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Current Signal Transduction Therapy


ISSN (Print): 1574-3624
ISSN (Online): 2212-389X

Research Article

The Effect of DNA Concentration on the HRM Performance in Detecting Jak2 p.V617F Variant in Patients with Myeloproliferative Neoplasms

Author(s): Atefeh Soltani, Aref Shariati, Mojtaba Didehdar, Ali Arash Anoushirvani, Seidamir Pasha Tabaeian and Alireza Moradabadi*

Volume 18, Issue 2, 2023

Published on: 31 July, 2023

Article ID: e090623217831 Pages: 7

DOI: 10.2174/1574362418666230609112551

Price: $65


Introduction: Janus kinase 2 (JAK2) is an intracellular signaling protein. JAK2 p. V617F is a common variant in normal karyotype myeloproliferative neoplasms (MPN). Highresolution melting (HRM) analysis is one of the essential methods for detecting the JAK2 p.V617F variant. In this study, we have investigated the effect of DNA concentration on detecting the JAK2 p.V617F variant using the HRM method.

Methods: Genomic DNA was extracted from human blood and diluted ten times in distilled water from 1 to 0.03; afterward, HRM was conducted for each dilution (triplicate). Using SPSS v.20.0 software, the mean Tm of each dilution was calculated and compared.

Results: The HRM results revealed the JAK2 wild type and variant to have Tms of 81/64°C and 80/76°C, respectively. At the endpoint of the pre-amplification, the dilutions had different emissions. The statistical analysis revealed no statistically significant differences in Tm between samples with varying DNA concentrations (P value > 0.05).

Conclusion: There have been no significant differences obtained in the analysis of JAK2 p.V617F point variant in different DNA dilutions, implying that the HRM analysis has no relation to DNA concentration.

Keywords: JAK2 p.V617F, HRM, DNA concentration, genomic, karyotype myeloproliferative neoplasms, pre-amplification.

Graphical Abstract
Socoro-Yuste N, de Peredo AG, Dagher M, Mondet J, Zaccaria A, Court M, et al. Ph(-) myeloproliferative neoplasm red blood cells display deregulation of IQGAP1-Rho GTPase signaling depending on CALR/JAK2 status. Biochim Biophys Acta 2016; 1863(11): 2758-65.
Milosevic Feenstra JD, Nivarthi H, Gisslinger H, et al. Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms. Blood 2016; 127(3): 325-32.
[] [PMID: 26423830]
Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005; 365(9464): 1054-61.
[] [PMID: 15781101]
Zhao R, Xing S, Li Z, et al. Identification of an acquired JAK2 mutation in polycythemia vera. J Biol Chem 2005; 280(24): 22788-92.
[] [PMID: 15863514]
Levine RL, Wadleigh M, Cools J, et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005; 7(4): 387-97.
[] [PMID: 15837627]
Kralovics R, Passamonti F, Buser AS, Teo S-S, Tiedt R, Passweg JR, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. NEMJ 2005; 352(17): 1779-90.
Er TK, Lin SF, Chang JG, et al. Detection of the JAK2 V617F missense mutation by high resolution melting analysis and its validation. Clin Chim Acta 2009; 408(1-2): 39-44.
[] [PMID: 19595684]
Moradabadi A, Farsinejad A, Khansarinejad B, Fatemi A. Development of a high resolution melting analysis assay for rapid identification of JAK2 V617F missense mutation and its validation. Exp Hematol Oncol 2019; 8(1): 10.
[] [PMID: 31165012]
Trifa AP, Popp RA, Cucuianu A, Dima D, Militaru MS, Paţiu M. JAK2 p. V617F mutation–tetra-primer PCR and PCR-RFLP comparative semiquantitative approaches for estimation of the mutant allele in myeloproliferative neoplasms. Revista Română de Medicină de Laborator 2009; 14(1)
Zhao C, Xie S, Wu H, et al. Quantification of allelic differential expression using a simple Fluorescence primer PCR-RFLP-based method. Sci Rep 2019; 9(1): 6334.
[] [PMID: 31004110]
Moradabadi AR, Farsinejad AR, Fatemi A. Modified PCR-RFLP for detection of JAK2V617F mutation in patients with myeloproliferative neoplasm. Sci J Iran Blood Transfus Organ 2017; 14(4): 289-94.
Di Francia R, Crisci S, Muto T, et al. Optimization of a low-cost, sensitive PNA clamping PCR method for JAK2 V617F variant detection. J Appl Lab Med 2020; 5(4): 643-55.
[] [PMID: 32407536]
Zaka Khosravi S, Moonesi M, Moradabadi A, Rajaeinejad M, Heidari MF, Noroozi-Aghideh A. Rapid detection of N-RAS gene common mutations in Acute Myeloid Leukemia (AML) using High Resolution Melting (HRM) method. Asian Pac J Cancer Prev 2022; 23(1): 125-30.
[] [PMID: 35092380]
Moonesi M, Zaka Khosravi S, Moradabadi A, et al. Comparison of High-Resolution Melting (HRM) analysis with direct sequencing for the detection of DNMT3A mutations in AML patients. Asian Pac J Cancer Prev 2022; 23(7): 2185-90.
[] [PMID: 35901322]
Mahmoudi A, Moradabadi A, Noroozi-aghideh A. Comparison of high-resolution melting analysis with direct sequencing for detection of FLT3-TKD, FLT3-ITD and WT1 mutations in acute myeloid leukemia. Cancer Treat Res Commun 2021; 28: 100432.
[] [PMID: 34303121]
Tahmasebi H, Dehbashi S, Arabestani MR. High resolution melting curve analysis method for detecting of carbapenemases producing pseudomonas aeruginosa. J Krishna Inst Med Sci Uni 2018; 7(4): 70-7.
Federici MTT, Branda Sica A, Artigas R, Briano C, Dutra F. Llambí S. High- resolution melting (HRM) curve analysis: New approach used to detect blad and dumps in uruguayan holstein breed. Arch Vet Sci 2018; 23(4)
Ritort F. Open questions about DNA melting. Phys Life Rev 2018; 25: 34-6.
[] [PMID: 29555152]
Vologodskii A, Frank-Kamenetskii MD. DNA melting and energetics of the double helix. Phys Life Rev 2018; 25: 1-21.
[] [PMID: 29170011]
Heideman DAM, Thunnissen FB, Doeleman M, et al. A panel of high resolution melting (HRM) technology-based assays with direct sequencing possibility for effective mutation screening of EGFR and K-ras genes. Anal Cell Pathol 2009; 31(5): 329-33.
[] [PMID: 19759413]
Milbury CA, Li J, Makrigiorgos GM. COLD-PCR-enhanced high-resolution melting enables rapid and selective identification of low-level unknown mutations. Clin Chem 2009; 55(12): 2130-43.
[] [PMID: 19815609]
Bilbao-Sieyro C, Santana G, Moreno M, et al. High resolution melting analysis: A rapid and accurate method to detect CALR mutations. PLoS One 2014; 9(7): e103511.
[] [PMID: 25068507]
Moradabadi A, Fatemi A, Noroozi-aghideh A. Analysis of the reannealing- instead of melting-curve in the detection of JAK2 V617F mutation by HRM method. J Blood Med 2019; 10: 235-41.
[] [PMID: 31413649]
Ebili H, Ilyas M. High resolution melt analysis, DNA template quantity disparities and result reliability. Clin Lab 2015; 61(01+02/2015): 155-9.
[] [PMID: 25807649]
Carillo S, Henry L, Lippert E, et al. Nested high-resolution melting curve analysis a highly sensitive, reliable, and simple method for detection of JAK2 exon 12 mutations-clinical relevance in the monitoring of polycythemia. J Mol Diagn 2011; 13(3): 263-70.
[] [PMID: 21497288]
Lin CY, Ho CM, Tamamyan G, Yang SF, Peng CT, Chang JG. Validating the sensitivity of high-resolution melting analysis for JAK2 V617F mutation in the clinical setting. J Clin Lab Anal 2016; 30(6): 838-44.
[] [PMID: 27169616]

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