Graduate Institute of Oncology, National Taiwan University College of Medicine, Taipei, Taiwan; Division of Hematology and Oncology, Department of Internal Medicine, Mackay Memorial Hospital, Taipei, Taiwan; Laboratory of Good Clinical Research Center, Department of Medical Research, Mackay Memorial Hospital, Tamsui District, New Taipei City, Taiwan; Department of Medicine, Mackay Medical College, New Taipei City, Taiwan.
Division of Hematology and Oncology, Department of Internal Medicine, Mackay Memorial Hospital, Taipei, Taiwan; Laboratory of Good Clinical Research Center, Department of Medical Research, Mackay Memorial Hospital, Tamsui District, New Taipei City, Taiwan.
Clin Chim Acta. 2015 Feb 2;440:133-9. doi: 10.1016/j.cca.2014.11.011. Epub 2014 Nov 15.
Somatic CALR exon 9 mutations have recently been identified in patients with JAK2/MPL-unmutated myeloproliferative neoplasm, and have become an important clonal marker for the diagnosis of essential thrombocythemia (ET) and primary myelofibrosis. In the present study, we sought to use high-resolution melting analysis (HRMA) as a screening method for the detection of CALR mutations.
32 JAK2/MPL-unmutated ET patients were retrospectively enrolled and 8 healthy adults were used as wild-type control. CALR exon 9 mutation was independently screened by HRMA with the CFX Connect real-time system and Sanger sequencing. TA-cloning was used to detect CALR exon 9 mutations in patients suspected to have low mutant allele burden.
The maximal sensitivity of HRMA in identifying both CALR type 1 and type 2 mutants from patients' genomic DNA was 2.5%. Twenty-two samples were found to have distinct melting curves from wild-type. The presence of CALR mutations in 16 of these 22 samples was confirmed by Sanger sequencing, while the other 6 samples were wild-type by sequencing. After TA-cloning, CALR mutations were detected in 5 of 6 patients from 1 (6%) of 16 clones to 1 (2%) of 50 clones. Therefore, HRMA identified CALR mutations in 21 (65.6%) of 32 ET patients compared to 16 (50%) patients by Sanger sequencing, with a false positive rate of 3% and no false negative.
The HRMA developed in our system is a rapid and sensitive technique for the detection of CALR exon 9 mutations.
最近在 JAK2/MPL 未突变的骨髓增殖性肿瘤患者中发现了体细胞 CALR 外显子 9 突变，并且已成为诊断原发性血小板增多症（ET）和原发性骨髓纤维化的重要克隆标志物。在本研究中，我们试图使用高分辨率熔解分析（HRMA）作为检测 CALR 突变的筛选方法。
回顾性纳入 32 例 JAK2/MPL 未突变的 ET 患者，8 例健康成年人作为野生型对照。通过 CFX Connect 实时系统和 Sanger 测序，使用 HRMA 独立筛选 CALR 外显子 9 突变。TA 克隆用于检测疑似低突变等位基因负担的患者的 CALR 外显子 9 突变。
HRMA 从患者基因组 DNA 中识别 CALR 1 型和 2 型突变体的最大灵敏度为 2.5%。22 个样本与野生型具有明显不同的熔解曲线。22 个样本中有 16 个通过 Sanger 测序证实存在 CALR 突变，而另外 6 个样本通过测序为野生型。经过 TA 克隆，在 6 例患者中的 5 例（6%）的 16 个克隆中检测到 1 个（2%）的 50 个克隆存在 CALR 突变。因此，与 Sanger 测序相比，HRMA 在 32 例 ET 患者中鉴定出 21 例（65.6%）CALR 突变，假阳性率为 3%，无假阴性。
我们的系统开发的 HRMA 是一种快速、敏感的 CALR 外显子 9 突变检测技术。
