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

Anna Gillio-Tos,Guglielmo Ronco,Valentina Fiano,Laura De Marco,Morena Trevisan,Chiara Grasso,Silvia Gori,Alessandra Mongia,

doi:10.1371/journal.pone.0194619

Abstract

BackgroundMeasuring 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.MethodsObjective 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.ResultsGeneration 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.ConclusionsThe 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.ImpactPotential application of these methylation protocols in screening settings can be explored to identify women with high probability of progression to high grade lesions.

Highlights

Persistent infection by oncogenic high risk human papillomavirus types drives the development of high-grade cervical intraepithelial neoplasia which may, if untreated, progress to invasive cervical cancer.In hrHPV positive women, measuring DNA methylation in the HPV genome has shown promise for accurate detection of hgCIN and cancer [1,2,3,4,5,6,7,8,9]
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

Summary

Introduction

Persistent infection by oncogenic high risk human papillomavirus (hrHPV) types drives the development of high-grade cervical intraepithelial neoplasia (hgCIN) which may, if untreated, progress to invasive cervical cancer.In hrHPV positive women, measuring DNA methylation in the HPV genome has shown promise for accurate detection of hgCIN and cancer [1,2,3,4,5,6,7,8,9]. Most studies employed whole genome sequencing, with the aim of assessing the methylation status of the majority of viral CpGs, which are widespread along the HPV genome and poorly clustered in islands [16]. This approach provided evidence that methylation events, which represent a defence mechanism by the host cell to silence viral DNA [17], largely involve most of the viral CpGs, single CpG sites can be methylated at different levels. 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

Results

Conclusion