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Introduction. Haplotype JAK2 46/1 is associated with more frequent development of Ph-negative myeloproliferative neoplasms (MPN) and with an increased detection rate of the JAK2 V617F mutation. At the same time, the molecular mechanisms of such associations remain unclear. Previously, there were no studies of regional, age and gender aspects of the predictive value of carriage of the 46/1 JAK2 haplotype, which could assess this relationship in some observations.Aim — to analyze the degree of association between 46/1 haplotype and the V617F mutation of the JAK2 gene depending on the sex, age, and place of residence of patients examined for suspected MPN.Methods. The study included 949 DNA samples from patients with suspected MPN. Samples of 150 volunteers and blood donors were included in the control group. Haplotype 46/1 (rs10974944), V617F mutation in the JAK2 gene, mutations in the CALR gene (type 1: c.1092_1143del; L367fs*46, COSV57116546; type 2: c.1154_1155insTTGTC; K385fs*47, COSV57116551) and in the MPL gene (W515K, W515L) were determined for all samples using real-time polymerase chain reaction (PCR-RT).Results. The 46/1 JAK2 haplotype were shown to be associated with a clinically significant level (> 2 %) of the allelic burden of the JAK2 V617F mutation. The odds ratio of the risk of developing a V617F positive MPN when carrying this haplotype variant did not depend on the main place of residence of the patients and was found to be most pronounced in men under 50 years of age. The odds ratio of the risk did not depend on the age of the examined women.Conclusion. The association of 46/1 haplotype with the presence of other drivers of MPN mutations in the CALR or MPL genes was also statistically significant, which confirms the hypothesis of “favorable soil” rather than “hypermutability” of the JAK2 gene.

Early diagnosis of tick-borne borreliosis determines the indications for etiotropic therapy, and the detection of borrelia in a tick that has bitten you serves as the basis for antibiotic prophylaxis. To determine the causative agent of borreliosis, PCR methods are most widely used, which requires special conditions for organizing the work of laboratories and the use of expensive equipment. In addition, the procedure for isolating bacterial DNA and subsequent amplification takes several hours of working time. At the same time, methods for detecting borrelia in the isothermal LAMP-reaction are described, which makes it possible to significantly speed up the diagnosis, does not require complex equipment and highly qualified personnel. It is also known that LAMP in some cases allows analysis without prior extraction of nucleic acids. The purpose was a development of a modified test for isothermal detection of DNA of borreliosis pathogens for an accelerated result and the possibility of excluding the stage of nucleic acid extraction. We used 40 samples of Borrelia DNA and 11 Ixodes persulcatus ticks. To shorten the detection time for Borrelia, the previously described LAMP method was modified by the introduction of additional loop primers. The copy number of the positive DNA sample of the borrelia plasmid was estimated using digital PCR. The results of the LAMP reaction were compared with those of the commercial PRC-RT test. The additional use of loop primers approximately halved the detection time for Borrelia DNA without affecting the comparative diagnostic efficiency. The analytical sensitivity limit of the modified LAMP method was 4 copies/μl or 21 molecules of the plasmid standard added to the reaction. In comparative testing with RT-PCR, the sensitivity of the LAMP method is 90%, and the specificity is 100%. The possibility of detecting borrelia in ticks without the stage of DNA extraction has been demonstrated for the first time. A modified isothermal method for the detection of pathogens of tick-borne borreliosis has been developed, which allows analysis within 20-30 minutes, including in ticks without preliminary DNA extraction.

Simultaneous quantitative measurement of mRNA of the WT1, BAALC, EVI1, PRAME and HMGA2 genes in whole blood samples reflects the specific pathological proliferative activity in acute leukemia and their ratio is promising as a diagnostic marker. The transcriptome profile of acute leukemia cells is usually assessed using NGS or microarray techniques after a preliminary procedure for isolation of mononuclear cells. However, the results of using the multiplex PCR reaction for the simultaneous determination of all above mRNAs in whole blood samples have not been published so far. Determination of mRNA of WT1, BAALC, EVI1, PRAME and HMGA2 genes in venous blood level samples by multiplex RT-PCR. The study included 127 blood samples from patients who diagnosis of acute leukemia was subsequently confirmed. In the comparison group, 87 samples of patients without oncohematological diagnosis were selected, including 31 samples (K1) with a normal blood formula and 56 samples (K2) with a violation of the cellular composition - anemia, leukocytosis and thrombocytopenia. RNA isolation and reverse transcription were performed using the Ribozol-D and Reverta-L kits (TsNIIE, Russia). Determination of the mRNA expression level of the WT1, BAALC, EVI1, PRAME and HMGA2 genes by multiplex real-time PCR using a homemade multiplex PCR kit. The mRNA level was characterized by high interindividual variation and did not correlate with the rate of circulating leukocytes or blood blasts. Expression of WT1 mRNA was observed in whole blood only in one patient from the control group and in 112 (88%) patients with leukemia and was combined with a decrease in the level of HMGA2 mRNA expression and BAALC mRNA values. In contrast to the control groups, patients with leukemia had higher levels of BAALC mRNA in AML and ALL, increased PRAME mRNA in AML and APL, but lower levels of HMGA2 in APL.

Overactive JAK pathway signaling is a hallmark of immune diseases and critically affects on inflammation and coagulation. A number of mutations in the JAK2 gene act as driving forces of myeloproliferative neoplasms (MPN), the pathogenesis of certain variants of acute leukemia, a number of solid malignancies and cardiovascular diseases. Assays for quantifying JAK2 mRNA in circulating blood cells can be used as a marker associated with the activity of this enzyme. Development of an original method for detecting JAK2 mRNA in venous blood and assessment of the possible diagnostic value in chronic oncohematological diseases. The development of an RT-PCR method for determining the expression of the JAK2 gene mRNA in venous blood samples was carried out in accordance with the MIQE requirements. Primers and TaqMan probes were designed using the Primer3 program, taking into account the possibility of excluding subsequent DNase treatment. The stability of the investigated mRNA was assessed in vacutainers with different anticoagulants and depending on the storage time of the samples. The study of the expression of JAK2 mRNA in blood leukocytes of 41 patients with B-CLL, 16 patients with CML, 12 patients with multiple myeloma and 39 donors using the developed "real-time" PCR method. The study revealed a decrease in the level of JAK2 mRNA in venous blood samples in patients with primary CLL, but not with CML or with multiple myeloma. The level of the marker in the majority of patients with CLL after the start of therapy returned to the range typical for healthy people. It has been shown that the values of the relative expression of JAK2 mRNA are most stable in the range of 2 - 7 hours after taking blood in a vacutainer with EDTA. An original RT-PCR method was developed for the quantitative determination of JAK2 mRNA in venous blood samples, which meets the requirements of the MIQE system. Determination of JAK2 mRNA can be useful for clarifying the pathogenesis features of certain diseases involving impaired Janus kinase activity and can become a promising marker for prognosis and assessment of the effectiveness of therapy.

A large-scale study of DNA sequencing databases (Jorgenson E., et al., 2018) to search for hereditary susceptibility to early erectile dysfunction revealed an association with the rs17185536-T polymorphism regulating the expression of topologically associating domain, among which is the SIM1 gene, which plays an important role in maintaining mass body and sexual function. The proximity of the rs17185536 locus to the CCNC (cyclin C), PRDM13 (histone methyltransferase) and USP45 (ubiquitin-specific peptidase 45) genes suggests the possible involvement of this polymorphism in the pathogenesis and other diseases associated with impairments of these genes.Materials and methods. The study included a total of 280 people: 81 of which were healthy donors, 116 pregnant women, 25 patients with polycythemia vera, 29 with essential thrombocythemia, and 29 with primary myelofibrosis.Results. Using the original method of determining the polymorphism rs17185536, the allele of specific PCR-RT showed that the prevalence of the T allele among all examined patients was 19 %, and variants of the genotypes: C/C with 73 %, C/T with 23 % and T/T with 4 %, which corresponds to Winkler e. a. data. The absence of statistically significant differences in the frequency of occurrence of this polymorphism in the pathology of pregnancy and in chronic myeloproliferative diseases indicates a low probability of involvement of rs17185536-T in the pathogenesis of these diseases. In the group of pregnant women, there is an association of the rs17185536-T allele with obesity, uterine fibroids and the development of preeclampsia.Conclusions. For the first time, using the original method of analysis, it was confirmed that the Russian population has a comparable prevalence of the gene of hereditary susceptibility to impotence. Despite the topographic proximity of the rs17185536 locus to the genes regulating the repair and functioning of DNA, its polymorphisms do not affect the risk of chronic myeloproliferative diseases developing. The association of the rs17185536-T allele with the risk of developing a pregnancy pathology requires further study.

—The influence of exposure to mobile phone radiation on in vitro platelet aggregation has been studied. It has been shown that a 30-min exposure of blood samples to mobile phone radio frequency waves increases ADP-induced platelet aggregation in groups of healthy volunteers and patients with stroke, whereas platelet aggregation in JAKV617F-positive patients with polycythemia vera is significantly suppressed and is eventually completely inhibited. The mechanisms that underlie this phenomenon may be associated with cell oxidative stress and anomalies the JAK2 signal transduction pathway in malignized blood cells. The results indicate that it is important to set safety standards for the use of mobile devices by patients with various diseases.

The JAK2 V617F somatic mutation is one of the most frequent markers of CHIP (clonal hematopoiesis of indeterminate potential). CHIP is characterized by the presence of a myeloid cells clone in peripheral blood in the absence of the sufficient reasons to diagnose the hematologic disease. The CHIP is proposed as a potential independent risk factor for vascular pathology. The aim of this study is to identify carriers of JAK2 V617F mutation among patients admitted for planned hospitalization at the Federal Center of Cardiovascular Surgery of Krasnoyarsk.The study included 930 venous blood samples. JAK2 V617F mutation was detected by using the allele - specific real time polymerase chain reaction.JAK2 V617F mutation was detected in 15 (1.6%) patients, but only two of them had blood cell count that could cause a hematological disease to be suspected.The inclusion of the JAK2 V617F mutation detection in the complex of laboratory tests of the cardiovascular patients can facilitate the timely identification of patients with increased thrombotic risk, as well as the timely diagnosis of myeloproliferative diseases.Соматическая мутация V617F в гене JAK2 является одним из наиболее частых маркеров клонального гемопоэза «неопределенного потенциала», так называемого синдрома CHIP (clonal hematopoiesis of indeterminate potential). Синдром CHIP характеризуется наличием в периферической крови клона миелоидных клеток при отсутствии достаточных оснований для диагноза онкогематологического заболевания. Наличие CHIP предложено рассматривать как потенциальный независимый фактор риска сосудистой патологии. Цель работы - выявление мутации V617F в гене JAK2 у пациентов, поступивших на плановую госпитализацию в ФГБУ «Федеральный центр сердечно - сосудистой хирургии» Красноярска. Материалы и методы. В исследование включено 930 образцов венозной крови пациентов. Выявление мутации V617F в гене JAK2 выполняли методом аллель - специфической полимеразной цепной реакции в режиме реального времени. Результаты. Среди обследованного контингента выявлены 15 (1,6%) пациентов с мутацией V617F в гене JAK2, при этом только у двух из них показатели гемограммы могли указать на возможность гематологического заболевания. Заключение. Включение теста выявления мутации V617F гена JAK2 в меню лабораторных исследований пациентов кардиологического профиля может способствовать своевременному выявлению больных с повышенным тромбогенным риском, а также своевременной диагностике миелопролиферативных заболеваний.

The evaluation was implemented concerning impact of anticoagulants used during venous blood sampling, on aggregation of thrombocytes and acetylsalicylic acid effect in vitro in 111 patients with suspicion to chronic myelo-proliferative tumors and 16 healthy volunteers. The vacutainers (Becton Dickinson) with 3.2% citrate, with heparin (Becton Dickinson) and S-Monovette system (Sarstedt AGCo) with recombinant herudin were applied. The analysis of aggregation was implemented using the technique of impedance in whole blood before and after preliminary incubation of blood samples with acetylsalicylic acid effect in 0.1 mM concentration. The induction was implemented by ATP in final concentration of 5 mkM. It is demonstrated that ATP-induced amplitude of aggregation under application of heparin and hirudin is significantly higher in comparison with its level in samples of citrate blood. At that, acetylsalicylic acid effect paradoxically increases amplitude of aggregation in samples with heparin but not in samples with citrate or hirudin. The application of hirudin permits evaluating impact of acetylsalicylic acid effect both at aggregation and disaggregation component of thrombocyte functions under erythrocytosis and thrombocytosis and can be recommended as a preferable approach in testing individual sensitivity of patients to acetylsalicylic acid effect.

The detection of somatic mutations in the 9 exon of the calreticulin gene (CALR) is regulated by the clinical recommendations as a diagnostic criterion for chronic Ph-negative myeloproliferative neoplasms (MPN). Some methods of nucleic acids testing are used to identify CALR gene mutations with different requirements for special skills of personnel and expensive equipment. The purpose of this work is to compare the results of the detection of CALR gene mutations in venous blood samples by allele-specific RT-PCR with subsequent electrophoresis, fragment analysis and Sanger- or pyro- sequencing. We used 1284 blood samples of patients with suspected MPN and 20 blood donor samples. Mutations in the CALR gene of the I and II type were identified using PCR-RT with the original primers and TaqMan probes. Also, all samples were tested for mutations in the CALR gene by electrophoretic detection of PCR results in an agarose gel. The use of allele-specific RT-PCR followed by electrophoretic detection made it possible to determine clinically significant mutations in the CALR gene in 81 venous blood samples of JAK2- and MPL-negative patients, including 42 cases of type I mutation, 33 cases of type II mutation and 8 rare CALR mutations. Mutations in the 9 exon of the CALR gene were not detected in any of the 20 blood donor samples or in 121 blood samples of patients with polycythemia vera. In randomly selected 20 negative samples, CALR gene mutations were also not detected using Sanger sequencing. All positive samples were confirmed by fragment analysis, as well as with Sanger- sequencing and pyro- sequencing. The described combined approach to detect mutations of the CALR gene in peripheral blood samples can be used in clinical diagnostic laboratories that have a standard set of equipment for electrophoresis of nucleic acids and a PCR-RT. We also propose a confirmatory test based on the pyrosequencing of DNA using the system of genetic analysis "PyroMark Q24".

The article presents results of identification of somatic mutation in in gene Junus kinase-2 (V617F JAK2) in blood samples of persons included in program of dispensarization of adult population and periodic preventive examinations of workers of railroad transport. The technology of allele-specific polymerase chain reaction in real-time in pooled trials from blood samples received by clinical diagnostic laboratory for hematological analysis was developed with purpose of carrying out study. The results of testing among 986 persons aged from 45 to 90 years (median - 69 years) permitted to establish 0.7% of patients (3 females and 4 males) with mutation V617F JAK2. The high thrombogenic potential of mutation V617F JAK2 and its involvement into pathogenesis of chronic myeloid tumors makes appropriate to include test on its detection into menu of laboratory analyses of program of dispensarization of population.

Background: Expression of mRNA thrombopoietin receptor (MPL) is detected in various tissues, but peripheral blood cells mature thrombopoietin receptor found in the platelet and mononuclear cells only (http://www.genecards.org/). It has previously been shown MPL mRNA regulates mobilization of bone marrow stem cells (Xiao N. et al., Blood, 2015) and activation of Th2-lymphocytes (Takyar S. et al., J Exp Med, 2013). MPL mRNA expression in bone marrow is associated with the progression of AML (Wetzler M. et al., J Clin Oncol, 1997) and increase of it expression in bone marrow megakaryocytes in Ph- negative myeloproliferative neoplasm (MPN) more pronounced in patients with primary myelofibrosis (PMF) (Bock O. et al., J Pathol, 2004) has been described. MPL mRNA level in venous blood leukocytes in patients with hematologic malignancies is unknown. Aims: Evaluate the MPL mRNA expression in venous blood leukocytes in patients with certain hematologic malignancies Methods: Real-time PCR was performed to detect of MPL mRNA transcripts levels in white blood cells 21 healthy volunteers, 28 patients with chronic myeloid neoplasm (MPN: PMF - 2, PV- 21 and ET - 5), 27 patients with chronic lymphocytic leukemia (CLL), 48 patients with chronic myeloid leukemia (CML), 3 patients with acute lymphocytic leukemia (ALL) and 8 patients with acute myeloid leukemia (AML). Venous blood were collected in tube with RNAse inhibitor. Total RNA was isolated using "Ribo-zol-D" (Aplisens). 10 µg of total RNA were transcribed using "Reverta-L" (Aplisens). PCR was optimized for the thermocycler iQ5 (Bio-Rad) under following condition: 40 cycle of 95 C for 15 sec and 60 C for 60 sec, reaction mixture contained 1X KCl PCR buffer, 2 mM MgCl2, 400 uM dNTP, 300 uM forward and reverse primers, 200 uM probe and 1 unit Taq-polymerase. The results were calculated utilizing the delta Ct method in the software package of "R". The threshold cycles (Ct) MPL gene and housekeeping gene GAPD determined using Cy0 method. The results was normalization with GAPDH reference gene and are presented as median and quartiles (25% and 75%). Results: mRNA MPL expression level in leukocytes venous blood decreased among PMF (0.024, 0.03) > ET (0.013; 0.003-0.016) = PV (0.010; 0.007-0.018) > CML (0.002; 0.0008-0.005) > CLL (0.0005; 0.0002-0.0016) = AML (0.0021; 0.0006-0.0027) = ALL (0.0018; 0.0009-0.0027). MPL mRNA expression level was no correlated with any blood cells count. Summary and Conclusions: Our study support the hypothesis of the independence certain functions mRNA MPL and functions mature thrombopoetin receptor in blood cells. Measurement of mRNA MPL can used as an additional diagnostic marker for the differentiation of MPN and evaluate progress in hematologic malignancies. Figure Expression levels mRNA MPL gene in explored patients groups Figure. Expression levels mRNA MPL gene in explored patients groups Disclosures No relevant conflicts of interest to declare.