Mutational analysis of the tyrosine kinome in colorectal cancers
Mutational analysis of the tyrosine kinome in colorectal cancers
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DOI：
10.1126/science.1082596
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发表时间：
2003-05-09
期刊：
影响因子：
56.9
通讯作者：
Velculescu, VE
中科院分区：
文献类型：
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作者：
Bardelli, A;Parsons, DW;Velculescu, VE
酪氨酸激酶（TK）是控制分化、转录、细胞周期进程、凋亡、运动和侵袭的信号传导途径的中心调节因子（1）。虽然一些TK基因已被证明在特定的人类癌症中发生突变改变（1），但尚不清楚在任何特定的癌症类型中TK基因家族的成员有多少或多久发生改变。在这项研究中，我们使用了高通量测序技术和人类基因组计划的生物信息学来解决这个问题。最近的一项分析将人类基因组的蛋白激酶补体（“激酶组”）组织成包含九大基因组的树状图（2）。我们选择了这个树状图的一个主要分支，包含九个主要组中的三个，用于突变分析。这些基因包括90个酪氨酸激酶基因（TK组）、43个酪氨酸激酶样基因（TKL组）和5个受体鸟苷酸环化酶基因（RGC组）。为了评估这些基因在结直肠癌中是否发生了遗传改变，我们首先分析了编码其预测激酶结构域的所有外显子。从基因组数据库中提取了来自所有注释的TK、TKL和RGC基因的总共819个外显子（3）。这些外显子通过聚合酶链反应（PCR）从35个结直肠癌细胞系的DNA中扩增，并直接测序（3）。从获得的4 Mb序列信息中，我们鉴定出14个基因，其激酶结构域中含有体细胞（即肿瘤特异性）突变。然后在另外147例结直肠癌中分析这些基因的突变（3）。我们在14个基因中鉴定了46个突变，其中2个是同义突变;其余的是非同义突变或剪接位点改变（表1;表S1）。所有这些突变在癌症中都被证明是体细胞的，可以通过对来自匹配的正常组织的DNA进行测序来评估。两个独立的观察结果支持了这一假设，即在我们的研究中，在七个基因中发现的突变在一个以上的肿瘤中发生突变。
Tyrosine kinases (TKs) are central regulators of signaling pathways that control differentiation, transcription, cell cycle progression, apoptosis, motility, and invasion (1). Although a few TK genes have been shown to be mutationally altered in specific human cancers (1), it is not known how many or how often members of the TK gene family are altered in any particular cancer type. In this study, we have used high-throughput sequencing technologies and bioinformatics from the human genome project to address this question. A recent analysis organized the protein kinase complement of the human genome (the “kinome”) into a dendrogram containing nine broad groups of genes (2). We selected one major branch of this dendrogram, containing three of the nine major groups, for mutational analysis. These included the 90 tyrosine kinase genes (TK group), the 43 tyrosine kinase–like genes (TKL group), and the 5 receptor guanylate cyclase genes (RGC group). To evaluate whether these genes were genetically altered in colorectal cancer, we initially analyzed all exons encoding their predicted kinase domains. A total of 819 exons from all annotated TK, TKL, and RGC genes containing this domain were extracted from genomic databases (3). These exons were polymerase chain reaction (PCR)-amplified from DNA derived from 35 colorectal cancer cell lines and were directly sequenced (3).From the 4 megabases (Mb) of sequence information obtained, we identified 14 genes that contained somatic (ie, tumor-specific) mutations within their kinase domains. These genes were then analyzed for mutations in another 147 colorectal cancers (3). We identified 46 mutations in the 14 genes, 2 of which were synonymous; the rest were either nonsynonymous or splice site alterations (Table 1; table S1). All of these mutations were shown to be somatic in the cancers that could be assessed by sequencing of DNA from matched normal tissue. Two independent observations support the hypothesis that the mutations found in the seven genes mutated in more than one tumor in our