G. Ronco and N. Segnan and R. Rizzolo and P. Giubilato and B. Ghiringhello and G. Maina and L. Pasero and A. Sapino and M.G. Accinelli and S. Girlando and M. Barbareschi and {Del Mistro}, A. and L. Baboci and H. Frayle and R. Trevisan and M. Zorzi and C. Fedato and S. Baracco and E. Insacco and D. Minucci and M. Matteucci and G.L. Onnis and M. Manfredi and G.P. Casadei and G. Collina and P. Schincaglia and M. Serafini and B. Vitali and M. Aldi and S. Folicaldi and R. Nannini and G. Galanti and {De Lillo}, M. and {Giorgi Rossi}, P. and C. Naldoni and F. Carozzi and M. Confortini and A. Iossa and C. Sani and M. Zappa and G.L. Taddei and S. Brezzi and P. Raggi and E. Gomes and A. Pellegrini and M.L. Schiboni and P. Borgia and F. Chini and Group, {New Technologies for Cervical Cancer Screening (NTCC) Working}",
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TY - JOUR

T1 - Assessment of viral methylation levels for high risk HPV types by newly designed consensus primers PCR and pyrosequencing

AU - Gillio-Tos, A.

AU - Fiano, V.

AU - Grasso, C.

AU - Trevisan, M.

AU - Gori, S.

AU - Mongia, A.

AU - De Marco, L.

AU - Ronco, G.

AU - Segnan, N.

AU - Rizzolo, R.

AU - Giubilato, P.

AU - Ghiringhello, B.

AU - Maina, G.

AU - Pasero, L.

AU - Sapino, A.

AU - Accinelli, M.G.

AU - Girlando, S.

AU - Barbareschi, M.

AU - Del Mistro, A.

AU - Baboci, L.

AU - Frayle, H.

AU - Trevisan, R.

AU - Zorzi, M.

AU - Fedato, C.

AU - Baracco, S.

AU - Insacco, E.

AU - Minucci, D.

AU - Matteucci, M.

AU - Onnis, G.L.

AU - Manfredi, M.

AU - Casadei, G.P.

AU - Collina, G.

AU - Schincaglia, P.

AU - Serafini, M.

AU - Vitali, B.

AU - Aldi, M.

AU - Folicaldi, S.

AU - Nannini, R.

AU - Galanti, G.

AU - De Lillo, M.

AU - Giorgi Rossi, P.

AU - Naldoni, C.

AU - Carozzi, F.

AU - Confortini, M.

AU - Iossa, A.

AU - Sani, C.

AU - Zappa, M.

AU - Taddei, G.L.

AU - Brezzi, S.

AU - Raggi, P.

AU - Gomes, E.

AU - Pellegrini, A.

AU - Schiboni, M.L.

AU - Borgia, P.

AU - Chini, F.

AU - Group, New Technologies for Cervical Cancer Screening (NTCC) Working

N1 - cited By 0

PY - 2018/3

Y1 - 2018/3

N2 - 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.

AB - 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.

KW - capsid protein

KW - primer DNA

KW - virus DNA, CpG island

KW - DNA methylation

KW - DNA sequence

KW - female

KW - genetics

KW - human

KW - isolation and purification

KW - metabolism

KW - Papillomaviridae

KW - papillomavirus infection

KW - polymerase chain reaction

KW - procedures

KW - reproducibility

KW - risk

KW - software

KW - virology, Capsid Proteins

KW - CpG Islands

KW - DNA Methylation

KW - DNA Primers

KW - DNA, Viral

KW - Female

KW - Humans

KW - Papillomavirus Infections

KW - Polymerase Chain Reaction

KW - Reproducibility of Results

KW - Risk

KW - Sequence Analysis, DNA

KW - Software

U2 - 10.1371/journal.pone.0194619

DO - 10.1371/journal.pone.0194619

M3 - Article

VL - 13

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 3

M1 - 0194619

ER -