Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis
Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis
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DOI：
10.2353/jmoldx.2007.060167
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发表时间：
2007-07-01
影响因子：
4.1
通讯作者：
Waha, Andreas
中科院分区：
文献类型：
--
作者：
Mikeska, Thomas;Bock, Christoph;Waha, Andreas
对化疗的耐药性是癌症患者治疗过程中的主要并发症。 MGMT基因的高甲基化会改变DNA的修复，并与用烷基化剂治疗的胶质母细胞瘤患者的生存率更长有关。因此，MGMT启动子甲基化作为化学疗法耐药性的预测生物标志物起着重要作用。为了将这种已建立的相关性用于分子诊断程序，我们比较并优化了三种实验技术[联合亚硫酸盐限制性分析，一种底漆扩展和基于高性性液态色谱法名为Sirph（snupe snupe snupe ion配对相位逆期高表现高表现）液相色谱）和焦磷酸测序]关于它们检测MGMT启动子甲基化的准确性。最初，使用亚硫酸盐测序来获得22个胶质母细胞瘤样品中的MGMT启动子区域和三种正常脑对照组的综合甲基化谱。接下来，我们从统计上确定了最能区分甲基化和未甲基化的MGMT启动子的CPG位点。然后将这些结果用于设计最佳的亚硫酸氢盐限制分析，SIRPH和焦磷酸测序测定法，以精确且具有成本效益的MGMT启动子甲基化评估。我们比较了所有三种技术的可靠性和对特征良好的肿瘤样品的可重复性。优化的焦磷酸测序；测定最佳，并提供了一种敏感，健壮且易于使用的方法，用于定量评估MGMT甲基化，以供Snap-Frozen和Popaffin Imbedded标本进行定量评估。
Resistance to chemotherapy is a major complication during treatment of cancer patients. Hypermethylation of the MGMT gene alters DNA repair and is associated with longer survival of glioblastoma patients treated with alkylating agents. Therefore, MGMT promoter methylation plays an important role as a predictive biomarker for chemotherapy resistance. To adopt this established correlation into a molecular diagnosis procedure, we compared and optimized three experimental techniques [combined bisulfite restriction analysis, a primer extension- and denaturing high-performance liquid chromatography-based method named SIRPH (SNuPE ion pair-reverse phase high-performance liquid chromatography), and pyrosequencing] with regard to their accuracy of detecting MGMT promoter methylation. Initially, bisulfite sequencing was used to obtain a comprehensive methylation profile of the MGMT promoter region in 22 glioblastoma samples and in three normal brain controls. Next, we statistically identified CpG sites that best discriminate between methylated and unmethylated MGMT promoters. These results were then used to design optimal combined bisulfite restriction analysis, SIRPH, and pyrosequencing assays for accurate and cost-efficient assessment of MGMT promoter methylation. We compared all three techniques with regard to their reliability and reproducibility on well-characterized tumor samples. The optimized pyrosequencing; assay performed best and provides a sensitive, robust, and easy-to-use method for quantitative assessment of MGMT methylation, for both snap-frozen and paraffin-embedded specimens.