A 115-bp MethyLight assay for detection of p16 (CDKN2A) methylation as a diagnostic biomarker in human tissues.
A 115-bp MethyLight assay for detection of p16 (CDKN2A) methylation as a diagnostic biomarker in human tissues.
复制标题
用于检测 p16 (CDKN2A) 甲基化作为人体组织中诊断生物标志物的 115-bp MethyLight 测定
DOI：
10.1186/1471-2350-12-67
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发表时间：
2011-05-13
影响因子：
--
通讯作者：
Deng D
中科院分区：
文献类型：
--
作者：
Zhou J;Cao J;Lu Z;Liu H;Deng D
背景
P16甲基化是预测上皮异型增生恶变的潜在生物标志物。一种基于探针的、定量的、甲基化特异性的聚合酶链式反应(MSP)称为甲基光，可能成为临床上检测该标志物的合格方法。我们研究了78例口腔粘膜上皮异型增生患者的口腔粘膜活检组织，这些患者登记在一项已发表的4年随访队列中，其中p16甲基化阳性异型增生患者的癌症风险显著高于不存在p16甲基化的患者(通过150个碱基MSP和亚硫酸氢盐测序；+133~+283，转录起始点，+1)。分别用70bp的经典(+238~+307)和115bp的新型(+157~+272)甲基化分析方法分析含有足够DNA的样本(N=102)中的p16甲基化状态。
结果
使用经典的甲基光检测方法，在75个样本中检测到p16甲基化。MSP阳性标本中p16甲基化阳性率和甲基化比例均高于MSP阴性标本(双侧：37/44 vs.38/58，P=0.035；比例[中位数]：0.78vs.0.02，P 0.007)。以已发表的MSP检测结果为金标准，我们发现该方法的敏感性、特异性和准确性分别为70.5%、84.5%和55.0%。由于经典的MethyLight方法的扩增子只与MSP扩增子部分重叠，因此我们进一步设计了一种115个碱基的新的MethyLight分析方法，其中正义链上的扩增子与反义链上的MSP扩增子完全重叠。在44例MSP阳性标本中有26例甲基化，58例MSP阴性标本中有2例甲基化(P=0.000)。克隆测序结果证实了这些结果。用115个碱基的甲基光试剂盒检测的敏感性、特异性和准确性分别为59.1%、98.3%和57.4%。在随访期间(≥60岁)，用115BP甲基化法检测到p16甲基化和不甲基化的患者的口腔癌发病率有显著差异(6/8比6/22，P=0.034，双侧)。
结论
115碱基的甲基化检测方法对临床检测p16甲基化具有很高的特异性，是一种实用、实用的检测方法。
Background
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.
Results
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).
Conclusions
The 115-bp MethyLight assay is a useful and practical assay with very high specificity for the detection of p16 methylation clinically.