The Pyrosequencing-Based Method for JAK2 Exon 12 Somatic Mutation Detection.Diagnostics (Basel) 2026 Jun 06; 16(12).D
Background: The detection of JAK2 exon 12 mutations is important for the differential diagnosis of myeloproliferative neoplasms (MPN) and is included in the World Health Organization's diagnostic criteria for polycythemia vera (PV). We developed and evaluated a pyrosequencing-based technique to detect somatic mutations in JAK2 exon 12 and tested the method in a group of PV patients. Methods: PCR and pyrosequencing primers were designed for region JAK2 exon 12; PCR conditions were optimized for subsequent qualitative and quantitative detection by pyrosequencing. Diagnostic specificity and sensitivity were determined using plasmid controls. Genomic DNA from 145 MPN patients was used to validate the method. Results: The analytical characteristics of the method were as follows: the limit of blank was 1.2-7.4% and the limit of detection was 3.4-9.9% depending on mutation type. DNA samples from Russian patients with clinical evidence of MPNs were analyzed. In 7 of 145 cases (4.8%) JAK2 exon 12 mutations were detected. One patient had the mutation I540-E543insdelTCAGAAATG<AAA. The A-homopolimer insertion in this complex mutation type could not be clearly identified by pyrosequencing, and Sanger confirmation was required. For another patient with N542-E543delAATGAA mutation, we observed the mutant allele burden growing from 15 to 55% between 2014 and 2018 and further reduction during treatment to 11%. Conclusions: The developed pyrosequencing-based method presents a simple solution to qualitative and quantitative JAK2 exon 12 mutations detection. However, some complex mutation types may require manual interpretation and Sanger confirmation.
