PCR-HRM for Detecting JAK2V617F Gene Mutation: Is It a Sensitive Assay?
-
Mitra Rezaei
,
Mihan PourAbdollah Toutkaboni
,
Babak Salimi
,
Sharareh Seifi
,
Fatemeh Maryam Sheikholeslami
Abstract
Background: A substitution of G to T at nucleotide 1849 in exon 14 of the Janus kinase2 (JAK2) gene is recognized to MPNs disorders. Based on WHO guidelines detection of the mutation is very important to confirm the disease in the suspected patients. Methods: Eighty-seven patients with different background diseases were tested for JAK2 V617F mutation by four different methods, including polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP), amplification refractory mutation system (ARMS), polymerase chain reaction-high resolution melting(PCR-HRM), and two different commercial kits. Results: The mean age of patients was 53.38±17.43 years, 72.4% were males, and 37.6% were females. JAK2 mutation was detected in 16 patients (18.3%). Of those, 7 (43.75%) suffered from PV, 5 (31.25%) from ET, 3 (18.75%) from PMF, and 1 (6.15%) from unclassified neoplastic disorders. The frequency of JAK2 mutationwas 71.4% (5/7) in PV, 80% (4/5) in ET, and 66.7% (2/3) in PMF patients. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and GE of PCR-HRM for detection of the JAK2 mutation was 86.7%, 100%, 100%, 97.3%, and 97.7%, respectively. While the sensitivity, specificity, PPV, NPV, andGE of PCR-RFLP were 93.3%, 80.5%, 50%, 98.3%, and 82.7%, respectively. On the other hand, the sensitivity, specificity, PPV, NPV, and GE of ARMS assays were evaluated by about 80%, 96%, 100%, 96%, and 96.5%, respectively. Conclusion: This study showed that PCR-HRM was a more sensitive assay to detect the JAK2 V617F mutation than the other assays. So, it can be used as a quick, easy, and effective method for screening the JAK2 V617F mutation in patients with MPNs disorders. PCR-RFLP must accompany it as a gold standard method for confirmation of the mutation of JAK2 V617F.
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12. Frantz C, Sekora DM, Henley DC, Huang CK, Pan Q, Quigley NB, et al. Comparative evaluation of three JAK2V617F mutation detection methods. American journal of clinical pathology. 2007;128(5):865-74.
13. Lay M, Mariappan R, Gotlib J, Dietz L, Sebastian S, Schrijver I, et al. Detection of the JAK2 V617F mutation by LightCycler PCR and probe dissociation analysis. The Journal of molecular diagnostics : JMD. 2006;8(3):330-4.
14. Vannucchi AM, Pancrazzi A, Bogani C, Antonioli E, Guglielmelli P. A quantitative assay for JAK2(V617F) mutation in myeloproliferative disorders by ARMS-PCR and capillary electrophoresis. Leukemia. 2006;20(6):1055-60.
15. 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. Experimental hematology & oncology. 2019;8:10.
16. Cankovic M, Whiteley L, Hawley RC, Zarbo RJ, Chitale D. Clinical performance of JAK2 V617F mutation detection assays in a molecular diagnostics laboratory: evaluation of screening and quantitation methods. American journal of clinical pathology. 2009;132(5):713-21.
17. McClure R, Mai M, Lasho T. Validation of two clinically useful assays for evaluation of JAK2 V617F mutation in chronic myeloproliferative disorders. Leukemia. 2006;20(1):168-71.
18. Warshawsky I, Mularo F, Hren C, Jakubowski M. Failure of the Ipsogen MutaScreen kit to detect the JAK2 617V>F mutation in samples with additional rare exon 14 mutations: implications for clinical testing and report of a novel 618C>F mutation in addition to 617V>F. Blood. 2010;115(15):3175-6.
2. Er TK, Lin SF, Chang JG, Hsieh LL, Lin SK, Wang LH, et al. Detection of the JAK2 V617F missense mutation by high resolution melting analysis and its validation. Clinica chimica acta; international journal of clinical chemistry. 2009;408(1-2):39-44.
3. Zhao R, Xing S, Li Z, Fu X, Li Q, Krantz SB, et al. Identification of an acquired JAK2 mutation in polycythemia vera. The Journal of biological chemistry. 2005;280(24):22788-92.
4. James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature. 2005;434(7037):1144-8.
5. Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, 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.
6. Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. The New England journal of medicine. 2005;352(17):1779-90.
7. Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 2005;365(9464):1054-61.
8. Tefferi A, Thiele J, Orazi A, Kvasnicka HM, Barbui T, Hanson CA, et al. Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc international expert panel. Blood. 2007;110(4):1092-7.
9. Jones AV, Kreil S, Zoi K, Waghorn K, Curtis C, Zhang L, et al. Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood. 2005;106(6):2162-8.
10. Melzner I, Weniger MA, Menz CK, Moller P. Absence of the JAK2 V617F activating mutation in classical Hodgkin lymphoma and primary mediastinal B-cell lymphoma. Leukemia. 2006;20(1):157-8.
11. Olsen RJ, Tang Z, Farkas DH, Bernard DW, Zu Y, Chang CC. Detection of the JAK2(V617F) mutation in myeloproliferative disorders by melting curve analysis using the LightCycler system. Archives of pathology & laboratory medicine. 2006;130(7):997-1003.
12. Frantz C, Sekora DM, Henley DC, Huang CK, Pan Q, Quigley NB, et al. Comparative evaluation of three JAK2V617F mutation detection methods. American journal of clinical pathology. 2007;128(5):865-74.
13. Lay M, Mariappan R, Gotlib J, Dietz L, Sebastian S, Schrijver I, et al. Detection of the JAK2 V617F mutation by LightCycler PCR and probe dissociation analysis. The Journal of molecular diagnostics : JMD. 2006;8(3):330-4.
14. Vannucchi AM, Pancrazzi A, Bogani C, Antonioli E, Guglielmelli P. A quantitative assay for JAK2(V617F) mutation in myeloproliferative disorders by ARMS-PCR and capillary electrophoresis. Leukemia. 2006;20(6):1055-60.
15. 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. Experimental hematology & oncology. 2019;8:10.
16. Cankovic M, Whiteley L, Hawley RC, Zarbo RJ, Chitale D. Clinical performance of JAK2 V617F mutation detection assays in a molecular diagnostics laboratory: evaluation of screening and quantitation methods. American journal of clinical pathology. 2009;132(5):713-21.
17. McClure R, Mai M, Lasho T. Validation of two clinically useful assays for evaluation of JAK2 V617F mutation in chronic myeloproliferative disorders. Leukemia. 2006;20(1):168-71.
18. Warshawsky I, Mularo F, Hren C, Jakubowski M. Failure of the Ipsogen MutaScreen kit to detect the JAK2 617V>F mutation in samples with additional rare exon 14 mutations: implications for clinical testing and report of a novel 618C>F mutation in addition to 617V>F. Blood. 2010;115(15):3175-6.
| Files | ||
| Issue | Vol 11 No 4 (2019) | |
| Section | Original Articles | |
| DOI | https://doi.org/10.18502/bccr.v11i4.5722 | |
| Keywords | ||
| JAK2 V617F mutation PCR-RFLP MPNs disorders Myeloproliferative Neoplastic Disorders | ||
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This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Rezaei M, PourAbdollah Toutkaboni M, Salimi B, Seifi S, Sheikholeslami FM. PCR-HRM for Detecting JAK2V617F Gene Mutation: Is It a Sensitive Assay?. Basic Clin Cancer Res. 2020;11(4):173-181.
