Rapid identification of IDH1 and IDH2 mutations in acute myeloid leukaemia using high resolution melting curve analysis-猫眼课题宝

DOI：

10.1111/j.1365-2141.2010.08423.x

发表时间：

2011-02

期刊：

British Journal of Haematology

影响因子：

6.5

通讯作者：

中科院分区：

医学2区

文献类型：

作者：

S. M. Noordermeer;E. Tönnissen;Inge Vissers;A. V. D. van der Heijden;L. T. van de Locht;P. Deutz‐Terlouw;E. Marijt;J. Jansen;B. A. van der Reijden

白血病复发突变的筛查正变得越来越重要，因为其中许多突变对疾病结局有影响。目前，突变检测主要是基于DNA测序的方法，或者利用等位基因特异的探针进行定量聚合酶链式反应。随着对癌症复发突变的识别越来越多，需要开发快速有效的突变筛查方法。另一种突变检测技术是高分辨率熔融(HRM)分析(Wittwer，2009)。HRM分析是一种快速的单井技术，使用嵌入DNA的荧光饱和染料进行聚合酶链式反应，然后进行熔化曲线分析。由于突变会导致不同的熔化性质，因此与未突变的样品相比，它们会被识别出来。HRM分析中的阳性样本可以随后进行测序，以确定准确的核苷酸变化。最近，在急性髓系白血病(AML)、骨髓增生异常综合征和骨髓增生性疾病中发现了依赖NADP的异柠檬酸脱氢酶基因IDH1和IDH2的突变(Mardis等人，2009；Abbas等人，2010；Chou等人，2010；Green&Beer，2010；Kosmider等人，2010；Marcucci等人，2010；Wagner等人，2010)。我们使用应用生物系统公司的实时聚合酶链式反应平台(美国加利福尼亚州卡尔斯巴德)，研究了HRM分析和直接测序在168例AML患者队列中筛查这些突变的应用。序列分析在32名患者(19%)中检测到IDH1或IDH2突变(表SI和SII)。使用数据S1中描述的方法，也可以通过HRM分析检测突变(所使用的引物组合如表SIII所示)。测试了带有和不带有M13序列的不同引物，显示了IDH1或IDH2突变的不同结果(图1)。使用HRM分析，没有遗漏突变。对于IDH1 R132，HRM和序列分析都发现了12个突变样本(7？1%)，另有3个阳性样本(1？8%)仅通过HRM分析发现。为了阐明HRM分析发现的额外突变是否是假阳性，我们比较了两种技术的敏感度。为此，突变样本的基因组DNA稀释序列通过HRM分析和直接测序进行了测量。HRM分析中的阳性丢失的等位基因频率约为9%，而通过测序仍可检测到4%的突变(图S1)。由于测序更敏感，这表明HRM分析发现的不包含任何SNP的额外阳性样本代表假阳性结果。序列分析发现19例IDH2 R140突变阳性(11.3%)。对该突变的初步HRM分析发现了许多假阳性结果。然而，排除低聚合酶链式反应产量的样本(Ct&gt；26？8，8个样本)显著改善了突变和野生型样本之间的区分。排除后，所有测序阳性标本均经HRM分析确认，但仍有2例假阳性。HRM和序列分析均发现2例患者存在R172个IDH2突变(1？2%)，HRM发现2例假阳性(1？2%)(表SI和SII)。1例患者同时显示IDH1R132C和IDH2R140Q突变。用焦磷酸测序法测定突变的等位基因频率(引物组合如表SIV所示)。IDH1R132C突变的等位基因频率为15.5%，而IDH2R140Q突变的等位基因频率更低(数据未显示)。由于IDH1和IDH2突变是典型的杂合性突变，并且两个突变的总等位基因频率都低于50%，因此我们无法断定这两个突变是代表一个相同的克隆还是独立的克隆。1例患者表现出IDH2 R140W纯合子突变，经序列和HRM分析证实(图S2)。SNP阵列分析表明，该纯合子突变是由15号染色体大部分区域的单亲二体(UPD)引起的，包括IDH2基因座(图S3)。在32名IDH突变患者中，我们发现与其他染色体异常的共存证实了其他研究的数据(Chou等人，2010；Kosmider等人，2010；Marcucci等人，2010；Paschka等人，2010；Wagner等人，2010)。此外，IDH1或IDH2突变患者中没有一例显示Mecom(也称为EVI1)过表达(表SI)。使用测序或HRM分析可能会遗漏低频率的突变等位基因。为了测试IDH1突变的发生频率是否低于HRM分析的检测下限，我们设计了针对最常见的IDH1 R132H突变的等位基因特异性QPCR(用于引物

Screening for recurrent mutations in leukaemia is becoming increasingly important because many of them have an impact on disease outcome. Nowadays, mutational detection is mostly based on DNA sequencing of PCR products or QPCR using allele specific probes. With the increasing identification of recurrent mutations in cancer, the development of fast and efficient approaches for mutational screening is needed. An alternative technique for mutation detection is high resolution melting (HRM) analysis (Wittwer, 2009). HRM analysis is a fast ‘single-well-technique’ combining PCR using a fluorescent saturating dye that is intercalated in the DNA, and melt curve analysis afterwards. As mutations cause different melting properties, they are identified when compared to non-mutated samples. Positive samples in HRM analysis can be sequenced subsequently to determine exact nucleotide changes. Recently, mutations in the NADP-dependent isocitrate dehydrogenase genes IDH1 and IDH2 were shown in acute myeloid leukaemia (AML), myeloid dysplastic syndromes and myeloproliferative diseases (Mardis et al, 2009; Abbas et al, 2010; Chou et al, 2010; Green & Beer, 2010; Kosmider et al, 2010; Marcucci et al, 2010; Wagner et al, 2010). We study the application of HRM analysis and direct sequencing to screen for these mutations in a cohort of 168 AML patients, using the real-time PCR platform of Applied Biosystems (Carlsbad, CA, USA). Sequence analysis detected IDH1 or IDH2 mutations in 32 patients (19%) (Tables SI and SII). Using the methods as described in the Data S1, mutations could also be detected by HRM analysis (used primer combinations are indicated in Table SIII). Different primers with and without M13sequences were tested, showing differential results for IDH1 or IDH2 mutations (Fig 1). Using HRM analysis, no mutations were missed. For IDH1 R132, 12 mutated samples (7Æ1%) were found both by HRM and sequence analysis while an additional three positive samples (1Æ8%) were found only by HRM analysis. To elucidate whether the extra mutations found by HRM analysis were false positives, we compared the sensitivity of both techniques. For this, a dilution series of genomic DNA from a mutated sample was measured by HRM analysis followed by direct sequencing. Positivity in HRM analysis was lost at an allele frequency of approximately 9%, whereas the mutation could still be detected at a percentage of 4% by sequencing (Fig S1). As sequencing was more sensitive, this suggests that the extra positive samples found by HRM analysis, that did not contain any SNPs, represent false positive results. Nineteen samples were found positive for the IDH2 R140 mutation (11Æ3%) by sequence analysis. Initial HRM analysis for this mutation identified many false positive results. However, exclusion of samples with low PCR yields (Ct>26Æ8, eight samples) significantly improved the discrimination between mutated and wild type samples. After exclusion, all positive samples found by sequencing were confirmed by HRM analysis while two cases remained false positive. R172 IDH2 mutations were found in two patients (1Æ2%) both by HRM and sequence analysis and two false positive samples were found by HRM (1Æ2%) (Tables SI and SII). One patient showed both an IDH1 R132C and IDH2 R140Q mutation. Allele frequencies of the mutations were measured by pyrosequencing (primer combinations are specified in Table SIV). The allele frequency of the IDH1 R132C mutation was 15Æ5%, while the IDH2 R140Q frequency was even lower (data not shown). Because IDH1 and IDH2 mutations are typically heterozygous and the total allele frequencies of both mutations was below 50%, we cannot conclude whether the mutations represent one and the same or independent clones. One patient exhibited a homozygous IDH2 R140W mutation, identified by both sequence and HRM analysis (Fig S2). SNP array analysis showed that the homozygous mutation was caused by uniparental disomy (UPD) on a large part of chromosome 15, including the IDH2 locus (Fig S3). Of the 32 patients with IDH mutations, we found cooccurrences with other chromosomal aberrations confirming data of other studies (Chou et al, 2010; Kosmider et al, 2010; Marcucci et al, 2010; Paschka et al, 2010; Wagner et al, 2010). Furthermore, none of the IDH1 or IDH2 mutated patients showed overexpression of MECOM (also known as EVI1) (Table SI). Low frequencies of mutated alleles may be missed using sequencing or HRM analysis. To test whether IDH1 mutations occur at frequencies under the detection limit of HRM analysis, we designed an allele specific QPCR for the most frequently occurring IDH1 R132H mutation (for primer

Rapid identification of IDH1 and IDH2 mutations in acute myeloid leukaemia using high resolution melting curve analysis

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