Methylation target array for rapid analysis of CpG island hype rmethyl ation in multiple tissue genomes
Methylation target array for rapid analysis of CpG island hype rmethyl ation in multiple tissue genomes
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DOI：
10.1016/s0002-9440(10)63628-0
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发表时间：
2003-07-01
影响因子：
6
通讯作者：
Huang, THM
中科院分区：
文献类型：
--
作者：
Chen, CM;Chen, HL;Huang, THM
多个CpG岛的超甲基化是癌症中的常见事件。为了评估这种表观遗传改变的预后价值，我们开发了甲基化靶阵列（MTA），来自组织微阵列的概念，用于同时分析数百个组织基因组中的DNA超甲基化。在NITA中，接头连接的CpG岛片段用甲基化敏感性核酸内切酶消化并用侧翼引物扩增。一组468个NITA扩增子，代表了93个乳腺肿瘤，20个正常乳腺组织和4个乳腺癌细胞系中甲基化CpG岛的全部谱，排列在尼龙膜上进行探针杂交。在肿瘤扩增子中检测到阳性杂交信号，但在正常扩增子中未检测到，表明肿瘤样品中存在异常的超甲基化。这是由于异常位点受到保护，不受甲基化敏感性限制，并在肿瘤样品中通过PCR扩增，而相同的位点受到限制，不能在正常样品中扩增。在乳腺肿瘤和癌细胞系中检测的10个基因的超甲基化频率为：GPC 3 60%，RASSF1A 58%，3OST 3B 32%，HOXA 5 30%，uPA 28%，WT 1 25%，BRCA 1 23%，DAPK 1 9%，KL 0%。此外，这些基因的5至7个位点的超甲基化与所分析的患者的激素受体状态、临床分期和诊断时的年龄显著相关。因此，这种新的方法提供了一个额外的途径来评估乳腺癌中DNA超甲基化的临床病理后果。
Hypermethylation of multiple CpG islands is a common event in cancer. To assess the prognostic values of this epigenetic alteration, we developed Methylation Target Array (MTA), derived from the concept of tissue microarray, for simultaneous analysis of DNA hypermethylation in hundreds of tissue genomes. in NITA, linker-ligated CpG island fragments were digested with methylation-sensitive endonucleases and amplified with flanking primers. A panel of 468 NITA amplicons, which represented the whole repertoire of methylated CpG islands in 93 breast tumors, 20 normal breast tissues, and 4 breast cancer cell lines, were arrayed on nylon membrane for probe hybridization. Positive hybridization signals detected in tumor amplicons, but not in normal amplicons, were indicative of aberrant hypermethylation in tumor samples. This is attributed to aberrant sites that were protected from methylation-sensitive restriction and were amplified by PCR in tumor samples, while the same sites were restricted and could not be amplified in normal samples. Hypermethylation frequencies of the 10 genes tested in breast tumors and cancer cell lines were 60% for GPC3, 58% for RASSF1A, 32% for 3OST3B, 30% for HOXA5, 28% for uPA, 25% for WT1, 23% for BRCA1, 9% for DAPK1, and 0% for KL. Furthermore, hypermethylation of 5 to 7 loci of these genes was significantly correlated with hormone receptor status, clinical stages, and ages at diagnosis of the patients analyzed. This novel approach thus provides an additional avenue for assessing clinicopathological consequences of DNA hypermethylation in breast cancer.