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Background. According to WHO guidelines, one of the criteria for diagnosis of polycythemia vera is the presence of somatic mutations in exon 12 of the JAK2 gene, but to date there is no universally accepted simple method to analyze these mutations. We have previously proposed two methods for screening such mutations based on heteroduplex and HRM (High Resolution Melt) assays, which are relatively cheap and fast compared to sequencing.Aim. To analyze the sensitivity of these screening methods.Materials and methods. The study used cloned DNA samples from 6 patients with various mutations in exon 12 of the JAK2 gene that we had previously identified, as well as a clone of the corresponding wild-type DNA segment. Dilution of the cloned mutant samples with wild-type clones was performed to obtain samples with different levels of allele burden: 100, 50, 25, 12.5, 6.25, 3.13, 1.56 and 0.78 %. Heteroduplex analysis followed by PAGE (polyacrylamide gel) and HRM analysis was then performed with the diluted samples.Results. The sensitivity threshold of the heteroduplex analysis was found to be between 3.13–6.25 % allele burdens depending on the specific mutation, the sensitivity threshold of the HRM assay was 6.25–12.5 % similarly.Conclusion. Our proposed methods of heteroduplex analysis followed by PAGE and HRM-analysis for the detection of polycythemia vera-specific mutations in exon 12 of the JAK2 gene allow increasing the efficiency of using different types of sequencing and can be used as simpler and less expensive methods of preliminary screening of these mutations.

Background. The development of myelofibrosis (MF) is driven by complex molecular genetic events that include driver somatic mutations responsible for the constitutive activation of the JAK/STAT signaling pathway (JAK2, CALR, and MPL), additional mutations affecting epigenetic regulators (TET2, ASXL1, IDH1/2, etc.) and RNA splicing (SRSF2, U2AF1, SF3B1, etc.), as well as genetic aberrations that contribute to genomic instability and disease progression.Aim. To analyze driver (JAK2, CALR, MPL) and prognostic (ASXL1) somatic mutations in patients with MF and evaluate their impact on survival.Materials and methods. The study included 29 patients diagnosed with MF, selected by hematologists from the City Clinical Hospital No. 7 and Regional Clinical Hospital (Krasnoyarsk).Results. 26 (89.6 %) out of 29 examined patients had some driver mutations in JAK2, CALR, MPL genes. The p.V617F mutation in the JAK2 gene was found in 20 (68.9 %) patients. Mutations in the CALR gene were detected in 4 (13.8 %) patients, mutations in the MPL gene were found in 3 patients (10.3 %). In 1 of 26 patients, 2 driver mutations were present simultaneously. 3 (10.3 %) patients were triple negative. Mutations in the ASXL1 gene were detected in 12 (41.4 %) out of 29 examined patients. Conducted targeted NGS (next generation sequencing) for 13 out of 29 patients revealed additional genetic variants that contribute to the understanding of the development mechanism and disease course. When evaluating the overall survival in the groups of patients diagnosed with MF examined by us, depending on the combination of driver (JAK2, CALR, MPL) and prognostic (ASXL1) mutations, no statistically significant differences were found (p = 0.12). This appears to be due to the small sample size. At the same time, assessment of patient survival depending on ASXL1 status showed that in the presence of mutations in the ASXL1 gene, the median survival was 45 months (range 7–120 months), while in the absence of mutations it was 48 months (range 21–359 months) (p = 0.03).Conclusion. The results obtained allow us to assume that the presence of mutations in the ASXL1 gene is an unfavorable factor in the course of the disease.