Centre for Genetic Consultation and Cancer Screening, 108 Institute of Clinical Medical and Pharmaceutical Sciences, Hanoi, Vietnam.
Vietnamese - German Center for Medical Research, 108 Institute of Clinical Medical and Pharmaceutical Sciences, No 1, Tran Hung Dao Street, Hai Ba Trung District, Hanoi, Vietnam.
BMC Med Genet. 2019 Jun 27;20(1):115. doi: 10.1186/s12881-019-0819-6.
Calreticulin (CALR) gene mutations are currently recommended as biomarkers in diagnosis of patients with myeloproliferative neoplasms (MPN) with Jak2 V617F negative phenotype. Our aim was to establish a rapid, low cost and sensitive assay for identification of CALR gene mutations and to validate the diagnostic performance of the established assay in a patient cohort with different clinical MPN phenotypes.
One hundred five Philadelphia-negative MPN patients, including polycythemia vera (PV), essential thrombocythaemia (ET), and primary myelofibrosis (PMF) were initially screened for JAK2 mutations by amplification-refractory mutation system (ARMS-PCR) methodology and were further subjected to detection of CALR gene mutations by our in-house assay, a PCR based amplicon length differentiation assay (PCR-ALDA). The PCR-ALDA methodology was compared with real time PCR and Sanger sequencing methods. Furthermore, the analytical sensitivity of the assay was established.
PCR - ALDA approach was able to detect and discriminate the pseudo-positive samples containing more than 1% CALR mutant alleles. CALR mutations were not detected in 63 Jak2 V617F positive cases in all three methods. In contrast, amongst 42 Jak2 V617F negative cases, both PCR-ALDA and Sanger sequencing coherently identified 12 CALR mutants compared to 10 CALR mutants detected by real-time PCR method.
PCR-ALDA can be utilized as an easy-to-use, rapid, low cost and sensitive tool in the detection of CALR mutations in Philadelphia-negative MPN patients.
钙网织蛋白（CALR）基因突变目前被推荐作为 Jak2 V617F 阴性表型骨髓增殖性肿瘤（MPN）患者诊断的生物标志物。我们的目的是建立一种快速、低成本和敏感的方法来鉴定 CALR 基因突变，并在具有不同临床 MPN 表型的患者队列中验证该方法的诊断性能。
105 例费城阴性 MPN 患者，包括真性红细胞增多症（PV）、特发性血小板增多症（ET）和原发性骨髓纤维化（PMF），最初通过扩增受阻突变系统（ARMS-PCR）方法筛选 JAK2 突变，然后通过我们的内部检测方法，即基于 PCR 的扩增子长度差异检测（PCR-ALDA）检测 CALR 基因突变。将 PCR-ALDA 方法与实时 PCR 和 Sanger 测序方法进行比较。此外，还确定了该方法的分析灵敏度。
PCR-ALDA 方法能够检测和区分含有 1%以上 CALR 突变等位基因的假阳性样本。在所有三种方法中，均未在 63 例 Jak2 V617F 阳性病例中检测到 CALR 突变。相比之下，在 42 例 Jak2 V617F 阴性病例中，PCR-ALDA 和 Sanger 测序均一致地鉴定出 12 个 CALR 突变，而实时 PCR 方法仅检测到 10 个 CALR 突变。
PCR-ALDA 可作为一种易于使用、快速、低成本和敏感的工具，用于检测费城阴性 MPN 患者的 CALR 突变。
