Identification of Chimeric NTRK3 Genes in Papillary Thyroid Cancer Cells by Analyzing the Imbalance of the Expression of 5' and 3' mRNA Fragments.

The standard for detecting chimeric genes of neurotrophic receptor tyrosine kinases (NTRK) is next generation sequencing (NGS). However, this analysis is expensive and takes several days. As a rapid screening method for the detection of NTRK3-dependent papillary thyroid cancer, an analysis of the expression imbalance between 5' and 3' NTRK3 mRNA fragments was used (5'/3' RT-PCR). The reference method for detection of NTRK3 rearrangements was fluorescent in situ hybridization (FISH), and the most frequent rearrangements in papillary thyroid cancer were tested using reverse transcription PCR (RT-PCR). Using 5'/3' RT-PCR, 18 samples of papillary thyroid cancer carrying chimeric transcripts of NTRK3 mRNA were detected. The sensitivity of the developed technique was 88.9% and specificity was 99.3%. Thus, a fast and cost-effective method of screening samples of papillary thyroid cancer in paraffin blocks is proposed with acceptable sensitivity and specificity.

Development of a Cell Line Containing the Chimeric ETV6-NTRK3 Gene. The Search for Mutations of the Tyrosine Kinase Chimeric Domain That Cause Resistance to Larotrectinib.

The development, registration, and further use of entrectinib and larotrectinib for the treatment of tumors resulting from oncogenic stimulation of chimeric neurotrophin receptors (TRK) attracted much interest to the mechanisms of tumor cells resistance to TRK inhibitors during treatment. In the presented study, a cell line carrying the chimeric gene ETV6-NTRK3 (HFF-EN) was created on the basis of human fibroblasts. The transcription level of the chimeric ETV6-NTRK3 gene in HFF-EN was comparable to the transcription level of the household ACTB gene, the expression of the ETV6-NTRKA protein was confirmed by immunoblotting. A comparison of the dose-effect curves of fibroblasts and HFF-EN cells showed a ~38-fold increase in the sensitivity of HFF-EN to larotrectinib. To obtain a cell model of the resistance to larotrectinib in NTRK-dependent cancer, we used cell passages with a gradually increasing concentration of larotrectinib and obtained six resistant clones. p.G623E c.1868G>A mutation was found in five clones, and p.R582W c.1744C>T mutation, previously not described as a resistance mutation, was found in one clone showing significantly less resistance. These results can be further used for more complete understanding of the mechanisms of the resistance to TRK inhibitors and for the development of new drugs.

Differences in Transcriptomic Profiles of Brain and Thyroid Tumors with NTRK Gene Rearrangement.

For tumors with chimeric NTRK genes, entrectinib and larotrectinib can be prescribed regardless of tumor localization. We compared changes in the transcriptional activity of genes in brain tumors (BT) and thyroid cancer (TC) with rearrangement (NTRK+) and without rearrangement (NTRK-) of the NTRK genes using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. We revealed an increase in the transcription of the JUN gene in NTRK+ samples in comparison with NTRK- samples: by 1.6 times for BT (p=0.239) and by 2.5 times for TC (p=0.003). The transcription of eight HOX genes in NTRK+ BT samples was also increased (by 85-725 times, p<0.05) in comparison with NTRK-. In NTRK+ TC samples, the level of miR-31 and miR-542 was statistically significantly higher (by 3 and 2.5 times, respectively) than in NTRK-samples. For the NTRK+ BT samples, the levels of miR-10b, miR-182, and miR-21 more than 5-fold surpassed the corresponding values in NTRK-samples (p<0.05). These findings reflect differences in activation of gene transcription resulting from NTRK gene rearrangement in BT and TC.

Increased Frequencies of the ‒174G and ‒572C IL6 Alleles in Populations of Indigenous Peoples of Siberia Compared to Russians.

Abstract-The study of immune response and inflammation gene polymorphisms in a genogeographic context is relevant in the study of human populations. Here, in the indigenous populations of Siberia the frequencies of polymorphic variants ‒174G/C (rs1800795) and ‒572C/G (rs1800796) of the IL6 gene encoding the proinflammatory cytokine IL-6 were determined. For the first time, it was shown that the frequencies of the ‒174G and ‒572C alleles, which determine increased inflammatory response and are also associated with several diseases were statistically significantly higher in ethnic groups of Buryats, Teleuts, Yakuts, Dolgans and Tuvinians than in Russians living in Siberia. These values were in the intermediate position between those in the European and East-Asian groups. We hypothesize an adaptive role of these IL6 genetic variants in human settlement from Africa to the Eurasian continent. However, due to the departure from the traditional way of life and the increasing anthropogenic environmental pollution, the risk of diseases whose pathogenesis is based on inflammation in indigenous Siberian populations is likely increased.

[Investigation of the mutational status of the FGFR3 gene in urothelial bladder carcinoma]. / Issledovanie mutatsionnogo statusa gena FGFR3 v urotelial'noi kartsinome mochevogo puzyrya.

OBJECTIVE: To study the somatic mutational status of the FGFR3 gene in urothelial bladder cancer (BC) and evaluate its relationship with the clinical and morphological characteristics of the tumor, deficiency of the DNA mismatch repair (dMMR), PD-L1 tumor status, and immunohistochemical (IHC) expression of the p16 protein. MATERIAL AND METHODS: Surgical material of 40 patients with BC, on which the mutational status of the FGFR3 gene was studied using the molecular genetic method, as well as the MMR status, PD-L1 and p16 expression by the IHC method. RESULTS: FGFR3 mutations, such as G370C, S249C, S371C/Y373C, R248C, were detected in 35.0% of the studied BC samples. FGFR3 status did not depend on the gender and age of patients, as well as on the degree of tumor lymphoid infiltration (TILs). Statistically significant differences were found in the analysis of FGFR3 status depending on the histological structure and degree of tumor differentiation, as well as on the pT stage. The FGFR3 status of BC was not associated with the IHC expression of the studied proteins of the MMR system, as well as with the PD-L1 status. Higher levels of PD-L1 expression were demonstrated by BC tumor cells, in which no aberrations in FGFR3 were detected. There was no significant association between p16 status and the presence of FGFR3 mutations, but for FGFR3-positive carcinomas, the basal pattern of p16 staining by IHC was noted. CONCLUSION: A positive somatic mutational status of the FGFR3 gene was statistically significantly more common in the group of papillary low-grade non-muscle-invasive BC, demonstrating basal p16 IHC staining. In the study sample, there was no statistically significant relationship between the FGFR3 status of BC and gender and age differences, TILs, MMR status, PD-L1 status (SP142 and 22C3), and p16 status. The results of the study indicate the need to determine the FGFR3 status in patients with BC for further prescription of personalized therapy.

[Increased Frequencies of the -174G and -572C IL6 Alleles in Populations of Indigenous Peoples of Siberia Compared to Russians].

The study of immune response and inflammation gene polymorphisms in a genogeographic context is relevant in the study of human populations. Here, in the indigenous populations of Siberia the frequencies of polymorphic variants -174G/C (rs1800795) and -572C/G (rs1800796) of the IL6 gene encoding the proinflammatory cytokine IL-6 were determined. For the first time, it was shown that the frequencies of the -174G and -572C alleles, which determine increased inflammatory response and are also associated with several diseases were statistically significantly higher in ethnic groups of Buryats, Teleuts, Yakuts, Dolgans and Tuvinians than in Russians living in Siberia. These values were in the intermediate position between those in the European and East-Asian groups. We hypothesize an adaptive role of these IL6 genetic variants in human settlement from Africa to the Eurasian continent. However, due to the departure from the traditional way of life and the increasing anthropogenic environmental pollution, the risk of diseases whose pathogenesis is based on inflammation in indigenous Siberian populations is likely increased.

Evaluation of Advantages of Lithium Chloride as a Precipitating Agent in RNA Isolation from Frozen Vein Segments.

Contamination of the sample with DNA is a problem when isolating RNA by phenol-chloroform extraction, and DNase treatment introduces an additional error in the analysis of gene expression. Bearing in mind the ability of LiCl to precipitate RNA selectively, we studied the possibility and advantage of using LiCl as a precipitation agent in the protocol for RNA precipitation [3] from frozen segments of large vessels. Combined use of LiCl with isopropanol as precipitating agents (optimal ratio 2.5 M and 40%, respectively) significantly eliminates negative effects of using this salt (inhibition of reverse transcription and low RNA yield).

Prediction of EVT6-NTRK3-Dependent Papillary Thyroid Cancer Using Minor Expression Profile.

Solid tumors resulting from oncogenic stimulation of neurotrophin receptors (TRK) by chimeric proteins are a group of rare tumors of various localization that respond to therapy with targeted drugs entrectinib and larotrectinib. The standard method for detecting chimeric TRK genes in tumor samples today is considered to be next generation sequencing with the determination of the prime structure of the chimeric transcripts. We hypothesized that expression of the chimeric tyrosine kinase proteins in tumors can determine the specific transcriptomic profile of tumor cells. We detected differentially expressed genes allowing distinguishing between TRK-dependent tumors papillary thyroid cancer (TC) from other molecular variants of tumors of this type. Using PCR with reverse transcription (RT-PCR), we identified 7 samples of papillary TC carrying a EVT6-NTRK3 rearrangement (7/215, 3.26%). Using machine learning and the data extracted from TCGA, we developed of a recognition function for predicting the presence of rearrangement in NTRK genes based on the expression of 10 key genes: AUTS2, DTNA, ERBB4, HDAC1, IGF1, KDR, NTRK1, PASK, PPP2R5B, and PRSS1. The recognition function was used to analyze the expression data of the above genes in 7 TRK-dependent and 10 TRK-independent thyroid tumors obtained by RT-PCR. On the test samples from TCGA, the sensitivity was 72.7%, the specificity - 99.6%. On our independent validation samples tested by RT-PCR, sensitivity was 100%, specificity - 70%. We proposed an mRNA profile of ten genes that can classify TC in relation to the presence of driver NTRK-chimeric TRK genes with acceptable sensitivity and specificity.

Development of a Test System to Detect the Omicron Variant of SARS-CoV-2 and the Frequency of Its Detection in Patients.

We developed a new test system to detect the omicron variant of SARS-CoV-2 using allele-specific reverse transcription PCR and estimated the frequency of its detection in patients living in the Novosibirsk Region. Clinical samples were divided into 3 groups: samples collected from December 1 to December 30, 2021 (group 1; n=66), from December 30, 2021 to January 10, 2022 (group 2; n=20), and from January 11 to January 22, 2022 (group 3; n=101). Based on the identification of 5 mutations specific to SARS-CoV-2 (B.1.1.529), two systems of oligonucleotide primers and probes were developed for detecting this coronavirus genotype in clinical samples. Limit of detection (LOD95) was 4×103 genome equivalents per 1 ml of clinical sample for the first test system and 2×103 for the for the second test system. The omicron variant of SARS-CoV-2 was absent in group 1 of studied samples, but was detected in 20% (4/20) of group 2 samples and 88% of group 2 samples collected within less than 2 weeks of January 2022. Using developed test system, we showed that in less than 2 weeks the omicron variant has become dominant in patients, which confirms previously published data on its exceptional contagiousness.

The Level of LINE-1 mRNA Is Increased in Extracellular Circulating Plasma RNA in Patients with Colorectal Cancer.

We performed a comparative quantitative analysis of LINE-1 mRNA levels in extracellular total plasma RNA in patients with colon cancer and practically healthy donors. Quantitative multiplex PCR with reverse transcription was used to assess the level of LINE-1 and 18S rRNA mRNA in extracellular total plasma RNA. The median of LINE-1 mRNA values in colon cancer patients (4.95) was significantly higher than in healthy donors (2.3) (p=0.037). It was shown for the first time that the level of LINE-1 mRNA in total RNA from blood plasma can be determined in the format of a liquid biopsy and serve as a new potential non-invasive marker of colon cancer.

[Features of the cell composition of inflammatory infiltrate in different phases of diffuse alveolar lung damage with COVID-19]. / Osobennosti kletochnogo sostava vospalitel'nogo infil'trata v raznye fazy diffuznogo al'veolyarnogo povrezhdeniya legkikh pri COVID-19.

BACKGROUND: Acute respiratory distress syndrome (ARDS) with COVID-19 has a worse prognosis than ARDS with other diseases. Mortality from ARDS with COVID-19 is 26.0 - 61.5%, and due to other causes - 35.3-37.2%. OBJECTIVE: To find of the correlation between polymorphonuclear leukocytes (PMNs), lymphocytes, and macrophages in the cellular composition of the inflammatory infiltrate at different stages and phases of diffuse alveolar damage (DAD) with COVID-19, analyzing the autopsy material. MATERIAL AND METHODS: The lung tissue of 25 patients who died from ARDS with COVID-19 without a secondary bacterial or mycotic infection, another thanatologically significant pathology of the lungs, was studied. To study the cellular composition of the inflammatory infiltrate and the dynamics of its changes a double immunohistochemical analysis of the expression of antibodies to CD15, CD3, and CD68 was used. RESULTS: The inflammatory infiltrate and intraalveolar exudate in the exudative phase of DAD was represented by 56.8% of PMNs (CD15-positive cells; hereinafter - the average value of the percentage of positive cells to the total number of cells of the inflammatory infiltrate), 6.9% - lymphocytes (CD3-positive cells) and 19.5% macrophages (CD68-positive cells). In the early stage of the proliferative phase: 14.1% PMNs, 38.7% lymphocytes and 13.5% macrophages. In the late stage of the proliferative phase: 11.3% PMNs, 14.5% lymphocytes and 39.3% macrophages. CONCLUSIONS: In the exudative phase of DAD a statistically significant predominance of PMN was revealed, which could determine the main volume of lung damage and the severity of ARDS with COVID-19. In the early stage of the proliferative phase of DAD, a statistically significant change in the composition of the inflammatory infiltrate was revealed to compare with the exudative phase: a significant decrease in the content of PMNs relative to the total number of cells in the inflammatory infiltrate; an increase in the number of lymphocytes, which is probably associated with the start of organization and repair processes. In the late stage of the proliferative phase of DAD, compared with its early stage, was revealed a statistically significant increase in the number of macrophages in ratio.

TCF7L2 gene polymorphism in populations of f ive Siberian ethnic groups.

Investigation of the frequencies of functionally signif icant gene variants in the context of medical biology and gene geography is a relevant issue for studying the genetic structure of human populations. The transition from a traditional to an urbanized lifestyle leads to a higher incidence of civilizational diseases associated with metabolic disorders, including type 2 diabetes mellitus. The goal of the present paper is to analyze the frequencies of functionally signif icant gene alleles in the metabolic prof iles of indigenous Siberian peoples to identify the gene pool resilience, evaluate the susceptibility of various ethnic groups to metabolic disorders under changing environmental conditions, and predict the epidemiological situation that may occur in the near future. The study was performed in the monoethnic samples of eastern and western Buryats, Teleuts, Dolgans, and two territorial groups of Yakuts. A real-time PCR was used to determine the frequencies of single nucleotide polymorphisms (SNPs) G103894T, rs12255372, and C53341T, rs7903146 in the TCF7L2 gene. The results obtained were compared to the frequencies identif ied for Russians from Eastern Siberia and the values available in the literature. The frequencies of the polymorphic variants studied in the samples from the indigenous Siberian peoples place them in between Caucasian and East Asian populations, following the geographic gradient of polymorphism distribution. A signif icantly lower occurrence of type 2 diabetes risk alleles TCF7L2 (103894T) and TCF7L2 (53341T) in the samples of indigenous Siberian peoples compared to Russians was observed, which agrees with their lower susceptibility to metabolic disorders compared to the newcomer Caucasian population. Taking into account urbanization, a reduced growth in type 2 diabetes incidence may be predicted in indigenous Siberian peoples, i. e. Buryats, Yakuts, Dolgans, and Teleuts, compared to the newcomer Caucasian population. A further study of population structure with respect to different metabolic prof ile genes is required to better understand the molecular genetic foundations of the adaptive potential of indigenous Siberian peoples.

Modification of isothermal test for detecting DNA of pathogens of tick-borne borreliosis.

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.

[N-aсetyltransferase (NAT2) gene polymorphism and gene network analysis]. / Polimorfizm variantov gena N-atsetiltransferazy 2 (NAT2) i analiz gennoi seti.

To search for new targets of therapy, it is necessary to reconstruct the gene network of the disease, and identify the interaction of genes, proteins, and drug compounds. Using the online bioinformatics tools we have analyzed the current data set related to the metabolism of xenobiotics, mediated by the N-acetyltransferase 2 (NAT2) gene. The study of allelic polymorphism of the NAT2 gene has a prognostic value, allowing to determine the risk of a number of oncological diseases, the degree of increased risk due to smoking and exposure to chemical carcinogens, including drugs. The aim of this study was to determine the frequencies of two important "slow" variants of the NAT2 gene (NAT2*5, rs1801280 and NAT2*7, rs1799931), which significantly affected the rate of xenobiotic acetylation among the indigenous Nenets population of Northern Siberia. The obtained frequencies of polymorphic variants among the Nenets occupy an intermediate value between those for Europeans and Asians, which might indicate specific features of adaptation. We present a model of the distribution of two polymorphic variants of the NAT2 gene involved in the biotransformation of xenobiotics to study the characteristics of their metabolism in the indigenous inhabitants of Yamal.

Comparison of polymerase chain reaction and flow cytometry for measuring telomere length of human leukocytes.

Telomere length can be measured by polymerase chain reaction (PCR), allowing to obtain the absolute length of telomeres (ALT) in base pair, and by flow cytometry, which can only estimate the relative telomere length. The aim of the study was to compare the results of the two methods and to develop an accurate and reliable way of converting the relative telomere length to absolute. The peripheral blood from 21 donors was analyzed. Measurement of leukocyte telomere length by flow cytometry was carried out using a commercial Telomere PNA Kit / FITC (Dako, Denmark) with two CytoFLEX flow cytometers (Beckman Coulter, China) and BD FACSCanto II (Becton Dickinson, USA), obtaining the molecular equivalent of fluorescence (MEF). To measure telomere length by real-time PCR, calibrators with a known number of telomeric repeats were prepared. Two quantitative PCRs were carried out: one for telomeric repeats, the other for determining the number of genome-equivalents of DNA, three times for each sample, which made it possible to calculate ALT. A strong direct relationship was found between the MEF obtained with BD FACSCanto II and CytoFLEX (r = 0.97). Analysis of PCR and flow cytometry results showed a significant correlation between ALT and MEF. We calculated the regression equations of ALT and MEF for CytoFLEX - y = 0.0043x (r = 0.84) and for BD FACSCanto II - y = 0.0051x (r = 0.82). Correlation analysis showed a high comparability of telomere lengths measured by two methods. The obtained regression equations allow converting the results of flow cytometry into absolute values, allowing the comparison of the results of different research groups and the use of this method in clinical trials.

[Characterization of Recombinant Endonuclease IV from Mycobacterium tuberculosis].

Mycobacterium tuberculosis cells contain two apurinic/apyrimidinic (AP) endonucleases, endonuclease IV (MtbEnd) and exonuclease III (MtbXthA), the former playing a dominant role in protecting mycobacterial DNA from oxidative stress. Mycobacterial endonuclease IV substantially differs from its homologs found in Escherichia coli and other proteobacteria in a number of conserved positions important for DNA binding and AP site recognition. The M. tuberculosis end gene was cloned, and recombinant MtbEnd purified and characterized. The protein efficiently hydrolyzed DNA at the natural AP site and its 1'-deoxy analog in the presence of divalent cations, of which Ca^(2+), Mn^(2+), and Co^(2+) supported the highest activity. Exonuclease activity was not detected in MtbEnt preparations. The pH optimum was estimated at 7.0-8.0; the ionic strength optimum, at ~50 mM NaCl. Enzymatic activity of MtbEnd was suppressed in the presence of methoxyamine, a chemotherapeutic agent that modifies AP sites. Based on the results, MtbEnd was assumed to provide a possible target for new anti-tuberculosis drugs.

Detection of Mutations in Mycobacterium tuberculosis pncA Gene by Modified High-Resolution Melting Curve Analysis of PCR Products.

We developed a protocol for detection of mutations in the pncA gene associated with M. tuberculosis resistance to pyrazinamide by analyzing melting curves of 7 overlapping amplicons with artificial heteroduplex formation (H-HRM) formed by co-amplification of wild-type DNA and test DNA and compared its efficiency and robustness with those of classical HRM analysis. Using HRM and H-HRM, we analyzed 35 PZAR DNA isolates carrying mutations in the pncA gene, 3 PZAR isolates without mutations in the pncA gene, and 20 PZAS isolates without mutations in the pncA gene were analyzed. The sensitivity and specificity of HRM for detection of mutations in the pncA gene were moderate: 88.57% (CI 73.26%-96.80%) and 82.61% (CI 61.22%-95.05%), respectively. The sensitivity of the H-HRM test was 97.14% (CI 85.08%-99.93%) and specificity was 95.65% (CI 78.05%-99.89%), with a significant improvement in accuracy - 96.55% vs. 93.85% for HRM. In general, despite addition stage of equalizing the concentrations of the test and control mycobacterial DNA, H-HRM showed greater stability and reproducibility at standard settings of the melting curve analysis software.

Soluble Ligand of the Immune Checkpoint Receptor (sPD-L1) in Blood Serum of Patients with Renal Cell Carcinoma.

The content of the soluble ligand of the immune checkpoint receptor (sPD-L1) was determined in the blood serum of 106 patients with renal cell carcinoma and 11 patients with benign kidney tumors by direct ELISA (Human sPD-L1 Platinum ELISA; Affimetrix, eBioscience). The control group included 19 healthy men and 18 women. Serum level of sPD-L1 significantly surpassed the control values in both patients with primary renal cancer (p<0.0001) and in patients examined during disease progression (p<0.05). In patients with benign kidney tumors, the level of this marker was significantly higher than in the control (p<0.05), but lower than in patients with renal cell carcinoma. The sPD-L1 level significantly increased with disease stage (p<0.001); it was higher in the presence of metastases in regional lymph nodes irrespective of their number (N1 or N2) than in the absence of metastases (N0); it was also increased in patients with distant metastases (M1) and patients with grade III-IV tumors in comparison with grade III-IV tumors (p<0.05). The highest sPD-L1 levels were recorded in patients with tumor size corresponding to T2 and T3 and decreased in patients with T4 tumors. Thus, sPD-L1 level in patients with renal cell carcinoma correlated with tumor grade and metastasizing and can be considered as a promising marker in monitoring of the effect of anti-PD1/PD-L1 therapy.

One-phase phenol-free method for microRNA isolation from blood plasma.

MicroRNA extraction is an essential procedure when discovering MicroRNA-based biomarkers and approaches. Here we describe a new method for microRNA isolation from human blood plasma, based on isopropanol precipitation from the one-phase lysate. We demonstrate that the use of more than four volumes of lysis buffer based on 5 M guanidine isothiocyanate prevents the formation of large, viscous, and hardly soluble precipitate. Applying widely used linear polyacrylamide (LPAA) as the only precipitating agent proved ineffective. At the same time, adding poly(A)RNA or tRNA with LPAA significantly increased the amount of microRNA obtained. Replacing ß-mercaptoethanol with less volatile dithiothreitol in lysis buffer did not lead to a decrease in the yield. We compared the method proposed with miRNeasy Mini Kit (Qiagen) for isolation of microRNA from human blood plasma. MicroRNA yield was evaluated by the difference in median Ct values obtained for exogenous cel-238 and endogenous microRNA-21 cDNA amplification. For both tested microRNA, the precipitation from one-phase lysate provided better recovery with lower Ct values (Δ median Ct 4.94 for cel-238, р = 1,0E-04 and Δ median Ct 2.18 for microRNA-21, р = 9,0E-04). Thus, the method we described showed high yield and operating convenience because it can be applied without special equipment.

Loss of Heterozygosity in BRCA1 and BRCA2 Genes in Patients with Ovarian Cancer and Probability of Its Use for Clinical Classification of Variations.

Changes (or variants) in BRCA1 and BRCA2 gene sequences can have different lengths and clinical significance: from single nucleotide variants (SNV) and short insertions/deletions (<50 bp) to extended deletions and duplications (so-called copy number variations, or CNV). According to their clinical significance, all variants can be divided into pathogenic, likely pathogenic, variants of uncertain significance, likely benign, and benign. Moreover, variants can be germinal (i.e. inherited from parents) and somatic (arising in the process of development of the organism). A specific somatic event is loss of heterozygosity (LOH), i.e. transition of one or many point and short variants from heterozygous to homozygous state. Such an event can be the key to the development of carcinogenesis for cells carrying a pathogenic variant, if we consider it within the framework of the Knudson's two-hit carcinogenesis theory. We studied the prevalence and nature of LOH in of ovarian cancer samples carrying or not carrying a pathogenic variant. To this end, a full coding sequence of BRCA1/2 genes was determined in 30 pairs of DNA samples isolated from blood cells and paraffinized histological blocks of patients on a MiSeq Illumina instrument. Analyss of the obtained reads revealed 9 pathogenic point and short variants (30% patients): 6 germinal (20%) and 3 somatic (10%), and 8 somatic CNV (3 deletions and 5 duplications of several or all exons of the BRCA1 gene). LOH was detected in 70% patients; among the carriers of pathogenic variants - in 83%. For pathogenic variants, the percentage of reads with the alternative allele increased more often than for benign variants located in another gene, or detected in other patients (67% vs. 44%). However, the difference was statistically insignificant, which can be due to insufficient number of patients. Only in 3 of 21 cases of LOH (14%), it can be attributed to CNV. In other cases, LOH is most likely determined by gene conversion, but further research is needed.