Simultaneous detection of mutations and copy number variation of NPM1 in the acute myeloid leukemia using multiplex ligation-dependent probe amplification
Simultaneous detection of mutations and copy number variation of NPM1 in the acute myeloid leukemia using multiplex ligation-dependent probe amplification
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DOI：
10.1016/j.mrfmmm.2016.02.001
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发表时间：
2016-04-01
影响因子：
2.3
通讯作者：
Kozlowski, Piotr
中科院分区：
文献类型：
--
作者：
Marcinkowska-Swojak, Malgorzata;Handschuh, Luiza;Kozlowski, Piotr
NPM1基因编码核磷蛋白，这是一种参与多种细胞功能和癌变的蛋白质。NPM1基因突变导致该蛋白脱位，是急性髓性白血病（AML）中最常见的遗传病变；它被认为是AML发病机制的创始事件，是一个有利的预后标志。此外，在实体肿瘤和一些白血病细胞系中，NPM1基因的过表达也很常见。因此，本研究的目的是开发一种检测NPM1突变并同时分析拷贝数改变（CNAs）的新方法，这可能是NPM1基因表达失调的基础。为了解决这两个问题，我们采用了一种基于多路连接相关探针放大（MLPA）的策略。设计的NPM1mut+检测能够检测三种最常见的NPM1突变：A， B和d。使用来自波兰AML和其他血液增生性疾病患者的83个样本来测试该检测的准确性。为了验证结果，我们使用了传统的Sanger测序和下一代转录组测序。使用NPM1mut+检测，我们分别在14个、1个和0个分析样本中检测到突变A、D和B。所有这些突变都是通过互补测序方法确认的，证明了所提出的测试的100%特异性和敏感性。测序分析允许鉴定两个额外的罕见突变（I和ZE）。所有的突变都是在AML病例中发现的，占这些病例的25%。我们在研究样本中没有观察到NPM1基因的任何CNAs（扩增），无论是否有突变。该方法简单、可靠、经济。它可以很容易地引入临床实践，或开发针对NPM1基因和其他癌症相关基因中不太常见的突变。(C) 2016 Elsevier B.V.版权所有
The NPM1 gene encodes nucleophosmin, a protein involved in multiple cell functions and carcinogenesis. Mutation of the NPM1 gene, causing delocalization of the protein, is the most frequent genetic lesion in acute myeloid leukemia (AML); it is considered a founder event in AML pathogenesis and serves as a favorable prognostic marker. Moreover, in solid tumors and some leukemia cell lines, overexpression of the NPM1 gene is commonly observed. Therefore, the purpose of this study was to develop a new method for the detection of NPM1 mutations and the simultaneous analysis of copy number alterations (CNAs), which may underlie NPM1 gene expression deregulation. To address both of the issues, we applied a strategy based on multiplex ligation-dependent probe amplification (MLPA). A designed NPM1mut+ assay enables the detection of three of the most frequent NPM1 mutations: A, B and D. The accuracy of the assay was tested using a group of 83 samples from Polish patients with AML and other blood-proliferative disorders. To verify the results, we employed traditional Sanger sequencing and next-generation transcriptome sequencing. With the use of the NPM1mut+ assay, we detected mutations A, D and B in 14, 1 and 0 of the analyzed samples, respectively. All of these mutations were confirmed by complementary sequencing approaches, proving the 100% specificity and sensitivity of the proposed test. The performed sequencing analysis allowed the identification of two additional rare mutations (I and ZE). All of the mutations were identified exclusively in AML cases, accounting for 25% of those cases. We did not observe any CNAs (amplifications) of the NPM1 gene in the studied samples, either with or without the mutation. The presented method is simple, reliable and cost-effective. It can be easily introduced into clinical practice or developed to target both less-frequent mutations in the NPM1 gene and other cancer-related genes. (C) 2016 Elsevier B.V. All rights reserved.