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

<div><p>Background</p><p>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.</p><p>Methods</p><p>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.</p><p>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.</p><p>PCR consensus primers were designed by PyroMark Assay Design software to be suitable for amplification of many different oncogenic HPV types.</p><p>Results</p><p>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.</p><p>Conclusions</p><p>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.</p><p>Impact</p><p>Potential application of these methylation protocols in screening settings can be explored to identify women with high probability of progression to high grade lesions.</p>