Searching HPV genome for methylation sites involved in molecular progression to cervical precancer

Abstract

Background: Human Papilloma Virus has been considered as the main cause for cervical cancer. In this study we investigated epigenetic changes and especially methylation of specific sites of HPV genome. The main goal was to correlate methylation status with histological grade as well as to determine its accuracy in predicting the disease severity by establishing optimum methylation cutoffs.Methods: In total, sections from 145 cases genotyped as HPV16 were obtained from formalin- fixed, paraffin-embedded tissue of cervical biopsies, conization or hysterectomy specimens. Highly accurate pyrosequencing of bisulfite converted DNA, was used to quantify the methylation percentages of UTR promoter, enhancer and 5' UTR, E6 CpGs 494, 502, 506 and E7 CpGs 765, 780, 790. The samples were separated in different groupings based on the histological outcome. Statistical analysis was performed by SAS 9.4 for Windows and methylation cutoffs were identified by MATLAB programming language.Results: The most important methylation sites were at the enhancer and especially UTR 7535 and 7553 sites. Specifically for CIN3+ (i.e. HSIL or SCC) discrimination, a balanced sensitivity vs. specificity (68.1%, 66.2% respectively) with positive predictive value (PPV) and negative predictive value (NPV) (66.2%, 68.2% respectively) was achieved for UTR 7535 methylation of 6.1% cutoff with overall accuracy 67.1%, while for UTR 7553 a sensitivity 60.9%, specificity 69.0%, PPV=65.6%, NPV=64.5% and overall accuracy=65.0% at threshold 10.1% was observed.Conclusion: Viral HPV16 genome was found methylated in NF-1 binding sites of UTR in cases with high grade disease. Methylation percentages of E6 and E7 CpG sites were elevated at the cancer group.

Genetic variability of the HPV16 early genes and LCR. Present and future perspectives

“…However, we cannot exclude the possibility that this theory might be applicable for HPV16 in the future. Additional factors should be taken into consideration in order to lead to more accurate conclusions about cervical cancer risk, such as multiple HR-HPV infections, viral DNA integration, the methylation status of HPV DNA and mutations into the host target proteins ( Refs 13,18,34,[158][159][160].…”

The Role of Methylation of Host and/or Human Papillomavirus (HPV) DNA in Management of Cervical Intraepithelial Neoplasia Grade 2 (CIN2) Lesions

“…An epigenetic change is defined as a change in the expression of host or viral genes without changes of the encoding DNA sequence [ 37 ]. DNA methylation is the most extensively studied epigenetic change in HPV-related cancers [ 50 ]. During DNA methylation, DNA methyltransferase enzymes covalently add methyl groups to cytosine preceding guanine (CpG) [ 51 ].…”

Epigenetic approaches for cervical neoplasia screening (Review)

“…Certain studies have demonstrated that methylation assays are a useful tool for triaging women with advanced HPV infections, although their performance is rather poor in on-going hrHPV infections that have not progressed yet to CIN3 or cervical cancer (97,(104)(105)(106). In addition, with advancements in technology, HPV DNA testing could be replaced by DNA methylation screening (95). At present, methylation testing is based on RT-qPCR to estimate the percentage of methylated DNA molecules in the sample for the targeted region (Fig.…”

“…In addition to host genes methylation, the methylation of viral genes exhibits high levels, particularly in capsid genes and late open reading frames. This has been associated with the progression of lesions from CIN2/3 to invasive cancer ( 95 , 96 ). In a study on HPV-infected Asian women with different grade of lesions, the methylation status of CpG islands in HPV16, 18, 52 and 58 was investigated.…”