Article

A 115-bp MethyLight assay for detection of p16 (CDKN2A) methylation as a diagnostic biomarker in human tissues

Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, Department of Aetiology, Peking University Cancer Hospital & Institute, Beijing, China.
BMC Medical Genetics (Impact Factor: 2.45). 05/2011; 12(1):67. DOI: 10.1186/1471-2350-12-67
Source: PubMed

ABSTRACT p16 Methylation is a potential biomarker for prediction of malignant transformation of epithelial dysplasia. A probe-based, quantitative, methylation-specific PCR (MSP) called MethyLight may become an eligible method for detecting this marker clinically. We studied oral mucosa biopsies with epithelial dysplasia from 78 patients enrolled in a published 4-years' followup cohort, in which cancer risk for patients with p16 methylation-positive dysplasia was significantly higher than those without p16 methylation (by 150-bp MSP and bisulfite sequencing; +133 ~ +283, transcription starting site, +1). The p16 methylation status in samples (N = 102) containing sufficient DNA was analyzed by the 70-bp classic (+238 ~ +307) and 115-bp novel (+157 ~ +272) MethyLight assays, respectively.
p16 Methylation was detectable in 75 samples using the classic MethyLight assay. The methylated-p16 positive rate and proportion of methylated-p16 by the MethyLight in MSP-positive samples were higher than those in MSP-negative samples (positive rate: 37/44 vs. 38/58, P=0.035, two-sided; proportion [median]: 0.78 vs. 0.02, P <0.007). Using the published results of MSP as a golden standard, we found sensitivity, specificity, and accuracy for this MethyLight assay to be 70.5%, 84.5%, and 55.0%, respectively. Because amplicon of the classic MethyLight procedure only partially overlapped with the MSP amplicon, we further designed a 115-bp novel MethyLight assay in which the amplicon on the sense-strand fully overlapped with the MSP amplicon on the antisense-strand. Using the 115-bp MethyLight assay, we observed methylated-p16 in 26 of 44 MSP-positive samples and 2 of 58 MSP-negative ones (P = 0.000). These results were confirmed with clone sequencing. Sensitivity, specificity, and accuracy using the 115-bp MethyLight assay were 59.1%, 98.3%, and 57.4%, respectively. Significant differences in the oral cancer rate were observed during the followup between patients (≥60 years) with and without methylated-p16 as detected by the 115-bp MethyLight assay (6/8 vs. 6/22, P = 0.034, two-sided).
The 115-bp MethyLight assay is a useful and practical assay with very high specificity for the detection of p16 methylation clinically.

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    • "The 115-bp sense-fragment is completely included within P16 exon-1, the most prevalent sequence investigated for P16 methylation. Methylation in this region is not only correlated with inactivation of this gene (Herman et al., 1995), but also associated with prognosis of epithelial dysplasia (Sun et al., 2004; Cao et al., 2009; Zhou et al., 2011). Briefly, the 115-bp methylated fragment in the sense-strand of P16 exon-1 was amplified using forward primer (5′-CgCggtCgtggttagttagt-3′), reverse primer (5′-tacGctcGacGactaCgaaa-3′), and P16-specific probe (6FAM-gttgtttttCgtCgtCggtt-TAMRA). "
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