Bettstetter M, Rümmele P, Hofstädter F, Dietmaier W
Institut für Pathologie, Universität Regensburg.
Verh Dtsch Ges Pathol. 2006;90:236-43.
Promoter hypermethylation is a common mechanism for epigenetic control of gene expression and occurs frequently in tumors silencing tumor suppressor genes. Our aim was to establish a quantitative and precise method to analyze promoter methylation of tumor samples in order to identify HNPCC candidates.
We established a new methylation specific relative quantitative real-time PCR technique for analysis of the methylation status of the hMLHI promoter in colorectal cancers (CRC). We determined methylation status of both the distal and proximal hMLH1-promoter region. The methylation quantification (MQ) was performed with cell line DNA and archival paraffinized tissue sections.
The accuracy of our analysis was validated with spiking experiments of methylated and unmethylated DNA. We assessed the hMLH1 methylation status 56 CRC patients with known microsatellite status and hMLH1 IHC. The methylation analysis divided the MSI-H CRC into two groups: Methylation positive sporadic CRC patients with a median age of 78.5 years and frequent BRAF mutations (82 %, p < 0.0001) and the unmethylated cancers from HNPCC candidates with a median age of 48 years. All hMLH1 positive sporadic MSS CRC were methylation negative. In all samples, the degree of methylation was mirrored by the shift of the melting points to higher temperatures.
In summary we introduced a quantitative and qualitative technique to analyze DNA methylation that can be performed with any dense CpG island. Our methylation analysis provides a potent diagnostic tool to differentiate between sporadic MSI-H cancers showing MLH1 methylation and MLH1 unmethylated HNPCC candidates.
启动子高甲基化是基因表达表观遗传调控的常见机制，在使肿瘤抑制基因沉默的肿瘤中频繁发生。我们的目的是建立一种定量且精确的方法来分析肿瘤样本的启动子甲基化，以识别遗传性非息肉病性结直肠癌（HNPCC）候选者。
我们建立了一种新的甲基化特异性相对定量实时聚合酶链反应（PCR）技术，用于分析结直肠癌（CRC）中hMLH1启动子的甲基化状态。我们测定了hMLH1启动子远端和近端区域的甲基化状态。甲基化定量（MQ）采用细胞系DNA和存档石蜡包埋组织切片进行。
通过甲基化和未甲基化DNA的加标实验验证了我们分析的准确性。我们评估了56例已知微卫星状态和hMLH1免疫组化的CRC患者的hMLH1甲基化状态。甲基化分析将微卫星高度不稳定（MSI-H）的CRC分为两组：甲基化阳性的散发性CRC患者，中位年龄78.5岁，BRAF突变频繁（82%，p<0.0001），以及来自HNPCC候选者的未甲基化癌症，中位年龄48岁。所有hMLH1阳性的散发性微卫星稳定（MSS）CRC均为甲基化阴性。在所有样本中，熔点向更高温度的偏移反映了甲基化程度。
总之，我们引入了一种定量和定性技术来分析DNA甲基化，该技术可用于任何密集的CpG岛。我们的甲基化分析提供了一种有效的诊断工具，用于区分显示MLH1甲基化的散发性MSI-H癌症和MLH1未甲基化的HNPCC候选者。
