ArticleInfectious agents and cancer2025
Methylation-specific qPCR for the EBV C promoter to quantify EBV methylation.
Article in Infectious agents and cancer, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
BACKGROUND/
objectivesEpstein-Barr Virus (EBV) is a ubiquitous virus associated with a variety of diseases including cancers. Evidence has emerged that the C promoter is methylated in many EBV-associated malignancies, whereas in free virion DNA it is unmethylated. We have developed and evaluated a methylation-specific PCR assay for the EBV C Promoter (MSPCP) that can be applied to human biological specimens to quantify EBV methylation.
methodsTwo sets of methylation-specific primers were designed to anneal to bisulfite-converted DNA sequences with 3 CpGs in the forward primer binding site, and 2 CpGs in the reverse primer binding site. We evaluated this method in synthetic oligonucleotides, DNA extracted from cell lines, virion supernatants, and a variety of clinical specimens. EBV methylation of Cp, as measured by MSPCP, was validated with two orthogonal methods in select samples.
resultsIn contrived samples, this method had a linear range between 0-100% methylation. Application of this assay to DNA extracted from 11 formalin-fixed paraffin-embedded biopsy specimens showed high-level C promoter methylation in EBV-associated tumors (94-100%) but not in EBV-associated lymphoid hyperplasia. High-level EBV methylation was also detected in cell-free DNA extracted from the plasma of 13 patients with EBV-associated Hodgkin lymphoma. In contrast, EBV methylation was either not-detected, or detected at very low levels, in saliva from 25 adults in a general university population consistent with the presence of virion DNA.
conclusionsMSPCP is a simple, rapid and accurate method that characterizes the methylation status of the EBV C promoter, which may be useful in a variety of research and clinical settings.
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