Methylation of multiple genes as molecular markers for diagnosis of a small, well-differentiated hepatocellular carcinoma
Methylation of multiple genes as molecular markers for diagnosis of a small, well-differentiated hepatocellular carcinoma
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DOI：
10.1002/ijc.24394
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发表时间：
2009-07-15
影响因子：
6.4
通讯作者：
Oka, Masaaki
中科院分区：
文献类型：
--
作者：
Moribe, Toyoki;Iizuka, Norio;Oka, Masaaki
目前的研究是为了确定强大的甲基化标记物及其组合，这些标记物可能被证明对早期肝细胞癌（HCC）的诊断有用。为了实现这一目标，我们在HCC和非HCC肝组织中进行了硅CpG定位，亚硫酸氢盐处理后的直接测序和焦磷酸测序，以及定量甲基化特异性PCR （MSP）。在筛选组（25例hcc）中，我们的直接测序分析显示，在计算机CpG定位列出的12个甲基化基因中，有7个基因（RASSF1A、CCND2、SPINT2、RUNX3、GSTP1、APC和CFTR）在I期和II期hcc中异常甲基化。在验证组（20对hcc和相应的非肿瘤肝组织）中，焦磷酸测序分析证实，这7个基因在早期hcc中异常且强烈甲基化，但在任何相应的非肿瘤肝组织中均未出现甲基化（p < 0.00001）。使用我们的新型定量MSP分析获得的结果与使用焦磷酸测序分析观察到的结果很好地相关。值得注意的是，在MSP实验中，RASSF1A在区分HCC和非HCC肝组织方面表现出最强大的性能。此外，RASSF1A、CCND2和SPINT2联合用于鉴别HCC和非HCC组织的敏感性为89-95%，特异性为91-100%，准确率为89-97%，正确诊断出所有早期HCC。这些结果表明，这3个基因的联合可能有助于早期HCC的准确诊断。(c) 2009年
The current study was conducted to identify robust methylation markers and their combinations that may prove useful for the diagnosis of early hepatocellular carcinoma (HCC). To achieve this, we performed in silico CpG mapping, direct sequencing and pyro-sequencing after bisulfite treatment, and quantitative methylation-specific PCR (MSP) in HCC and non-HCC liver tissues. In the filtering group (25 HCCs), our direct sequencing analysis showed that, among the 12 methylation genes listed by in silico CpG mapping, 7 genes (RASSF1A, CCND2, SPINT2, RUNX3, GSTP1, APC and CFTR) were aberrantly inethylated in stages I and II HCCs. In the validation group (20 pairs of HCCs and the corresponding non-tumor liver tissues), pyrosequencing analysis confirmed that the 7 genes were aberrantly and strongly methylated in early HCCs, but not in any of the corresponding non-tumor liver tissues (p < 0.00001). The results obtained using our novel quantitative MSP assay correlated well with those observed using the pyrosequencing analysis. Notably, in MSP assay, RASSF1A showed the most robust performance for the discrimination of HCC and non-HCC liver tissues. Furthermore, a combination of RASSF1A, CCND2 and SPINT2 showed 89-95% sensitivity, 91-100% specificity and 89-97% accuracy in discriminating between HCC and non-HCC tissues, and correctly diagnosed all early HCCs. These results indicate that the combination of these 3 genes may aid in the accurate diagnosis of early HCC. (C) 2009 UICC