Search

Methylation systems in the cells play an important role in the metabolic processes such as purine nucleotide biosynthesis and gene and protein activity regulation. An imbalance between entities in folic acid metabolism can adversely affect nucleotide synthesis and the DNA repair and methylation system, which can cause genome instability and impairments in chromosome segregation, and lead to abnormal expression of proto-oncogenes and inactivation of tumor suppressor genes. These processes may underlie the development of a range of cancer disease, including Non-Hodgkin’s lymphomas (NHL). Quite a few studies investigating the association of SNPs in the folate-metabolizing genes with NHL risk in populations of different ethnic origin are available to date. Because the low prevalence of this disease makes sampling difficult, most of these studies have small sizes, which may be one of the reasons why results obtained are often conflicting. The aim of this study was to investigate the role of some SNPs in folate genes (the C677T and A1298C SNPs in the MTHFR gene, A2756G in MTR, A66G in SHMT1, G1958A in MTHFD1 and 844ins68 in CBS) in genetic susceptibility to non-Hodgkin’s malignant lymphoma in the west-Siberian region. Methods: 146 unrelated patients from the Haematological Center (Novosibirsk city) with various types of NHL were investigated. Genomic DNA was isolated from leukocytes in venous blood and from buccal epithelium, using the standard methods of DNA separation. A PCR-restriction fragment length polymorphism (RFLP) assay was used to detect the MTHFD1 G1958A and CBS 844ins68 SNPs. Genotyping of the MTHFR, MTR, MTRR and SHMT1 gene SNPs was carried out by real-time PCR allelic discrimination with TaqMan probes. The alleles and genotypes distribution of SNPs in patients were compared with their distribution in healthy white Russian subjects from Novosibirsk. Results: We determined the allele and genotype frequencies for seven SNPs in folate metabolism in NHL and control groups. For all these SNPs, the genotype frequencies were in Hardy–Weinberg equilibrium in the control group. There were no statistically significant differences in the frequencies of alleles and genotypes of polymorphic loci of MTHFR, MTRR, CBS, SHMT1 genes between patients with NHL and controls. However, theG1958A MTHFD1 polymorphism showed a significant association with aggressive NHL. The 1958A allele (OR = 0.578, C.I. [0.415–0.805], p

The incidence of homozygote deletion of glutathione S-transferase genes M1 and T1 (null genotypes; or GSTM1“-” and GSTT1“-”) was studied in breast cancer patients living in Altai Krai. DNA was isolated from blood samples of 695 breast cancer patients (291 patients with familial cancer and 404 patients with sporadic cancer) and 263 women without history of tumor diseases. The frequency of GSTM1“-” and с GSTT1“-” genotypes was estimated in breast can cer patients (47.2 and 19.1%, respectively) and non-cancer participants (46.8 and 19.0%, respectively). No differences were found in the frequency of genotypes. The frequency of genotype combination GSTM1“-”+GSTT1“-” in patients with sporadic breast cancer (11.6%, 47 of 404 patients) was higher than in the control (6.1%, 16 of 263 patients; OR=2.03; 95% CI=2.09-3.83; p=0.02). The genotype frequency of genes in the control group did not differ from that in European residents of the Caucasian race. [ABSTRACT FROM AUTHOR]

The W-Beijing family is a widespread Mycobacterium tuberculosis clonal lineage that frequently causes epidemic outbreaks. This family is genetically homogeneous and conserved, so ETR-VNTR (exact tandem repeat-variable number of tandem repeats) typing is insufficient for strain differentiation, due to a common ETR-A to E profile (42435). This leads to the false clustering in molecular epidemiological studies, especially in the regions of predominance of the W-Beijing family. In this study, we searched for VNTR loci with a high evolutionary rate of polymorphism in the W-Beijing genome. Here we further evaluated VNTR typing on a set of 99 Mycobacterium tuberculosis clinical isolates and reference strains. These isolates were characterized and classified into several genotype families based on three ETR loci (A, C, E) and eight additional loci [previously described as QUB (Queen’s University Belfast) or MIRU (Mycobacterial Interspersed Repetitive Units) or Mtubs]. Ninety-nine strains were divided into 74 VNTR-types, 51 isolates of the W-Beijing family identified by IS6110 RFLP-typing (the restriction fragment length polymorphism-typing) and/or spoligotyping were subdivided into 30 VNTR-types. HGDI (the Hunter–Gaston discriminatory index) for all studied loci was close to that of IS6110 RFLP typing, a “gold standard” method for subtyping M. tuberculosis complex strains. The QUB 26 and QUB 18 loci located in the PPE genes were highly polymorphic and more discriminative than other loci (HGDI is 0.8). Statistically significant increase of tandem repeats number in loci ETR-A, -E, QUB 26, QUB 18, QUB 11B, Mtub21 was revealed in the W-Beijing group compared to genetically divergent non-W-Beijing strains. Thirty-six isolates were subjected to IS6110 RFLP typing. The congruence between results of the IS6110 RFLP typing and 11-loci VNTR typing was estimated on 23 isolates of the W-Beijing family. These isolates were subdivided into 9 IS6110-RFLP types and 13 VNTR types. The poor profiles correlation (0.767) reflects the differences in the rate and type of evolution between genome regions targeted by IS6110-RFLP and VNTR typing. VNTR typing in proposed format is powerful tool for discrimination of M. tuberculosis strains with different level of genetic relationship. [ABSTRACT FROM AUTHOR]

Exposure to time-varying magnetic fields causes shielding currents to flow beneath the surface of a superconductor up to a field-dependent penetration depth. In trapped field applications of bulk superconductors, in which the decay of trapped field due to external AC magnetic fields is caused by current redistribution (and not by helating and temperature rise), this penetration depth determines the degree of current redistribution in the superconductor and, in turn, the degree of decay of trapped field. In this study we propose and validate experimentally a model to explain the rate of decay of trapped field in a single grain bulk GdBa2Cu3O7−δ (GdBCO) superconductor exposed to an AC magnetic field in a crossed-field configuration. The model is based on calculating the time dependence of the trapped field using the Biot–Savart law and assuming that the time dependence of the current density changes at the depth of penetration of the induced shielding currents. Inside the superconductor, where the crossed-field has not penetrated, the time dependence is assumed to be logarithmic and the decay of current density due to flux creep, whereas within the penetration depth of the surface the time dependence is assumed to be exponential and the decay of current density due to its redistribution. The penetration depth was measured separately using SQUID magnetometry and used as an input parameter to the model. The model was compared subsequently with measurements of the decay of trapped field and found to be in excellent agreement with the observed behaviour. [ABSTRACT FROM AUTHOR]

The Beijing genotype is the most distributed M. tuberculosis family in Kazakhstan. In this study, we identified dominant Beijing clusters in Kazakhstan and assessed their drug susceptibility profiles and association with the most widely spread mutation Ser531Leu of the rpoB gene and the mutation Ser315Thr of the katG gene associated with resistance to rifampicin and isoniazid, respectively. M. tuberculosis isolates ( n = 540) from new TB cases were included in the study. MIRU-VNTR genotyping was performed for 540 clinical isolates to determine M. tuberculosis families using 24 loci. RD analysis was additionally performed for the Beijing isolates. The identification of mutations in the drug-resistance genes of M. tuberculosis was performed with allele-specific real-time PCR and Sanger sequencing. The Beijing genotype was identified in 60% (324/540) of the clinical isolates. Central Asian/Russian cluster 94-32 was the most distributed cluster among the Beijing isolates (50.3%; 163/324). Three other dominant Beijing clusters were identified as 94-33 (3.4%; 11/324), 100-32 (3.1%; 10/324) and 99-32 (3.1%; 10/324). The Beijing genotype was associated with drug-resistant TB (p < 0.0001), including multidrug-resistant TB (p < 0.0001), in our study. An association of the mutation Ser531Leu of the rpoB gene with the Beijing genotype was found ( p < 0.0001; OR = 16.0000; 95%CI: 4.9161-52.0740). Among the Beijing isolates, cluster 94-32 showed an association with MDR-TB ( p = 0.021). This is why the evaluation of the Beijing genotype and its clusters is needed to control MDR-TB in Kazakhstan.

Detecting copy number variations (CNVs) and alterations (CNAs) in the BRCA1 and BRCA2 genes is essential for testing patients for targeted therapy applicability. However, the available bioinformatics tools were initially designed for identifying CNVs/CNAs in whole-genome or -exome (WES) NGS data or targeted NGS data without adaptation to the BRCA1/2 genes. Most of these tools were tested on sample cohorts of limited size, with their use restricted to specific library preparation kits or sequencing platforms. We developed BRACNAC, a new tool for detecting CNVs and CNAs in the BRCA1 and BRCA2 genes in NGS data of different origin. The underlying mechanism of this tool involves various coverage normalization steps complemented by CNV probability evaluation. We estimated the sensitivity and specificity of our tool to be 100% and 94%, respectively, with an area under the curve (AUC) of 94%. The estimation was performed using the NGS data obtained from 213 ovarian and prostate cancer samples tested with in-house and commercially available library preparation kits and additionally using multiplex ligation-dependent probe amplification (MLPA) (12 CNV-positive samples). Using freely available WES and targeted NGS data from other research groups, we demonstrated that BRACNAC could also be used for these two types of data, with an AUC of up to 99.9%. In addition, we determined the limitations of the tool in terms of the minimum number of samples per NGS run (≥20 samples) and the minimum expected percentage of CNV-negative samples (≥80%). We expect that our findings will improve the efficacy of BRCA1/2 diagnostics. BRACNAC is freely available at the GitHub server., Competing Interests: The authors declare no conflict of interest.

Purpose: To develop a method for testing the MSI based on targeted NGS., Methods: Based on the results of previous studies, 81 microsatellite loci with high variability in MSI-H tumors were selected, and a method for calculating the MSI score was developed. Using the MSI score, we defined the MSI status in endometral (162), colon (153), and stomach (190) cancers. Accuracy of the MSI scores was evaluated by comparison with MMR immunohistochemistry for 137 endometrium (63 dMMR and 74 pMMR), 76 colon (29 dMMR and 47 pMMR), and 81 stomach (8 dMMR and 73 pMMR) cancers., Results: Classification of MSS and MSI-H tumors was performed with AUC (0.99), sensitivity (92%), and specificity (98%) for all tumors without division into types. The accuracy of MSI testing in endometrial cancer was lower than for stomach and colon cancer (0.98, 87%, and 100%, respectively). The use of 27 loci only, the most informative for endometrial cancer, increased the overall accuracy (1.00, 99%, and 99%). Comparison of MSI score values in 505 tumors showed that MSI score is significantly higher in colon ( p < 10 -5 ) and stomach ( p = 0.008) cancer compared with endometrial cancer., Conclusion: The MSI score accurately determines MSI status for endometrial, colon, and stomach cancers and can be used to quantify the degree of MSI.

DNA polymerases are a superfamily of enzymes synthesizing DNA using DNA as a template. They are essential for nucleic acid metabolism and for DNA replication and repair. Modern biotechnology and molecular diagnostics rely heavily on DNA polymerases in analyzing nucleic acids. Among a variety of discovered DNA polymerases, Bst polymerase, a large fragment of DNA polymerase I from Geobacillus stearothermophilus, is one of the most commonly used but is not as well studied as Taq polymerase. The ability of Bst polymerase to displace an upstream DNA strand during synthesis, coupled with its moderate thermal stability, has provided the basis for several isothermal DNA amplification methods, including LAMP, WGA, RCA, and many others. Bst polymerase is one of the key components defining the robustness and analytical characteristics of diagnostic test systems based on isothermal amplification. Here, we present an overview of the biochemical and structural features of Bst polymerase and provide information on its mutated analogs., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results., (© 2023 The Authors.)

IS6110 insertion sequence is a frequently used target for Mycobacterium tuberculosis detection. However, its sequence variability is studied insufficiently. We aimed to identify the most conservative and variable regions in IS6110 sequences and develop qPCR and LAMP oligonucleotide sets for the conservative regions. Using in-house Python scripts, 3609 M. tuberculosis genome sequences from the NCBI database were aligned; conservative regions were identified to design oligonucleotide sets. IS6110 fragments located within the 31-231 bp region were the most conservative and represented in genomes and were used to design qPCR and LAMP oligonucleotides. The in silico sensitivity of the qPCR oligonucleotides on the whole genome set was 99.1% and 98.4%. For the LAMP primers developed, the sensitivity was 96.9%. For qPCR, the limit of detection with 95% confidence (LoD 95% ) was four IS6110 copies per reaction, with LoD 90% being 200 BCG cells per ml of artificial sputum. For LAMP, LoD 95% was 16 copies per reaction, with LoD 90% being 400 Mycobacterium bovis Bacille Calmette-Guerin (BCG) cells per ml of artificial sputum. We have demonstrated the IS6110 sequence variability and designed highly sensitive qPCR and LAMP oligonucleotides to detect M. tuberculosis., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: Pathogenic variants (PVs) in BRCA1 and BRCA2 genes are essential biomarkers of an increased breast and ovarian cancer risk and tumor sensitivity to poly ADP ribose polymerase inhibitors. In Russia, eight PVs were thought to be the most common, among which BRCA1 c.5266dup is the most frequently identified one., Methods: We show the distribution of BRCA1/2 PVs identified with quantitative PCR and targeted next-generation sequencing in 1399 ovarian cancer patients recruited into the study from 72 Russian regions in 2015-2021., Results: The most abundant PVs were c.5266dup (41.0%), c.4035del (7.0%), c.1961del (6.3%), c.181 T > G (5.2%), c.3756&#95;3759del (1.8%), c.3700&#95;3704del (1.5%), and c.68&#95;69del (1.5%), all found in BRCA1 and known to be recurrent in Russia. Several other frequent PVs were identified: c.5152 + 1G > T (1.2%), c.1687C > T (1.0%), c.4689C > G (0.9%), c.1510del (0.6%), c.2285&#95;2286del (0.6%) in the BRCA1 gene; and c.5286 T > G (1.2%), c.2808&#95;2811del (0.8%), c.3847&#95;3848del (0.8%), c.658&#95;659del (0.7%), c.7879A > T (0.6%), in the BRCA2 gene. For the most common PV in the BRCA2 gene c.5286 T > G, we suggested that it arose about 700 years ago and is a new founder mutation., Conclusion: This study extends our knowledge about the BRCA1 and BRCA2 pathogenic variants variability., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Mutations in homologous recombination (HR) and Fanconi anemia (FA) genes may predispose to pancreatic cancer (PC) and enable the prediction of sensitivity to platinum-based chemotherapy. FOLFIRINOX is a standard treatment option for non-selected PC patients and could be effective due to undiagnosed DNA repair deficiency. Here, we aimed to determine the frequency of mutations in genes involved in the HR and FA pathways, evaluate their clinical implications, and determine the objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) of PC patients treated with platinum., Methods: We performed targeted DNA sequencing of 30 genes ( ABRAXAS1 , ATM , ATR , BARD1 , BLM , BRCA1 , BRCA2 , BRIP1 , CDKN2A , CHEK1 , CHEK2 , FANCC , FANCF , FANCG , FANCI , FANCL , FANCM , MRE11A , NBN , PALB2 , PTEN , RAD50 , RAD51C , RAD51D , RAD52 , RAD54B , RBBP8 , RINT1 , SLX4 , and XRCC2 ) for 543 PC patients., Results: In BRCA/PALB2 -mutated patients with advanced PC (33 patients, 6.1%), the PFS and OS were higher for first-line platinum therapy than for non-platinum therapy [PFS: HR = 0.28, 95% confidence interval (CI) = 0.10-0.81, p  = 0.02; OS: HR = 0.31, 95% CI = 0.08-1.16, p  = 0.08]. Among 93 patients (17.1%) with mutations in other HR/FA genes, no statistically significant difference in PFS and OS was observed between first-line platinum therapy and non-platinum therapy (PFS: HR = 0.83, 95% CI = 0.43-1.62, p  = 0.59; OS: HR = 0.58, 95% CI = 0.28-1.22, p  = 0.15). For patients with early PC, no prognostic value was observed for BRCA1/2 , PALB2 , or other HR/FA genes mutations. Moreover, a personal history of breast, ovarian, pancreatic, or prostate cancer was identified as the only independent predictor of the risk of BRCA/PALB2 mutations (HR = 5.83, 95% CI = 2.16-15.73, p  < 0.01)., Conclusion: Mutations in the BRCA1/2 and PALB2 genes increase the sensitivity of PC to platinum agents. Thus, alterations in these genes in PC patients must be determined prior to anticancer therapy., Competing Interests: Conflict of interest statement: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s), 2022.)

There are hundreds of coronaviruses, most of which circulate among animals, yet there are seven types that infect humans. Three of them can cause severe acute respiratory illness-SARS-CoV, SARS-CoV-2, and MERS-CoV. Other HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1 usually cause only mild to moderate upper respiratory tract infections. These four coronaviruses are called seasonal, because they are continuously circulating among human population and are responsible for up to 30% of all respiratory tract infections. Genetically, these low-pathogenic types are related to SARS-CoV-2. That is why questions concerning the cross-reactivity and cross-neutralization between antibodies against different types of coronaviruses have been raised. We addressed these questions by using enzyme-linked immunosorbent assays and targeted next-generation sequencing (NGS). We established the upper respiratory infection etiology for three patients who had been vaccinated with Sputnik V and tested positive on anti-SARS-CoV-2 antibodies. The symptoms included sore throat, nasal congestion, and myalgia. Their blood serum was analyzed for anti-SARS-CoV-2 antibodies in dynamics: before vaccination, and after the first and second dose of the vaccine. After the second dose, all patients were positive for IgG antibodies against SARS-CoV-2. The targeted NGS panel sequencing data analysis showed that these patients were infected with common coronavirus HCoV-OC43. These results suggest that S protein-targeted vaccine-induced antibodies against SARS-CoV-2 are not protective against seasonal coronavirus HCoV-OC43.

Next-generation sequencing (NGS)-library preparation for whole-genome sequencing (WGS) starts with DNA fragmentation, and sonication is a physical approach used most often due to its simplicity and reproducibility. However, the commercially available Covaris instrument has a high price for both the device and consumables. Here, we describe our in-house method of DNA shearing by sonication with small (100-600 µm) glass beads and an ultrasonic bath. The fragmentation conditions were optimized for the bacterial WGS with ∼550-bp fragment size (the ultrasonic bath water temperature 5-10°C, glass beads 0.06 g, the fragmentation time 50 s) and for human DNA with ∼250 bp (fragmentation with the same parameters for 4 min). Fragmentation results were compared with the Covaris instrument for preparing several bacterial NGS libraries for Illumina NGS platforms by several characteristics. We obtained close mean fragment lengths (523-623 versus 480-646), similar mono- and dinucleotide specificity of shearing, and comparable indicators of read alignment and de novo assembly for both methods. Thus, the described method is a new fast, and effective DNA fragmentation approach that can be used in different WGS applications., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Currently, several materials for the closure of the dura mater (DM) defects are known. However, the long-term results of their usage reveal a number of disadvantages. The use of antibiotics and chitosan is one of the major trends in solving the problems associated with infectious after-operational complications. This work compares the mechanical properties of samples of bacterial nanocellulose (BNC) impregnated with Novochizol™ and vancomycin with native BNC and preserved and native human DM. An assessment of the possibility of controling the mechanical properties of these materials by changing their thickness has been performed by statistical analysis methods. A total of 80 specimens of comparable samples were investigated. During the analysis, the results obtained, the factor of Novochizol™ addition has provided a statistically significant impact on the strength properties (Fisher Criteria p -value 0.00509 for stress and 0.00112 for deformation). Moreover, a stronger relationship between the thickness of the samples and their ultimate load was shown: R2=0.236 for BNC + Novochizol™ + vancomycin, compared to R2=0.0405 for native BNC. Using factor analysis, it was possible to show a significant effect of modified chitosan (Novochizol™) on the ultimate stress ( p -value = 0.005).

Mutations in the BRCA1 and BRCA2 genes are known risk factors and drivers of breast and ovarian cancers. So far, few studies have been focused on understanding the differences in transcriptome and functional landscapes associated with the disease (breast vs. ovarian cancers), gene ( BRCA1 vs. BRCA2 ), and mutation type (germline vs. somatic). In this study, we were aimed at systemic evaluation of the association of BRCA1 and BRCA2 germline and somatic mutations with gene expression, disease clinical features, outcome, and treatment. We performed BRCA1/2 mutation centered RNA-seq data analysis of breast and ovarian cancers from the TCGA repository using transcriptome and phenotype "portrayal" with multi-layer self-organizing maps and functional annotation. The results revealed considerable differences in BRCA1 - and BRCA2 -dependent transcriptome landscapes in the studied cancers. Furthermore, our data indicated that somatic and germline mutations for both genes are characterized by deregulation of different biological functions and differential associations with phenotype characteristics and poly(ADP-ribose) polymerase (PARP)-inhibitor gene signatures. Overall, this study demonstrates considerable variation in transcriptomic landscapes of breast and ovarian cancers associated with the affected gene ( BRCA1 vs. BRCA2 ), as well as the mutation type (somatic vs. germline). These results warrant further investigations with larger groups of mutation carriers aimed at refining the understanding of molecular mechanisms of breast and ovarian cancers.

Multiplex polymerase chain reaction (PCR) has multiple applications in molecular biology, including developing new targeted next-generation sequencing (NGS) panels. We present NGS-PrimerPlex, an efficient and versatile command-line application that designs primers for different refined types of amplicon-based genome target enrichment. It supports nested and anchored multiplex PCR, redistribution among multiplex reactions of primers constructed earlier, and extension of existing NGS-panels. The primer design process takes into consideration the formation of secondary structures, non-target amplicons between all primers of a pool, primers and high-frequent genome single-nucleotide polymorphisms (SNPs) overlapping. Moreover, users of NGS-PrimerPlex are free from manually defining input genome regions, because it can be done automatically from a list of genes or their parts like exon or codon numbers. Using the program, the NGS-panel for sequencing the LRRK2 gene coding regions was created, and 354 DNA samples were studied successfully with a median coverage of 97.4% of target regions by at least 30 reads. To show that NGS-PrimerPlex can also be applied for bacterial genomes, we designed primers to detect foodborne pathogens Salmonella enterica, Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus considering variable positions of the genomes., Competing Interests: The authors have declared that no competing interests exist.

Immunoglobulin G (IgG) is the most abundant serum antibody which structural characteristics and effector functions are modulated through the attachment of various sugar moieties called glycans. Composition of the IgG N-glycome changes with age of an individual and in different diseases. Variability of IgG glycosylation within a population is well studied and is known to be affected by both genetic and environmental factors. However, global inter-population differences in IgG glycosylation have never been properly addressed. Here we present population-specific N-glycosylation patterns of IgG, analyzed in 5 different populations totaling 10,482 IgG glycomes, and of IgG's fragment crystallizable region (Fc), analyzed in 2,579 samples from 27 populations sampled across the world. Country of residence associated with many N-glycan features and the strongest association was with monogalactosylation where it explained 38% of variability. IgG monogalactosylation strongly correlated with the development level of a country, defined by United Nations health and socioeconomic development indicators, and with the expected lifespan. Subjects from developing countries had low levels of IgG galactosylation, characteristic for inflammation and ageing. Our results suggest that citizens of developing countries may be exposed to environmental factors that can cause low-grade chronic inflammation and the apparent increase in biological age.

Nowadays the advent of innovative high-throughput sequencing allows obtaining high-quality microbiome profiling. However, PCR-based tests are still considered the "golden standard" for many clinical applications. Here, we designed a qPCR-based platform with fluorescent-labeled oligonucleotide probes for assessing human gut microbiome composition. The system allows conducting qualitative and semiquantitative analysis for 12 prokaryotic taxa that are prevalent in the human gut and associated with diseases, diet, age and other factors. The platform was validated by comparing microbiome profile data obtained with two different methods - the platform and high-throughput 16S rRNA sequencing - across 42 stool samples. The test can form the basis for precise and cost-efficient microbiome assay for large-scale surveys including clinical trials with interventions related to diet and disease risks., Competing Interests: Decalartion of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Primary immunodeficiency diseases (PID) area heterogeneous group of disorders caused by genetic defects of the immune system, which manifest clinically as recurrent infections, autoimmune diseases or malignancies. Early detection of PID remains a challenge, particularly in older children with milder and less specific symptoms. This study aimed to assess TREC and KREC diagnostic ability in PID. Data from children assessed by clinical immunologists at Speransky Children's Hospital, Moscow, Russia with suspected immunodeficiencies were analyzed between May 2013 and August 2016. Peripheral blood samples were sent for TREC/KREC, flow cytometry (CD3, CD4, CD8 and CD19), IgA and IgG analysis. A total of 434 children [189 healthy, 97 with group I and II PID (combined T and B cell immunodeficiencies & well-defined syndromes with immunodeficiency) and 148 group III PID (predominantly antibody deficiencies)] were included. Area under the curve (AUC) for TREC in PID groups I and II diagnosis reached 0.82 (CI = 0.75-0.90), with best model providing sensitivity of 65% and specificity of 92%. Neither TREC, nor KREC had added value in PID group III diagnosis. In this study, the predictive value of TREC and KREC in PID diagnosis was examined. We found that the TREC had some diagnostic utility for groups I and II PID. Possibly, addition of TREC measurements to existing clinical diagnostic algorithms may improve their predictive value. Further investigations on a larger cohort are needed to evaluate TREC/KREC abilities to be used as diagnostic tools on a wider scale., (Copyright © 2020 Korsunskiy, Blyuss, Gordukova, Davydova, Zaikin, Zinovieva, Zimin, Molchanov, Salpagarova, Eremeeva, Filipenko, Prodeus, Korsunskiy, Hsu and Munblit.)

Purpose: Germinal and somatic rearrangements in BRCA1 gene play a significant role in carcinogenesis of breast and ovarian cancer. The present study is dedicated to the development of multiplex droplet digital PCR (ddPCR) assay for detecting large deletions and duplications in the BRCA1 gene., Methods: In-house tetraplex ddPCR assay for BRCA1 gene analysis was used for testing of DNA samples with BRCA1 status., Results: DNA specimens were purified from 24 individuals. The presence of BRCA1 rearrangements in samples was confirmed by a commercial MLPA-based kit. An amplitude-based multiplex ddPCR assay was developed: 8 multiplexes, each containing primers and probes to amplify 3 BRCA1 exons and 1 reference gene (ALB or RPP30). A novel assay demonstrated 100% concordance with the commercial MLPA-based kit, identifying 9 specimens with different deletions in BRCA1, 1 with duplication, and 14 with the wild-type BRCA1., Conclusions: We have designed a simple, precise, and cost-effective assay for BRCA1 rearrangement testing, based on ddPCR. The developed assay is the first multiplex ddPCR-based test that provides results in accordance with MLPA and can be used for routine clinical screening.

We describe the genetic diversity of 1327 Brucella strains from human patients in Kazakhstan using multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA). All strains were assigned to the Brucella melitensis East Mediterranean group and clustered into 16 MLVA11 genotypes, nine of which are reported for the first time. MLVA11 genotype 116 predominates (86.8%) and is present all over Kazakhstan indicating existence and temporary preservation of a "founder effect" among B. melitensis strains circulating in Central Eurasia. The diversity pattern observed in humans is highly similar to the pattern previously reported in animals. The diversity observed by MLVA suggested that the epidemiological status of brucellosis in Kazakhstan is the result of the introduction of a few lineages, which have subsequently diversified at the most unstable tandem repeat loci. This investigation will allow to select the most relevant strains for testing these hypotheses via whole genome sequencing and to subsequently adjust the genotyping scheme to the Kazakhstan epidemiological situation.

Background: The search for molecular biomarkers of early-onset colorectal cancer (CRC) is an important but still quite challenging and unsolved task. Detection of CpG methylation in human DNA obtained from blood or stool has been proposed as a promising approach to a noninvasive early diagnosis of CRC. Thousands of abnormally methylated CpG positions in CRC genomes are often located in non-coding parts of genes. Novel bioinformatic methods are thus urgently needed for multi-omics data analysis to reveal causative biomarkers with a potential driver role in early stages of cancer., Methods: We have developed a method for finding potential causal relationships between epigenetic changes (DNA methylations) in gene regulatory regions that affect transcription factor binding sites (TFBS) and gene expression changes. This method also considers the topology of the involved signal transduction pathways and searches for positive feedback loops that may cause the carcinogenic aberrations in gene expression. We call this method "Walking pathways", since it searches for potential rewiring mechanisms in cancer pathways due to dynamic changes in the DNA methylation status of important gene regulatory regions ("epigenomic walking")., Results: In this paper, we analysed an extensive collection of full genome gene-expression data (RNA-seq) and DNA methylation data of genomic CpG islands (using Illumina methylation arrays) generated from a sample of tumor and normal gut epithelial tissues of 300 patients with colorectal cancer (at different stages of the disease) (data generated in the EU-supported SysCol project). Identification of potential epigenetic biomarkers of DNA methylation was performed using the fully automatic multi-omics analysis web service "My Genome Enhancer" (MGE) (my-genome-enhancer.com). MGE uses the database on gene regulation TRANSFAC®, the signal transduction pathways database TRANSPATH®, and software that employs AI (artificial intelligence) methods for the analysis of cancer-specific enhancers., Conclusions: The identified biomarkers underwent experimental testing on an independent set of blood samples from patients with colorectal cancer. As a result, using advanced methods of statistics and machine learning, a minimum set of 6 biomarkers was selected, which together achieve the best cancer detection potential. The markers include hypermethylated positions in regulatory regions of the following genes: CALCA, ENO1, MYC, PDX1, TCF7, ZNF43.

Primary immunodeficiency diseases (PID) is a heterogeneous group of disorders caused by genetic defects of the immune system, which manifests clinically as recurrent infections, autoimmune diseases, or malignancies. Early detection of other PID remains a challenge, particularly in older children due to milder and less specific symptoms, a low level of clinician PID awareness and poor provision of hospital laboratories with appropriate devices. T-cell recombination excision circles (TREC) and kappa-deleting element recombination circle (KREC) in a dried blood spot and in peripheral blood using real-time polymerase chain reaction (PCR) are used as a tool for severe combined immune deficiency but not in PID. They represent an attractive and cheap target for a more extensive use in clinical practice. This study aimed to assess TREC/KREC correspondence with lymphocyte subpopulations, measured by flow cytometry and evaluate correlations between TREC/KREC, lymphocyte subpopulations and immunoglobulins. We carried out analysis of data from children assessed by clinical immunologists at Speransky Children's Hospital, Moscow, Russia with suspected immunodeficiencies between May 2013 and August 2016. Peripheral blood samples were sent for TREC/KREC, flow cytometry (CD3, CD4, CD8, and CD19), IgA, IgM, and IgG analysis. A total of 839 samples were analyzed for using TREC assay and flow cytometry and 931 KREC/flow cytometry. TREC demonstrated an AUC of 0.73 (95% CI 0.70-0.76) for CD3, 0.74 (95% CI 0.71-0.77) for CD4 and 0.67 (95% CI 0.63-0.70) for CD8, respectively, while KREC demonstrated an AUC of 0.72 (95% CI 0.69-0.76) for CD19. Moderate correlation was found between the levels of TREC and CD4 ( r = 0.55, p < 0.01) and KREC with CD19 ( r = 0.56, p < 0.01). In this study, promising prediction models were tested. We found that TREC and KREC are able to moderately detect abnormal levels of individual lymphocyte subpopulations. Future research should assess associations between TREC/KREC and other lymphocyte subpopulations and approach TREC/KREC use in PID diagnosis.

The use of targeted next-generation sequencing (NGS) provides great new opportunities for molecular and medical genetics. However, in order to take advantage of these opportunities, we need to have reliable tools for extracting the necessary information from the huge amount of data generated by NGS. Here we present our automatic multithreaded workflow for processing NGS data of BRCA1 and BRCA2 genes obtained with NGS technology named BRCA-analyzer. Optimizing it on the sequencing data of 899 samples from 693 patients, we were able to find the most reliable tools and adjust their parameters in such a way that all pathogenic variants found were confirmed by Sanger's sequencing. For 82 and 24 DNA samples from blood and formalin-fixed paraffin-embedded blocks, NGS libraries were prepared with GeneRead BRCA panel v2 (Qiagen). The reads obtained were processed with BRCA-analyzer and Qiagen GeneRead Data analysis workflow. In total 27 pathogenic variants were found and confirmed by Sanger's sequencing, with all of them determined with BRCA-analyzer. Qiagen GeneRead Data analysis discarded 5 true pathogenic variants due to their location in homopolymeric sequence stretches. For other 793 samples, libraries were prepared by the in-house method, and NGS data were analyzed by BRCA-analyzer in comparison to another free automatic amplicon NGS workflow Canary. From total 137 pathogenic variations, BRCA-analyzer found 135 and Canary 123. Mutations were missed by BRCA-analyzer due to the trimming primer sequences from reads before mapping to be fixed in the next version. On the freely available NGS data, we showed that BRCA-analyzer could also be used for hybrid capture gene panels, although it needs more extensive testing on such library preparation methods. Thus, BRCA-analyzer is an automatic workflow for processing NGS data of BRCA1/2 genes with variant filters adapted to amplicon-based targeted NGS data. BRCA-analyzer can be used to identify germline as well as somatic mutations. BRCA-analyzer is freely available at https://github.com/aakechin/BRCA-analyzer., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Heredity plays an important role in the etiology of varicose veins (VVs). However, the genetic basis underlying this condition remains poorly understood. Our aim was to replicate top association signals from genome-wide association studies (GWASs) for VVs of lower extremities using 2 independent datasets-our sample of ethnic Russian individuals (709 cases and 278 controls) and a large cohort of British residents from UK Biobank (10 861 cases and 397 594 controls). Associations of polymorphisms rs11121615, rs6712038, rs507666, rs966562, rs7111987, rs6062618, and rs6905288 were validated in the UK Biobank individuals at a Bonferroni-corrected significance level. In Russian cohort, only rs11121615 reached a nominal significance level of P < .05. Results of original GWAS and replication studies were combined by a meta-analysis, and polymorphisms listed above as well as rs111434909 and rs4463578 passed a genome-wide significant threshold. Notably, the majority of these polymorphisms were located within or near genes involved in vascular development and remodeling, and regulation of inflammatory response. Our results confirm the role of these polymorphisms in genetic susceptibility to VVs and indicate the revealed genomic regions as good candidates for further fine-mapping studies and functional analysis. Moreover, our findings implicate inflammation and abnormal vascular architecture in VVs pathogenesis., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Heredity is a well-known risk factor for varicose veins, but genetic basis of this condition remains poorly studied. Our aim was to conduct a large-scale genetic association study for primary varicose veins (PVVs) in the population of ethnic Russians. An initial scan using Illumina HumanExome-12 v1.0 BeadChip was performed for 273 patients with PVVs and 250 controls without a history of chronic venous disease and other venous disorders. After quality control and removal of monomorphic markers, 25,424 common and 48,232 rare variants were included in the analysis. 42 single nucleotide polymorphisms (SNPs) were genotyped in the independent replication cohort of 447 PVVs patients and 443 controls. Association of common variants with PVVs was investigated by logistic regression, and the impact of rare variants was analyzed using sequence kernel association test. No effect of low frequency alleles has been revealed in our study. Common variant analysis identified a promising signal at chromosome 6 within classical major histocompatibility complex (MHC) class III subregion. The most strongly associated SNP in a combined analysis that reached a suggestive significance level of 3.2e-05 was polymorphism rs4151657 in the complement factor B gene. Testing for potential pleiotropy with other traits indicated that the same causal variant in this region increases the risk of rheumatoid arthritis and has a negative impact on human height. Our results provide suggestive evidence for the involvement of the MHC class III genes in the pathogenesis of PVVs. Further independent studies are needed to confirm our pilot findings., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Small molecule Nutlin-3 reactivates p53 in cancer cells by interacting with the complex between p53 and its repressor Mdm-2 and causing an increase in cancer cell apoptosis. Therefore, Nutlin-3 has potent anticancer properties. Clinical and experimental studies of Nutlin-3 showed that some cancer cells may lose sensitivity to this compound. Here we analyze possible mechanisms for insensitivity of cancer cells to Nutlin-3., Methods: We applied upstream analysis approach implemented in geneXplain platform ( genexplain.com ) using TRANSFAC® database of transcription factors and their binding sites in genome and using TRANSPATH® database of signal transduction network with associated software such as Match™ and Composite Module Analyst (CMA)., Results: Using genome-wide gene expression profiling we compared several lung cancer cell lines and showed that expression programs executed in Nutlin-3 insensitive cell lines significantly differ from that of Nutlin-3 sensitive cell lines. Using artificial intelligence approach embed in CMA software, we identified a set of transcription factors cooperatively binding to the promoters of genes up-regulated in the Nutlin-3 insensitive cell lines. Graph analysis of signal transduction network upstream of these transcription factors allowed us to identify potential master-regulators responsible for maintaining such low sensitivity to Nutlin-3 with the most promising candidate mTOR, which acts in the context of activated PI3K pathway. These finding were validated experimentally using an array of chemical inhibitors., Conclusions: We showed that the Nutlin-3 insensitive cell lines are actually highly sensitive to the dual PI3K/mTOR inhibitor NVP-BEZ235, while no responding to either PI3K -specific LY294002 nor Bcl-XL specific 2,3-DCPE compounds.

Inflammation was shown to be activated in varicose veins, although its role in the development of vein wall transformation remains inconclusive. We aimed to investigate the influence of 13 inflammation-related single nucleotide polymorphisms (SNPs) TNF rs1800629 and rs3093661, IL1A rs1800587, IL1RN rs4251961, IL6 rs1800795 and rs1800796, IFNG rs2430561, IL10 rs1800896, TGFB1 rs1800469, HIF1A rs11549465, NFKB1 rs28362491, and rs4648068 on the risk of primary varicose veins (PVVs) in ethnic Russians. We genotyped 709 patients with PVVs and 278 individuals without a history of chronic venous disease and performed a single SNP and a haplotype analysis. Several associations with P < 0.05 were revealed in our study. Variant allele HIF1A rs11549465 T, TNF rs3093661 A, and NFKB1 rs28362491 ATTG deletion showed the reverse association with PVV risk, and allele IL6 rs1800795 C was associated with the increased risk of the studied pathology. Haplotype analysis revealed associations of TNF haplotypes rs3093661 A-rs1800629 G and IL6 rs1800795 C-rs1800796 G with the decreased and the increased risk of PVVs, correspondingly. However, all the observed associations failed to reach statistical significance after the correction for multiple testing, which was set at a level of 10 -3 due to many tests performed. Our study therefore provides evidence that investigated polymorphisms do not play a major role in susceptibility to PVVs.

Cutting of primers from reads is an important step of processing targeted amplicon-based next generation sequencing data. Existing tools are adapted for cutting of one or several primer/adapter sequences from reads and removing all of their occurrences. Also most of the existing tools use kmers and may cut only part of primers or primers with studied sequence of gene. Because of this, use of such programs leads to incorrect trimming, reduction of coverage, and increase in the number of false-positive and/or false-negative results. We have developed a new tool named cutPrimers for accurate cutting of any number of primers from reads. Using sequencing reads that were obtained during study of BRCA1/2 genes, we compared it with cutadapt, AlienTrimmer, and BBDuk. All of them trimmed reads in such a way that coverage of at least two amplicons decreased to unacceptable level (<30 reads) comparing with reads trimmed with cutPrimers. At the same time, Trimmomatic and AlienTrimmer cut all occurrences of primer sequences, so the length of the remaining reads was less than prospective.

Background: Analysis of EGFR mutations is becoming a routine clinical practice but the optimal EGFR mutation testing method is still to be determined., Methods: We determined the nucleotide sequence of deletions located in exon 19 of the EGFR gene in lung tumor samples of patients residing in different regions of Russia (153 tumor DNA specimens), using Sanger sequencing. We developed a droplet digital polymerase chain reaction assay capable of detecting all common EGFR deletions in exon 19. We also compared the therascreen amplification refractory mutation system assay with a droplet digital polymerase chain reaction assay for the detection of all the deletions in our study., Results: The droplet digital polymerase chain reaction assay demonstrated 100% sensitivity against polymerase chain reaction fragment length analysis and detected all possible types of deletions revealed in our study (22 types). At the same time, the therascreen EGFR RGQ PCR Kit was not able to detect deletions c.2252-2276>A and c.2253-2276 and showed low performance for another long deletion., Conclusion: Thus, we can conclude that the extraordinary length of deletions and their atypical locations (shift at the 3'-region compared to known deletions) could be problematic for the therascreen EGFR RGQ PCR Kit and should be taken into account during targeted mutation test development. However, droplet digital polymerase chain reaction is a promising and reliable assay that can be used as a diagnostic tool to genotype formalin-fixed paraffin-embedded cancer samples for EGFR or another clinically relevant somatic mutation.

Two hundred and twenty eight ethnic Russian individuals from Moscow, Russia, were genotyped at 14 single nucleotide polymorphisms CCL2 A-2578G; VEGFA C-2578A, G-634C, and C+936T; TNF G+419A and G-308A; IL1A G-889A; IL1RN T+1018C; IL6G-174C and G-572C; IFNG T+874A; IL1B C-511T; IL10 A+1082G; TGFB1 C-509T. Genotypes were determined using real-time polymerase chain reaction with TaqMan probes and polymerase chain reaction followed by melting analysis of dual-labeled probe. Genotype distribution was in accordance with Hardy-Weinberg equilibrium for all studied polymorphisms. Genotype data are available in the Allele Frequencies Net Database under identifier AFND 3367 and the population name "Russia Moscow Cytokine"., (Copyright © 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Brucellosis is a major zoonotic infection in Kazakhstan. However, there is limited data on its incidence in humans and animals, and the genetic diversity of prevalent strains is virtually unstudied. Additionally, there is no detailed overview of Kazakhstan brucellosis control and eradication programs. Here, we analyzed brucellosis epidemiological data, and assessed the effectiveness of eradication strategies employed over the past 70 years to counteract this infection. We also conducted multiple loci variable-number tandem repeat analysis (MLVA) of Brucella abortus strains found in Kazakhstan. We analyzed official data on the incidence of animal brucellosis in Kazakhstan. The records span more than 70 years of anti-brucellosis campaigns, and contain a brief description of the applied control strategies, their effectiveness, and their impact on the incidence in humans. The MLVA-16 method was used to type 94 strains of B. abortus and serial passages of B. abortus 82, a strain used in vaccines. MLVA-8 and MLVA-11 analyses clustered strains into a total of four and seven genotypes, respectively; it is the first time that four of these genotypes have been described. MLVA-16 analysis divided strains into 28 distinct genotypes having genetic similarity coefficient that varies from 60 to100% and a Hunter & Gaston diversity index of 0.871. MST analysis reconstruction revealed clustering into "Kazakhstani-Chinese (Central Asian)", "European" and "American" lines. Detection of multiple genotypes in a single outbreak confirms that poorly controlled trade of livestock plays a crucial role in the spread of infection. Notably, the MLVA-16 profile of the B. abortus 82 strain was unique and did not change during 33 serial passages. MLVA genotyping may thus be useful for epidemiological monitoring of brucellosis, and for tracking the source(s) of infection. We suggest that countrywide application of MLVA genotyping would improve the control of brucellosis in Kazakhstan., Competing Interests: The authors have declared that no competing interests.