title: Methylation enrichment pyrosequencing: combining the specificity of MSP with validation by pyrosequencing.
abstract: It has been suggested that detection of aberrant DNA methylation in clinical specimens such as sputum or saliva may be a valuable tumour biomarker. Any clinically applicable detection technique must combine high sensitivity with high specificity. In this study we describe methylation enrichment pyrosequencing (MEP), which benefits from the high sensitivity and specificity of methylation-specific PCR (MSP) but has a second, confirmatory, pyrosequencing step. The pyrosequencing reaction is rapid, relatively inexpensive and offers significant logistical advantages over previously described validation methods. As proof of principle, we illustrate MEP using assays of p16 and cyclin A1 promoters in a methylated DNA dilution matrix and also in a clinical setting using paired saliva and oral tumour specimens. Our results confirm that mis-priming of MSP, with subsequent false positive results, can occur frequently (perhaps 10%) in assays combining high numbers of PCR cycles and low concentrations of starting DNA. In our clinical example, MEP of saliva-derived DNA was more sensitive than standard non-methylation-specific pyrosequencing as illustrated using p16 and cyclin A1 promoter methylation assays.
authors.affiliation: Molecular Genetics and Oncology Group, School of Dental Sciences, University of Liverpool, Liverpool L69 3GN, UK.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Shaw
authors.forename: Emily K
authors.initials: EK
authors.lastname: Akufo-Tetteh
authors.forename: Janet M
authors.initials: JM
authors.lastname: Risk
authors.forename: John K
authors.initials: JK
authors.lastname: Field
authors.forename: Triantafillos
authors.initials: T
authors.lastname: Liloglou
mesh_terms: D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000235:genetics; D019925:Cyclin A / Q000235:genetics; D056750:Cyclin A1; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000032:analysis* / Q000378:metabolism; D006258:Head and Neck Neoplasms / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D012463:Saliva / Q000737:chemistry; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D014408:Biomarkers, Tumor; C503745:CCNA1 protein, human; D019925:Cyclin A; D056750:Cyclin A1; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004273:DNA, Neoplasm