engleski

Human Papillomavirus (HPV) Type 16 Variability in Croatia Assessed by High Resolution Melting (HRM) Method

Background: Viral genome variants, diverging by 2% within a viral type, contribute to the carcinogenic potential of HPV types presumably altering its transforming properties and/or immunogenicity. Most of the studies on HPV16 variability were concerned with E6 and E7 regions that encode for well known HPV oncogenes. However, studies of viral variants are very laborious and expensive as several genomic regions of each sample need to be sequenced to detect the few differing nucleotides that identify variants. Objectives: To simplify and reduce the costs of variant identification we investigated the possible application of HRM analysis to determine HPV16 variants of the E6/E7 region. Methods: The HRM analysis enables the distinction of PCR amplicons differing in even a sole nucleotide, and as such should be capable of easily indicate HPV variants. We have previously confirmed this assumption on a subset of samples. In this study, the performance of optimised HRM analysis was evaluated on 230 HPV16 positive cervical samples. The E6 region of HPV16 was amplified by a nested PCR and then melted. Samples giving an ambiguous result were additionally remelted in the presence of wild type HPV16 DNA to confirm the melting result. To evaluate the HRM method performance, a subset of samples was sequenced. Results: In about half of Croatian women, the European German variant as indicated by the presence of E6-350G mutation was found. In addition, it appears that no other variants are common. Conclusions: Our results indicate that HRM approach can indeed be informative, while saving time and significant cost of sequencing of each sample and it is interesting in low resource setting or where cost efficiency is the issue.

Human papillomaviruses (HPV); High Resolution Melting (HRM)

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