Detection of the hypennethylation of MLH1 promoter and its clinical application in endometrial cancer screening

启动子超甲基化导致MLH 1基因沉默是子宫内膜癌微卫星不稳定（MSI）表型的主要机制。MSI在子宫内膜癌的发生中起着重要作用，涉及多个基因的突变，我们建立了一种简便、灵敏的检测MLH 1上游700 bp区域48个CpG位点的甲基化的方法。这些位点的甲基化已通过硫酸氢盐测序证实。甲基化状态分为完全（超过80%的CpG被甲基化）、部分（10-80%）或非甲基化（小于10%）。在检查的子宫内膜癌中，30%完全甲基化，25%部分甲基化，45%未甲基化。MLH 1的免疫组化方法和MSI的分析表明，的程度，而不是区域特异性甲基化的CpG岛是MLH 1表达和MSI表型的减少至关重要。在甲基化癌症患者中，几乎一半的患者在其正常膀胱中含有甲基化启动子，其特征与癌性病变的特征相似，并且这些与MSI表型密切相关。相比之下，只有少数情况下，正常子宫内膜癌患者没有窝藏甲基化的启动子。目前的研究表明，MLH 1启动子的高甲基化是常见的在组织学证实的正常子宫内膜癌病变附近，支持的概念，即DNA错配修复基因的高甲基化是第一步，触发以下各种遗传事件在子宫内膜癌的发生。当然，这些基因事件也可能成为诊断和治疗中的分子靶点，在微小的临床样本中检测出某些分子靶点可能有助于癌症筛查。

Silencing of the MLH1 gene by promoter hypermethylation is the main mechanism underlying the microsatellite instability(MSI) phenotype in endometrial cancers. MSI has a key role in the endometrial carcinogesis where mutations of multiple genes have involved.We have developed the convenient and sensitive method for the detection of promoter hypermethylation in the region 700bp upstream of MLH1 covering 48 CpG sites. The metylation of these sites has been confirmed by bisulfate sequencing. Metylation status was classified as full(over 80% of CpGs are methylated), partial(10-80%) or nonmethylation(less than 10%). Of endometrial cancers examined, 30% were fully methylated, 25% were partially methylated and 45% were not methylated. Analysis of MLH1 by immunohistochemical methods and of MSI revealed that the degree, rather than region-specific methylation of CpG island is critical for decreased MLH1 expression and the MSI phenotype. Among patients with methylated cancers, almost half patients have contained methylated promoters in their normal endometria with profiles similar to those of cancerous lesions, and these were closely associated with the MSI phenotype. In contrast, only a few cases of normal endometria from patients without endometrial malignancies harbored methylated promoters. The present study suggests that hypermetylation of the MLH1 promoter is frequent in the histologically-cofirmed normal endometrium adjacent to cancerous lesions, supporting the notion that hypermethylation of DNA-mismatch repair genes is the initial step that triggers the following various genetic events in the endometrial carcinogenesis. Of course, the genetic events could be candidates for molecular targets in the diagnosis and treatment.Detection of some molecular targets in a tiny clinical sample might be a useful diagnostic aid in cancer screening.