Comparison of Sanger sequencing and next generation sequencing methods for investigation of JAK2 Exon 12 mutations in follow-up of patients with chronic myeloproliferative disease and JAK2 V617F non-mutation

Year 2024, Volume: 33 Issue: 2, 67 - 80, 25.12.2024

Mustafa Arslan , Büşranur Çavdarlı , Vehap Topçu , Hakkı Taştan

https://doi.org/10.53447/communc.1407463

Abstract

Myeloproliferative neoplasms (MPNs) are clonal disorders of hematopoietic stem cells with increased proliferation and efficient maturation of myeloid cells, leading to peripheral blood leukocytosis and excess erythrocytes or platelets. Mutations of the JAK2 V617F, CALR and MPL genes confirm the diagnosis of myeloproliferative neoplasm (MPN). Mutations in JAK2 have been identified in the majority of patients with PV, ET and PM, highlighting the importance of constitutive activation of JAK2 signaling induced by mutations. In our study, Sanger Sequencing and Next Generation Sequencing methods were used to search for JAK2 Exon 12 mutations in 100 individuals who suffered from Chronic Myeloproliferative Disease and did not have JAK2 V617F mutation by Real-Time PCR method, and the results were examined comparatively. The examination was made with DNA material isolated from peripheral blood samples taken from patients who were referred to Ankara Numune Training and Research Hospital (ANEAH) Genetic Diseases Diagnosis Center. First of all, individuals who have with negative JAK2 V617F RT-PCR test results were selected. PCR was performed by adjusting sufficient amounts and concentrations from the DNA samples obtained from the peripheral blood of these patients. After the PCR process, the JAK2 Exon 12 regions were sequenced and examined using the Sanger sequencing method. A Next Generation Sequencing (NGS) study was performed by creating libraries from the DNA of the patients whose JAK2 Exon 12 region was negative, and the results were analyzed using the database. Some of the studies were conducted at the ANEAH Genetic Diseases Diagnostic Center, and the other 46 patients were performed at the Intergen Genetic Diseases Diagnosis Center within the scope of NGS study service procurement. According to the analyzes made, the results of Sanger Sequencing and Next Generation Sequencing studies showed similarity. Despite the deep bottom readings, a different result could not be obtained from the Sanger Sequencing method in the NGS study.

Keywords

Chronic myeloproliferative disease, Sanger sequencing, next generation sequencing, Janus Kinas 2, Real-Time PCR

Supporting Institution

Gazi University

Project Number

5850

Thanks

The study was supported by Gazi University Scientific Research Projects Coordination Unit with ID number 5850.

References

• Tiziano, B., Jürgen, T., Heinz, G., The 2016 WHO classification and diagnostic criteria for myeloproliferative neoplasms: document summary and in-depth discussion, Blood Cancer Journal, 8 (15) (2018), 15-21. https://doi:10.1038/s41408-018-0054
• Zulkeflee, R. H., Zulkafli, Z., Johan, M. F., Husin, A., Islam, A., Hassan, R., Clinical and laboratory features of JAK2 V617F, CALR, and MPL mutations in Malaysian patients with classical Myeloproliferative Neoplasm (MPN), International Journal of Environmental Research and Public Health, 18 (14) (2021), 7582. https://doi:10.3390/ijerph18147582
• Tefferi, A., Vardiman, J. W., Classification and diagnosis of myeloproliferative neoplasms: The 2008 World Health Organization criteria and point-of-care diagnostic algorithms, Leukemia, 22 (1) (2008), 14-22. https://doi:10.1038/sj.leu.2404955
• Üstek, D., Abacı, N., Sırma, S., Çakiris, A., New generation DNA sequencing, Journal of Experimental Medicine Research Institute, 1 (1) (2011), 11-18.
• Chloé, J., Valérie, U., A unique clonal JAK2 mutation leading to constitutive signaling causes polycythaemia vera, Nature, 434 (2005), 1144–1148. https://doi.org/10.1038/nature03546
• Tefferi, A., The history of myeloproliferative disorders: before and after Dameshek, Leukemia, 22 (1) (2008), 3-13. https://doi.org/10.1038/sj.leu.2404946
• Harrison, C.J., Philadelphia Chromosome, Encyclopedia of Genetics, 23 (2001), 1449-1450. https://doi.org/10.1006/rwgn.2001.0991
• Karakuş, S., Chronic neutrophilic leukemia BCR-ABL negative chronic myeloid leukemia and other atypical myeloproliferative diseases, Blood, 2-1, (2012), 122-130.
• Arslan, S., Screening of BCR/ABL fusion gene mutations in patients diagnosed with CML and ALL, American Journal of Hematology, 93 (3) (2014), 442-459. https://doi.org/10.1002/ajh.25011
• Tefferi A., Primary myelofibrosis: 2021 update on diagnosis, risk-stratification, and management, American Journal of Hematology, 96 (1) (2021), 801-821. https://doi.org/10.1002/ajh.26050
• Hu, X., Li, J., Fu, M., Zhao, X., Wang, W., The JAK/STAT signaling pathway: from bench to clinic, Signal Transduction and Targeted Therapy, 6 (1) (2021), 402. https://doi.org/10.1038/s41392-021-00791-1
• Godfrey A. L., Myeloproliferative neoplasms (MPNs), Blood Reviews, 42 (2020), 100717. https://doi.org/10.1016/j.blre.2020.100717
• Baumeister, J., Chatain, N., Sofias, A. M., Lammers, T., Koschmieder, S., Progression of myeloproliferative neoplasms (MPN): Diagnostic and therapeutic perspectives, Cells, 10 (12) (2021), 3551. https://doi.org/10.3390/cells10123551
• Sawyers, C.L., Chronic myeloid leukemia, New English Journal of Medicine, 340 (17) (1999), 1330-1340. https://doi.org/10.1056/NEJM199904293401706
• El-Sharkawy F., Margolskee E., Pediatric myeloproliferative neoplasms, Clinical Laboratory Medicine, 41 (3) (2021), 529-540. https://doi.org/10.1016/j.cll.2021.04.010
• Liu, J., Wang, F., Luo, F., The role of JAK/STAT pathway in fibrotic diseases: Molecular and cellular mechanisms, Biomolecules, 13 (1) (2023), 119. https://doi.org/10.3390/biom13010119
• Barbui, T, Barosi, G, Birgeggard, G, et al., Philadelphia-negative classical myeloproliferative neoplasms: Critical concepts and management recommendations from european leukemianet, Journal Clinical Oncology, 29 (6) (2011), 761-770. https://doi.org/10.1200/jco.2010.31.8436
• Anía, BJ, Suman, VJ, Sobell, JL, Codd, MB, Silverstein, MN, Melton LJ, Trends in the incidence of polycythemia vera among Olmsted County, Minnesota residents, 1935-1989, American Journal of Hematology, 47 (2) (1994), 89-93. https://doi.org/10.1002/ajh.2830470205
• Shawky, A.M,, Almalki, F.A,, Abdalla, A.N,, Abdelazeem, A.H, Gouda A.M., A comprehensive overview of globally approved JAK inhibitors, Pharmaceutics, 14 (5) (2022), 1001. https://doi.org/10.3390/pharmaceutics14051001
• Godfrey, A.L., Green, A.C., Harrison, C.N., Essential thrombocythemia: challenges in clinical practice and future prospects, Blood, 141 (16) (2023), 1943-1953. https://doi.org/10.1182/blood.2022017625
• Tefferi, A., Pardanani, A., Essential thrombocythemia, New England Journal of Medicine, 381 (22) (2019), 2135-2144. https://doi.org/10.1056/NEJMcp1816082
• Gianelli, U., Thiele, J., Orazi, A., Gangat, N., Vannucchi, A.M., Tefferi, A., Kvasnicka, H.M., International consensus classification of myeloid and lymphoid neoplasms: myeloproliferative neoplasms, Virchows Archiv, 482 (1) (2023), 53-68. https://doi.org/10.1007/s00428-022-03480-8
• Gotlib, J., Chronic eosinophilic leukemia/hypereosinophilic syndrome, Cancer Treatment Research, 142 (2008), 69-106. PMID: 18283783
• Green, M. R., Sambrook, J., The basic polymerase chain reaction (PCR), Cold Spring Harbor Protocols, (2018), 436-456. https://doi.org/10.1101/pdb.prot095117
• Crossley, B. M., Bai, J., Glaser, A., Maes, R., Porter, E., Killian, M. L., Clement, T., Toohey-Kurth, K., Guidelines for sanger sequencing and molecular assay monitoring, Journal of Veterian Diagnostic Investigation, 32 (6) (2020), 767-775. https://doi.org/10.1177/1040638720905833
• Levy, S. E., Boone, B. E., Next-Generation sequencing strategies, Cold Spring Harbol Perspectives Medicine. 9 (7) (2019), 791. https://doi.org/10.1101/cshperspect.a025791
• Hu, T., Chitnis, N., Monos, D., Dinh, A., Next-generation sequencing technologies: An overview, Human Immunology, 82 (11) (2021), 801-811. https://doi.org/10.1016/j.humimm.2021.02.012
• Schnittger, S., Bacher, U., Haferlach, C., et al., Detection of JAK2 exon 12 mutations in 15 patients with JAK2V617F negative polycythemia vera, Haematologica, 94 (3) (2009), 414-418. https://doi.org/10.3324/haematol.13223