Detection of MPL Mutations by a Novel Allele-Specific PCR-Based Strategy
Detection of MPL Mutations by a Novel Allele-Specific PCR-Based Strategy
复制标题
DOI：
10.1016/j.jmoldx.2013.07.006
复制
发表时间：
2013-11-01
影响因子：
4.1
通讯作者：
Betz, Bryan L.
中科院分区：
文献类型：
--
作者：
Furtado, Larissa V.;Weigelin, Helmut C.;Betz, Bryan L.
MPL突变检测推荐用于缺乏JAK2 V617F突变的疑似原发性骨髓纤维化或原发性血小板增多症患者。MPL突变可发生在等位基因水平低于15%，这可能逃脱常用的突变筛查方法，如桑格测序。我们发展了一种新的多重等位基因特异性聚合酶链式反应方法，能够检测与原发性骨髓纤维化和原发性血小板增多症(W515L、W515K、W515A和S505N)相关的大多数复发的MPL外显子10突变，敏感度低至2.5%的突变等位基因。对15个参考病例和1380个连续样本的检测结果进行了回顾，这些样本被转至我室进行检测。对一系列58例MPL突变样本的检测性能与Sanger测序进行了比较。W515L阳性45例，S505N阳性6例，W515K阳性5例，W515A阳性1例，W515L和S505N同时阳性1例。有7例突变低于5%，Sanger测序未检测到。另外10例的突变水平在5%到15%之间，测序没有一致地检测到这些突变。所有结果在等位基因特异性检验中都很容易解释。该方法为MPL基因突变检测提供了一种灵敏可靠的解决方案。Sanger测序似乎不够灵敏，无法进行可靠的MPL突变检测。我们的数据还表明，S505N突变的相对频率可能被低估了，这突显了在MPL测试平台中包括这种突变的必要性。
MPL mutation testing is recommended in patients with suspected primary myelofibrosis or essential thrombocythemia who lack the JAK2 V617F mutation. MPL mutations can occur at allelic levels below 15%, which may escape detection by commonly used mutation screening methods such as Sanger sequencing. We developed a novel multiplexed allele-specific PCR assay capable of detecting most recurrent MPL exon 10 mutations associated with primary myelofibrosis and essential thrombocythemia (W515L, W515K, W515A, and S505N) down to a sensitivity of 2.5% mutant allele. Test results were reviewed from 15 reference cases and 1380 consecutive specimens referred to our laboratory for testing. Assay performance was compared to Sanger sequencing across a series of 58 specimens with MPL mutations. Positive cases consisted of 45 with W515L, 6 with S505N, 5 with W515K, 1 with W515A, and 1 with both W515L and S505N. Seven cases had mutations below 5% that were undetected by Sanger sequencing. Ten additional cases had mutation levels between 5% and 15% that were not consistently detected by sequencing. ALL results were easily interpreted in the allele-specific test. This assay offers a sensitive and reliable solution for MPL mutation testing. Sanger sequencing appears insufficiently sensitive for robust MPL mutation detection. Our data also suggest the relative frequency of S505N mutations may be underestimated, highlighting the necessity for inclusion of this mutation in MPL test platforms.