Rapid detection of JAK2 V617F mutation using high-resolution melting analysis with LightScanner platform
ABSTRACT Detection of the JAK2 mutation has recently been included under the essential diagnostic criteria for myeloproliferative neoplasm (MPN). High-resolution melt (HRM) curve analysis, a nongel-based, automated system, is introduced as a means of mutation scanning without the requirement of any post-PCR handling.
We studied the sensitivity and reproducibility of LightScanner™ platform in the detection of JAK2 V617F mutation and the availability for diagnostic use in MPN.
The reproducible sensitivity of HRM analysis with LightScanner™ platform was 5% for the detection of JAK2 V617F mutation. In the test of blind screening of 105 samples (48 Ph- MPN and 57 Ph+ chronic myeloid leukemia), the identical judgement was interpreted by two blinded investigators. HRM analysis of all cases was fully concordant with the results of PCR-RFLP and direct sequencing.
The HRM method developed here is an extremely sensitive, accurate and reliable technique and allows high-throughput, fast pre-screening to select for sequencing only those specimens that most likely contain mutant JAK2 V617F allele(s).
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ABSTRACT: Hotspot mutations of serine/arginine-rich splicing factor 2 (SRSF2) gene have been identified in a proportion of hematologic malignancies including myelodysplastic syndrome (MDS). The aim of the present study was to develop a new approach to screen SRSF2 mutation and analyze the clinical relevance of SRSF2 mutations in Chinese MDS. A protocol based on high-resolution melting analysis (HRMA) was established to screen SRSF2-P95 mutation in 108 MDS patients and was compared with Sanger sequencing. The clinical relevance of SRSF2 mutations was further evaluated. HRMA identified five (4.6%) cases with SRSF2 mutation, completely validated by Sanger sequencing without false positive or negative results. The sensitivities of HRMA and Sanger sequencing were 10% and 25% for the detection of SRSF2-P95H mutation, respectively, against the background of wild-type DNA. Patients with SRSF2 mutation had shorter overall survival time than those with wild-type SRSF2 in both the whole cohort of cases and those with normal karyotype (P = 0.069 and 0.023, respectively). Multivariate analysis confirmed SRSF2 mutation as an independent risk factor in both patient populations. We established a fast, high-throughput, and inexpensive HRMA-based method to screen SRSF2 mutation, which could be used in clinical diagnostic laboratories. SRSF2 mutations were significantly associated with mortality rate in the MDS affected Chinese.PLoS ONE 12/2014; 9(12):e115693. DOI:10.1371/journal.pone.0115693 · 3.53 Impact Factor
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ABSTRACT: 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. Copyright © 2014 Elsevier B.V. All rights reserved.Clinica Chimica Acta 11/2014; 440C:133-139. DOI:10.1016/j.cca.2014.11.011 · 2.76 Impact Factor
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ABSTRACT: A multiplex snapback primer system was developed for the simultaneous detection of JAK2 V617F and MPL W515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets for JAK2 and MPL mutation enrichment and detection, respectively. Linear-After exponential (LATE) PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS), quantitative PCR (qPCR) and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process.03/2014; 2014:458457. DOI:10.1155/2014/458457