Development of a robust and sensitive pyrosequencing assay for the detection of IDH1/2 mutations in gliomas
Development of a robust and sensitive pyrosequencing assay for the detection of IDH1/2 mutations in gliomas
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DOI：
10.1007/s10014-014-0186-0
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发表时间：
2015-01-01
影响因子：
3.3
通讯作者：
Ichimura, Koichi
中科院分区：
文献类型：
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作者：
Arita, Hideyuki;Narita, Yoshitaka;Ichimura, Koichi
异柠檬酸脱氢酶1/2（IDH 1/2）基因突变状态的评估已成为标准诊断程序的一个组成部分，因此，需要准确。然而，这可能受到各种因素的影响，包括分析方法和低肿瘤细胞含量。我们已经开发了一种快速，灵敏和稳健的检测方法，使用焦磷酸测序检测IDH 1或IDH 2中的所有类型的突变。使用一组代表所有不同类型IDH 1/2突变的对照质粒和一组160个肿瘤标本来评价检测突变的功效。通过对含有不同比例的野生型和突变等位基因的样品进行系列稀释分析，检查测定的灵敏度。对于大多数突变类型，焦磷酸测序测定检测到低至5%的突变等位基因，而常规桑格测序需要存在至少20%的突变等位基因以鉴定突变。焦磷酸测序法在三个样品中检测到IDH 1/2突变，这三个样品由于它们的低肿瘤细胞含量而被桑格测序遗漏。我们的检测方法特别适用于分析大量样本，例如在回顾性临床研究中。
Assessment of the mutational status of the isocitrate dehydrogenase 1/2 (IDH1/2) gene has become an integral part of the standard diagnostic procedure and, therefore, needs to be accurate. This may, however, be compromised by various factors including the method of analysis and a low tumor cell content. We have developed a rapid, sensitive and robust assay to detect all types of mutation in either IDH1 or IDH2 using pyrosequencing. The efficacy of detecting mutation was evaluated using a panel of control plasmids representing all the different types of IDH1/2 mutation and a set of 160 tumor specimens. The sensitivity of the assays was examined by a serial dilution analysis performed on samples containing various ratios of wild-type and mutant alleles. The pyrosequencing assay detected as little as 5 % of mutant alleles for most mutation types, while conventional Sanger sequencing required the presence of at least 20 % of mutant alleles for identifying mutations. The pyrosequencing assay detected IDH1/2 mutations in three samples which were missed by Sanger sequencing due to their low tumor cell contents. Our assay is particularly useful for the analysis of a large number of specimens as in a retrospective clinical study for example.