Detection of the JAK2V617F in Phildelphia-negative Myeloproliferative Neoplasms patients: Comparison of the PCR-RFLP and Nucleotide sequencing

Abstract

Abstract Introduction: Myeloproliferative Neoplasms (MPNs) are a group of hematological malignancies, characterized by the hyperplasia of progenitor hematopoietic cells in the bone marrow and increased circulation of mature cells in the peripheral blood. Methods: This study investigated the occurrence of the c.617Val>Phe variant in patients with myeloproliferative neoplasms in the state of Amazonas-Brazil. As well, to compare the molecular analysis of JAK2V617F by PCR-Restriction Fragment Length Polymorphism (RFLP) and Sanger Sequencing. The online application OpenEpi calculated the accuracy, sensibility, and specificity of the test. Results: Samples of 97 patients with hematological diseases were analyzed by PCR- RFLP methodology, 14 (14.43%) JAK2V617F-positiveand 83 (85.57%) were negative for variant. By Sanger sequencing, 13 patients were positive for the mutation (8 with GT genotype [heterozygote] and 3 with TT genotype [homozygous mutant]. Conclusion: Using the Sanger Sequencing results as a reference for mutation detection, the performance of the RFLP-PCR assay demonstrated similar results, with a sensitivity of 92.31%, specificity of 97.63%, and diagnostic accuracy of 96.91%. Both techniques are appropriate for detecting JAK2V617F.In summary, PCR-RFLP is the most suitable approach for quick and efficient V617F detection, may be used as a screening technique in routine laboratory.