Comparison of molecular genetic methods of detection of mutations in the CALR gene in myeloproliferative disorders

Molecular genetic detection of CALR gene somatic mutations is required for myeloproliferative neoplasms diagnosis and treatment according to the novel WHO clinical recommendations. CALR mutations are found in approximately 25–35 % cases of essential thrombocythemia and primary myelofibrosis and they are associated with benign clinical outcome. In this study we have compared sensitivity and selectivity of seve ral different options of CALR mutation molecular genetic detection in blood samples of 379 CMD patients and 17 healthy donors. Among methods compared in our study there have been conventional polymerase chain reaction with electrophoretic detection, real-time quantitative polymerase chain reaction, direct Sanger sequencing of polymerase chain reaction fragments and polymerase chain reaction high resolution melting curve analysis. By means of melting curve analysis CALR mutations have been found in 97 (25.5 %) patients, whereas in the cases of Sanger sequencing and polymerase chain reaction there have been 87 (23.0 %) and 84 (22.1 %) CALR mutation positive patients respectively.

CALR, polycythemia vera, essential thrombocythemia, primary myelofibrosis

1. Misyurin A.V. Molecular pathogenesis of the myeloproliferative diseases. Klinicheskaya onkogematologiya. Fundamental’nye issledovaniya i klinicheskaya praktika = Clinical Oncohematology. Basic Research and Clinical Practice 2009;2(3):211–21. (In Russ.).

2. Chi J., Pierides Ch., Mitsidou A. et al. A sensitive detection method for MPLW515L or MPLW515K mutation in myeloproliferative disorders. Euro J Exp Bio 2014;4(5):33–6.

3. Tutaeva V., Misurin A.V., Michiels J.J. et al. Application of PRV-1 mRNA expression level and JAK2V617F mutation for the differentiating between polycytemia vera and secondary erythrocytosis and assessment of treatment by interferon or hydroxyurea. Hematology 2007;12(6):473–9. DOI: 10.1080/10245330701384005.

4. Klampfl T., Gisslinger H., Harutyunyan A.S. Somatic mutations of ca lreticulin in myeloproliferative neoplasms. N Engl J Med 2013;369(25):2379–89. DOI: 10.1056/NEJMoa1311347.

5. Nangalia J., Massie C.E., Baxter E.J. et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med 2013;369(25):2391–405. DOI: 10.1056/NEJMoa1312542.

6. Michalak M., Corbett E.F., Mesaeli N. et al. Calreticulin: one protein, one gene, many functions. Biochem J 1999;2:281–92.

7. Zapun A., Darby N.J., Tessier D.C. Enhanced catalysis of ribonuclease B folding by the interaction of calnexin or calreticulin with ERp57. J Biol Chem 1998;273(11):6009–12. DOI: 10.1074/jbc.273.11.6009.

8. Ning L., Qui-Mei Y., Gale R.P. et al. Frequency and allele burden of CALR mutations in Chinese with essential thrombocythemia and primary myelofibrosis without JAK2(V617F) or MPL mutations. Leuk Rec 2015;39(5):510–4.

9. Jones A.V., Ward D., Lyon M. et al. Evaluation of methods to detect CALR mutations in myeloproliferative neoplasms. Leuk Res 2015;39(1):82–7. DOI: 10.1016/j.leukres.2014.11.019. Available at: https://www.lrjournal.com/ article/S0145-2126(14)00371-3/abstract.

10. Luo W., Zhongxin Yu.Z. Calreticulin (CALR) mutation in myeloproliferative neoplasms (MPNs). Stem Cell Investig 2015;2:16. DOI: 10.3978/j.issn.23069759.2015.08.01. Available at: http://sci. amegroups.com/article/view/7264/8051.

11. Reed G.H., Kent J.O., Witter C.T. Highresolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics 2007;8(6):597–608. DOI: 10.2217/14622416.8.6.597