Chen Chuan-Mu, Chen Hsiao-Ling, Hsiau Timothy H-C, Hsiau Andrew H-A, Shi Huidong, Brock Graham J R, Wei Susan H, Caldwell Charles W, Yan Pearlly S, Huang Tim Hui-Ming
Department of Life Sciences, National Chung Hsing University, Taiwan, Republic of China.
Am J Pathol. 2003 Jul;163(1):37-45. doi: 10.1016/S0002-9440(10)63628-0.
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 MTA, linker-ligated CpG island fragments were digested with methylation-sensitive endonucleases and amplified with flanking primers. A panel of 468 MTA 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.
多个CpG岛的高甲基化是癌症中的常见现象。为了评估这种表观遗传改变的预后价值，我们基于组织微阵列的概念开发了甲基化靶向阵列（MTA），用于同时分析数百个组织基因组中的DNA高甲基化。在MTA中，连接子连接的CpG岛片段用甲基化敏感的核酸内切酶消化，并用侧翼引物扩增。一组468个MTA扩增子，代表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岛的全部组成，被排列在尼龙膜上用于探针杂交。在肿瘤扩增子中检测到的阳性杂交信号，但在正常扩增子中未检测到，表明肿瘤样本中存在异常高甲基化。这归因于肿瘤样本中免受甲基化敏感限制并通过PCR扩增的异常位点，而相同位点在正常样本中受到限制且无法扩增。在乳腺肿瘤和癌细胞系中检测的10个基因的高甲基化频率分别为：GPC3为60%，RASSF1A为58%，3OST3B为32%，HOXA5为30%，uPA为28%，WT1为25%，BRCA1为23%，DAPK1为9%，KL为0%。此外，这些基因5至7个位点的高甲基化与所分析患者的激素受体状态、临床分期和诊断年龄显著相关。因此，这种新方法为评估乳腺癌中DNA高甲基化的临床病理后果提供了一条新途径。
