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.

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.

[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.

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.

[The first results of a combined all-Russian study on clinical genetics of multiple sclerosis].

Multiple sclerosis is a classic multifactorial disease in which etiology interaction of external factors and structural features of a large number of genes plays an important role. Identifying risk factors for multiple sclerosis and creating an integrated model of pathogenesis are urgent tasks of neurology. Revealing true risk factors is possible only in studies with sufficient statistical power, so with a large amount of samples. We conducted the association study of CD40 gene's polymorphisms and multiple sclerosis among residents of the Russian Federation. The results demonstrated the need to combine data from different researchers in clinical studies to increase the power of the study.