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

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. Objective of this methodological study was to set...

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Published in:PloS one 2018-03, Vol.13 (3), p.e0194619-e0194619
Main Authors: Gillio-Tos, Anna, Fiano, Valentina, Grasso, Chiara, Trevisan, Morena, Gori, Silvia, Mongia, Alessandra, De Marco, Laura, Ronco, Guglielmo
Format: Article
Language:English
Subjects:
Biology and life sciences
Biomarkers
Cancer
Capsid Proteins - genetics
Cervical cancer
Cervix
Correlation coefficient
Correlation coefficients
CpG Islands
Deoxyribonucleic acid
Diagnosis
DNA
DNA Methylation
DNA Primers - metabolism
DNA, Viral - genetics
DNA, Viral - metabolism
Epidemiology
Female
Genetic aspects
Genetic testing
Genomes
Human papillomavirus
Humans
Infections
Lesions
Medical screening
Medicine and Health Sciences
Methods
Methylation
Papillomaviridae - genetics
Papillomaviridae - isolation & purification
Papillomavirus infections
Papillomavirus Infections - diagnosis
Papillomavirus Infections - virology
Physical Sciences
Pilot projects
Polymerase chain reaction
Polymerase Chain Reaction - methods
Primers
Reproducibility of Results
Research and Analysis Methods
Risk
Risk factors
Sequence Analysis, DNA - methods
Software
Studies
Working groups
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cited_by cdi_FETCH-LOGICAL-c692t-b28fab8bdbff56e7be0f3c3df0afb9e4748feeece2ffcad9dd69d7aa5186ec693
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creator Gillio-Tos, Anna
Fiano, Valentina
Grasso, Chiara
Trevisan, Morena
Gori, Silvia
Mongia, Alessandra
De Marco, Laura
Ronco, Guglielmo
description 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. 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. 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. 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. Potential application of these methylation protocols in screening settings can be explored to identify women with high probability of progression to high grade lesions.
doi_str_mv 10.1371/journal.pone.0194619
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Methylation status has been widely investigated for HPV16, sporadically for other HPV types. 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. 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. 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. Potential application of these methylation protocols in screening settings can be explored to identify women with high probability of progression to high grade lesions.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>29579066</pmid><doi>10.1371/journal.pone.0194619</doi><tpages>e0194619</tpages><orcidid>https://orcid.org/0000-0001-6553-5362</orcidid><oa>free_for_read</oa></addata></record>
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identifier ISSN: 1932-6203
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source Publicly Available Content Database; PubMed Central
subjects Biology and life sciences
Biomarkers
Cancer
Capsid Proteins - genetics
Cervical cancer
Cervix
Correlation coefficient
Correlation coefficients
CpG Islands
Deoxyribonucleic acid
Diagnosis
DNA
DNA Methylation
DNA Primers - metabolism
DNA, Viral - genetics
DNA, Viral - metabolism
Epidemiology
Female
Genetic aspects
Genetic testing
Genomes
Human papillomavirus
Humans
Infections
Lesions
Medical screening
Medicine and Health Sciences
Methods
Methylation
Papillomaviridae - genetics
Papillomaviridae - isolation & purification
Papillomavirus infections
Papillomavirus Infections - diagnosis
Papillomavirus Infections - virology
Physical Sciences
Pilot projects
Polymerase chain reaction
Polymerase Chain Reaction - methods
Primers
Reproducibility of Results
Research and Analysis Methods
Risk
Risk factors
Sequence Analysis, DNA - methods
Software
Studies
Working groups
title Assessment of viral methylation levels for high risk HPV types by newly designed consensus primers PCR and pyrosequencing
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