Fak.NT Life Sciences, Department of Genetics/Epigenetics, Saarland University, Campus, Building A2 4, 66041, Saarbrücken, Germany.
Institute of Neuropathology, Medical Faculty of the Saarland University, Homburg, Germany.
Clin Epigenetics. 2022 Feb 18;14(1):26. doi: 10.1186/s13148-022-01244-4.
Promoter methylation of the DNA repair gene O-methylguanine-DNA methyltransferase (MGMT) is an acknowledged predictive epigenetic marker in glioblastoma multiforme and anaplastic astrocytoma. Patients with methylated CpGs in the MGMT promoter benefit from treatment with alkylating agents, such as temozolomide, and show an improved overall survival and progression-free interval. A precise determination of MGMT promoter methylation is of importance for diagnostic decisions. We experienced that different methods show partially divergent results in a daily routine. For an integrated neuropathological diagnosis of malignant gliomas, we therefore currently apply a combination of methylation-specific PCR assays and pyrosequencing.
To better rationalize the variation across assays, we compared these standard techniques and assays to deep bisulfite sequencing results in a cohort of 80 malignant astrocytomas. Our deep analysis covers 49 CpG sites of the expanded MGMT promoter, including exon 1, parts of intron 1 and a region upstream of the transcription start site (TSS). We observed that deep sequencing data are in general in agreement with CpG-specific pyrosequencing, while the most widely used MSP assays published by Esteller et al. (N Engl J Med 343(19):1350-1354, 2000. https://doi.org/10.1056/NEJM200011093431901 ) and Felsberg et al. (Clin Cancer Res 15(21):6683-6693, 2009. https://doi.org/10.1158/1078-0432.CCR-08-2801 ) resulted in partially discordant results in 22 tumors (27.5%). Local deep bisulfite sequencing (LDBS) revealed that CpGs located in exon 1 are suited best to discriminate methylated from unmethylated samples. Based on LDBS data, we propose an optimized MSP primer pair with 83% and 85% concordance to pyrosequencing and LDBS data. A hitherto neglected region upstream of the TSS, with an overall higher methylation compared to exon 1 and intron 1 of MGMT, is also able to discriminate the methylation status.
Our integrated analysis allows to evaluate and redefine co-methylation domains within the MGMT promoter and to rationalize the practical impact on assays used in daily routine diagnostics.
DNA 修复基因 O-甲基鸟嘌呤-DNA 甲基转移酶（MGMT）启动子的甲基化是多形性胶质母细胞瘤和间变性星形细胞瘤中公认的预测性表观遗传标志物。MGMT 启动子中 CpG 被甲基化的患者受益于烷基化剂（如替莫唑胺）治疗，表现出改善的总生存期和无进展生存期。因此，MGMT 启动子甲基化的精确测定对于诊断决策具有重要意义。我们发现不同的方法在日常工作中会产生部分不同的结果。为了对恶性胶质瘤进行综合神经病理学诊断，我们目前将甲基化特异性 PCR 检测和焦磷酸测序相结合。
为了更好地解释检测之间的差异，我们将这些标准技术和检测方法与 80 例恶性星形细胞瘤的深度亚硫酸氢盐测序结果进行了比较。我们的深度分析涵盖了扩展的 MGMT 启动子的 49 个 CpG 位点，包括外显子 1、内含子 1 的部分和转录起始位点（TSS）上游区域。我们观察到，深度测序数据通常与 CpG 特异性焦磷酸测序一致，而 Esteller 等人发表的最广泛使用的 MSP 检测（N Engl J Med 343(19):1350-1354, 2000. https://doi.org/10.1056/NEJM200011093431901 ）和 Felsberg 等人发表的结果（Clin Cancer Res 15(21):6683-6693, 2009. https://doi.org/10.1158/1078-0432.CCR-08-2801 ）在 22 个肿瘤（27.5%）中产生了部分不一致的结果。局部深度亚硫酸氢盐测序（LDBS）显示，位于外显子 1 中的 CpG 最适合区分甲基化和非甲基化样本。基于 LDBS 数据，我们提出了一个优化的 MSP 引物对，与焦磷酸测序和 LDBS 数据的一致性分别为 83%和 85%。MGMT 启动子上游一个被忽视的区域，其整体甲基化水平高于外显子 1 和内含子 1，也能够区分甲基化状态。
我们的综合分析允许评估和重新定义 MGMT 启动子内的共甲基化区域，并使日常诊断中使用的检测方法的实际影响合理化。
