片段分析与PCR电泳检测急性髓性白血病患者FLT3 - ITD突变的比较

Q4 Medicine
I. Maslyukova, D. Kurochkin, E. Martynova, V. Bakhtina, T. Subbotina
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引用次数: 0

摘要

背景。AML患者中FLT3-ITD突变的存在作为预后不良的标志,已被纳入ELN 2017风险分层指南。根据预测结果对患者进行分组的主要标准是等位基因比(AR),截断值为0.5:AR值<0.5为低,≥0.5为高。同时,如果确定AR的重要性毋庸置疑,那么关于重复长度和定位信息的价值仍然存在争议。有两种常见的FLT3-ITD筛查方法。第一种方法是更容易获得和更便宜的pCR电泳法,第二种方法是片段分析法,它更昂贵,需要特殊的设备,它不仅可以检测突变和确定重复长度,还可以量化或计算AR.Aim。比较片段分析和pCR电泳在AML患者dNA样本中寻找FLT3-ITD突变的效果。材料和方法。在2020-2022年期间,使用片段分析和pCR电泳分析了在地区临床医院(克拉斯诺亚尔斯克)接受治疗的45名确诊为AML的患者的血液和/或骨髓样本。通过Sanger测序证实和鉴定FLT3-ITD突变。两种方法均发现45例患者中有11例(24.45%)患者存在FLT3-ITD突变。片段分析结果显示,中位重复长度为42.70碱基对(26.01 ~ 99.84碱基对),AR为0.532(0.027 ~ 3.328),等位基因频率(Af)为34.71(2.67 ~ 76.90)%。在一个样品中鉴定出三种不同的过渡段。Sanger测序在11例患者中鉴定出9例突变。片段分析和pCR电泳在分析不同过渡段长度和不同等位基因比例的样品时结果相似。但可以假设,在ITd较小,AR和Af值较低的情况下,使用pCR电泳时,突变等位基因将无法显示,从而可能导致假阴性结果。使用pCR电泳方法的缺点还在于,如果不使用允许确定突变等位基因对应的条带大小和强度的特殊程序,则无法确定对AML风险分层很重要的AR值。因此,对于FLT3-ITD的检测,我们推荐使用片段分析法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Comparison of fragment analysis and PCR electrophoresis methods for the detection of FLT3‑ITD mutations in patients with acute myeloid leukemia
Background. The presence of the FLT3-ITD mutations in patients with AML serves as a marker of poor prognosis, which is included in the ELN 2017 risk stratification guideline. The main criterion for dividing patients into groups according to the predicted outcomes was the allelic ratio (AR) with a cutoff of 0.5: an AR value <0.5 is considered low, and ≥0.5 is considered high. At the same time, if the importance of AR determination is beyond doubt, the value of information about the length of the repeat and localization is still controversial. There are two common approaches for FLT3-ITD screening. The first, more accessible and cheaper method is the method of pCR electrophoresis and the second, more expensive and requiring special equipment, is the fragment analysis method, which allows not only to detect a mutation and determine the repeat length, but also to quantify or calculate AR.Aim. To compare fragment analysis and pCR electrophoresis in the search for the FLT3-ITD mutations in dNA samples from AML patients.Materials and methods. for the period of 2020–2022 fragment analysis and pCR electrophoresis were used to analyze blood and/or bone marrow samples taken from 45 patients with a confirmed diagnosis of AML who were treated at the Regional Clinical Hospital (Krasnoyarsk). Confirmation and identification of the FLT3-ITD mutations was performed by means of Sanger sequencing.Results. both methods revealed the FLT3-ITD mutations in 11 (24.45 %) patients among the 45 patients studied. According to the results of fragment analysis, the median repeat length was 42.70 base pairs (range 26.01–99.84 base pairs), AR was 0.532 (0.027–3.328), and the allelic frequency (Af) was 34.71 (2.67–76.90) %. Three different ITds were identified in one sample. Sanger sequencing identified mutations in 9 of 11 patients.Conclusion. fragment analysis and pCR electrophoresis showed similar results when analyzing samples with different ITd lengths and with different allelic ratios. but it can be assumed that in the case of a small ITd and low AR and Af values, when using pCR electrophoresis, the mutant allele will not be visualized, which can lead to a false negative result. The disadvantage of using the pCR electrophoresis method is also that without the use of special programs that allow determining the size and intensity of the band corresponding to the mutant allele, it is impossible to determine the AR value, which is important for AML risk stratification. Thus, for detection of the FLT3-ITD we recommend using the fragment analysis method.
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