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首页> 外文期刊>The Journal of molecular diagnostics: JMD >Microfluidic deletion/insertion analysis for rapid screening of KIT and PDGFRA mutations in CD117-positive gastrointestinal stromal tumors: diagnostic applications and report of a new KIT mutation.
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Microfluidic deletion/insertion analysis for rapid screening of KIT and PDGFRA mutations in CD117-positive gastrointestinal stromal tumors: diagnostic applications and report of a new KIT mutation.

机译：快速筛选CD117阳性胃肠道间质瘤中KIT和PDGFRA突变的微流控缺失/插入分析：诊断应用和新KIT突变的报告。

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摘要

Gastrointestinal stromal tumors (GISTs) frequently harbor mutations in the KIT and PDGFRA genes, the presence and type of which correlate with the response to the kinase inhibitor imatinib mesylate. Because most GIST mutations are deletions/insertions, we used a microfluidic apparatus to detect these size variations in polymerase chain reaction-amplified DNA. This approach, termed microfluidic deletion/insertion analysis (MIDIA), identified mutations in 30 of 50 DNA samples from paraffin-embedded CD117-positive GISTs (60%), comprising 25 deletions and five insertions. Sequencing of 14 MIDIA-positive samples confirmed the deletions/insertions, including two 3-bp alterations. Sequencing of all 20 MIDIA-negative samples also showed highly consistent results with MIDIA because 10 cases were wild type and eight displayed a single base substitution in which detection by MIDIA was not expected. Sequencing also revealed a 3-bp deletion undetected by MIDIA, thus establishing the resolution limit of MIDIA at deletions/insertions >or=3 bp. Denaturing high-pressure liquid chromatography analysis confirmed all mutations detected by MIDIA and sequencing. We pro-pose MIDIA as the first step in mutational screening of GIST because it allowed the detection of 75% of mutated cases (94% of deletions/insertions) in less than 30 minutes after polymerase chain reaction amplification and at a lower cost compared with denaturing high-pressure liquid chromatography and sequencing, which might then be used only for MIDIA-negative cases.
机译：胃肠道间质瘤（GIST）经常在KIT和PDGFRA基因中包含突变，突变的存在和类型与对激酶抑制剂甲磺酸伊马替尼的反应相关。因为大多数GIST突变是缺失/插入，所以我们使用微流控设备检测聚合酶链反应扩增的DNA中的这些大小变化。这种被称为微流体缺失/插入分析（MIDIA）的方法从石蜡包埋的CD117阳性GIST（50％）中鉴定出50个DNA样品中的30个突变（60％），包括25个缺失和5个插入。对14个MIDIA阳性样品的测序证实了缺失/插入，包括两个3 bp的变化。对所有20个MIDIA阴性样本的测序也显示了与MIDIA高度一致的结果，因为10例是野生型，其中8例显示了一个单碱基替代，其中预期不会被MIDIA检测到。测序还揭示了MIDIA未检测到的3 bp缺失，从而在≥3 bp的缺失/插入处建立了MIDIA的分辨率极限。变性高压液相色谱分析证实了MIDIA和测序检测到的所有突变。我们建议将MIDIA作为GIST突变筛选的第一步，因为它允许在聚合酶链反应扩增后不到30分钟的时间内检测到75％的突变病例（94％的缺失/插入），并且成本较低。变性高压液相色谱和测序，然后仅可用于MIDIA阴性情况。

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