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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.

JAK2 mutations can be associated with any phenotypic form of chronic myeloproliferative neoplasia, while MPL and CALR mutations occur, as a rule, in cases of essential thrombocythemia and primary myelofibrosis and they are not observed in polycythemia vera. In this article we describe a clinical case of CALR mutation (c.1154_1155insGTGTC; p.E386fs*46) presence in a JAK2-negative polycythemia vera patient at age 36. In January 2018 changes in his hemogramm were recorded for the first time. In June 2018, based on a diagnostic study of bone marrow trepanobiopsy, a diagnosis of polycythemia vera was made. Molecular genetic study of the patient’s DNA didn’t reveal mutations in the JAK2 (12 and 14 exons) and the MPL genes. CALR mutation was revealed during the screening by heteroduplex analysis with the electrophoresis in polyacrylamide gel. Then the mutation was identified by Sanger’s DNA sequencing as с.1154_1155insGTGTC; p.E386fs*46. The allelic burden level as determined by pyrosequencing was 20 % (June 2018). In conclusion we can suppose that the revealed CALR mutation с.1154_1155insGTGTC; p.E386fs*46 plays its role in our patient’s polycythemia phenotype.

Background. In accordance with the World health organization clinical guidelines, the analysis of somatic mutations in the CALR gene, as well as mutations in the JAK2 and MPL genes, are included in the list of criteria for the Ph-myeloproliferative neoplasms diagnosis.More than 50 different mutation variants have been found in the CALR gene, among which the most frequent are a 52 bp deletion (c.1092_1143del), also called type 1, and a 5 bp insertion (c.1154_1155insTTGTC), also called type 2 (88 %).The remaining 12 % are other type less frequent indels or combinations thereof.It is most convenient to use sequencing methods to identify all possible variants of CALR mutations. It is also important to develop inexpensive screening test that can detect any mutations in the analyzed DNA fragment of CALR gene. This method can be heteroduplex analysis followed by electrophoresis on polyacrylamide gel (PAGE).The objective: to develop and demonstrate the feasibility of using heteroduplex analysis with separation of the PCR product by electrophoresis on non-denaturing PAGE for the CALR exon 9 mutations detection as the screening test. Materials and methods. DNA samples of 13 CALR-positive patients with different phenotypic variants of Ph-myeloproliferative neoplasms were screened by heteroduplex analysis. For the most common variants of CALR mutations (c.1092_1143del and c.1154_1155insTTGTC), a threshold determination of the mutant allele presence was analyzed.Nucleotide sequence of exon 9 fragment was determined using Sanger sequencing. Also, all 13 samples were analyzed using the pyrosequencing method to assess the allelic burden level.Results. Heteroduplex analysis revealed mutations in exon 9 of the CALR gene in all 13 patients. The threshold determinations of the method in the case of the c.1154_1155insTTGTC and c.1092_1143del analysis are 6.25 % and 3.13 % of the mutant allele presence in the patient sample, respectively.Conclusion. The proposed variant of the heteroduplex analysis with separation of the PCR product by electrophoresis on non-denaturing PAGE can be recommended for use as the preliminary screening test which is carried out before the confirming sequencing methods for the different indels (or combinations thereof) CALR mutations determine.The presence of heteroduplexes indicates the presence of a mutation, even if the mutant product is not visualized (in case of small mutations).

Background. There are problems related to both quantitative assessment of an allele burden level of a mutant gene and interpretation of results in DNA samples with the burden level of the mutant allele less than 15–20 %, when using Sanger sequencing for analyzing somatic mutations. Applied Biosystems (USA) has developed new software Minor Variant Finder, which allows determining mutations with the allele burden level from 5 %.The objective: to determine the allele burden level and identification of minor variants of somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene using Minor Variant Finder software in patients with myeloproliferative neoplasms.Materials and methods. The level of mutant allele burden for 15 patients with myeloproliferative neoplasms was determined by the identified mutations using the Minor Variant Finder software, after analysis of point somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene by Sanger sequencing.Results. The allele burden level in all 5 ASXL1-positive samples and BCR-ABL-positive sample was determined as higher than 20 % using the Minor Variant Finder software. The allele burden level in 2 cases was higher than 20 % and in 7 cases lower than 20 %, when we analyzed 9 JAK2-positive samples.Conclusion. Minor Variant Finder software can be used to estimate the allele burden level and to identify minor variants of somatic mutations in the ASXL, JAK2 and BCR-ABL genes.