Department of Microbiology and Immunology, Weill Cornell Medicine, New York, New York.
Department of Mechanical Engineering, The University of Kansas, Lawrence, Kansas.
J Mol Diagn. 2020 Jul;22(7):885-900. doi: 10.1016/j.jmoldx.2020.03.009. Epub 2020 May 12.
The analysis of CpG methylation in circulating tumor DNA fragments has emerged as a promising approach for the noninvasive early detection of solid tumors, including colorectal cancer (CRC). The most commonly employed assay involves bisulfite conversion of circulating tumor DNA, followed by targeted PCR, then real-time quantitative PCR (alias methylation-specific PCR). This report demonstrates the ability of a multiplex bisulfite PCR-ligase detection reaction-real-time quantitative PCR assay to detect seven methylated CpG markers (CRC or colon specific), in both simulated (approximately 30 copies of fragmented CRC cell line DNA mixed with approximately 3000 copies of fragmented peripheral blood DNA) and CRC patient-derived cell-free DNAs. This scalable assay is designed for multiplexing and incorporates steps for improved sensitivity and specificity, including the enrichment of methylated CpG fragments, ligase detection reaction, the incorporation of ribose bases in primers, and use of uracil DNA glycosylase. Six of the seven CpG markers (located in promoter regions of PPP1R16B, KCNA3, CLIP4, GDF6, SEPT9, and GSG1L) were identified through integrated analyses of genome-wide methylation data sets for 31 different types of cancer. These markers were mapped to CpG sites at the promoter region of VIM; VIM and SEPT9 are established epigenetic markers of CRC. Additional bioinformatics analyses show that the methylation at these CpG sites negatively correlates with the transcription of their corresponding genes.
循环肿瘤 DNA 片段中的 CpG 甲基化分析已成为一种很有前途的方法，可用于非侵入性早期检测实体瘤，包括结直肠癌 (CRC)。最常用的检测方法包括将循环肿瘤 DNA 进行亚硫酸氢盐转化，然后进行靶向 PCR，再进行实时定量 PCR（也称为甲基化特异性 PCR）。本报告展示了一种多重亚硫酸氢盐 PCR-连接酶检测反应-实时定量 PCR 检测方法的能力，该方法可检测七种甲基化 CpG 标志物（CRC 或结肠特异性），包括模拟（约 30 个片段化 CRC 细胞系 DNA 与约 3000 个片段化外周血 DNA 混合）和 CRC 患者来源的无细胞游离 DNA。这种可扩展的检测方法设计用于多重检测，并包含提高灵敏度和特异性的步骤，包括甲基化 CpG 片段的富集、连接酶检测反应、在引物中掺入核糖碱基以及使用尿嘧啶 DNA 糖基化酶。七个 CpG 标志物中的六个（位于 PPP1R16B、KCNA3、CLIP4、GDF6、SEPT9 和 GSG1L 基因的启动子区域）是通过对 31 种不同类型癌症的全基因组甲基化数据集的综合分析确定的。这些标志物被映射到 VIM 启动子区域的 CpG 位点；VIM 和 SEPT9 是 CRC 的既定表观遗传标志物。进一步的生物信息学分析表明，这些 CpG 位点的甲基化与相应基因的转录呈负相关。
