BRACNAC: A BRCA1 and BRCA2 Copy Number Alteration Caller from Next-Generation Sequencing Data.

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.

Detecting Microsatellite Instability in Endometrial, Colon, and Stomach Cancers Using Targeted NGS.

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.

A spectrum of BRCA1 and BRCA2 germline deleterious variants in ovarian cancer in Russia.

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_3759del (1.8%), c.3700_3704del (1.5%), and c.68_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_2286del (0.6%) in the BRCA1 gene; and c.5286 T > G (1.2%), c.2808_2811del (0.8%), c.3847_3848del (0.8%), c.658_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.

Multiplex Droplet Digital PCR Assay for Detection of MET and HER2 Genes Amplification in Non-Small Cell Lung Cancer.

Non-small-cell lung cancer (NSCLC), a subtype of lung cancer, remains one of the most common tumors with a high mortality and morbidity rate. Numerous targeted drugs were implemented or are now developed for the treatment of NSCLC. Two genes, HER2 and MET, are among targets for these specific therapeutic agents. Alterations in HER2 and MET could lead to primary or acquired resistance to commonly used anti-EGFR drugs. Using current methods for detecting HER2 and MET amplifications is time and labor-consuming; alternative methods are required for HER2 and MET testing. We developed the first multiplex droplet digital PCR assay for the simultaneous detection of MET and HER2 amplification in NSCLC samples. The suitability of qPCR was assessed for the optimization of multiplex ddPCR. The optimal elongation temperature, reference genes for DNA quantification, and amplicon length were selected. The developed ddPCR was validated on control samples with various DNA concentrations and ratios of MET and HER2 genes. Using ddPCR, 436 EGFR-negative NSCLC samples were analyzed. Among the tested samples, five specimens (1.15%) showed a higher ratio of MET, and six samples (1.38%) showed a higher ratio of HER2. The reported multiplex ddPCR assay could be used for the routine screening of MET and HER2 amplification in NSCLC samples.

An inexpensive, simple and effective method of genome DNA fragmentation for NGS libraries.

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.

NGS-PrimerPlex: High-throughput primer design for multiplex polymerase chain reactions.

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.

The attachment of a DNA-binding Sso7d-like protein improves processivity and resistance to inhibitors of M-MuLV reverse transcriptase.

Reverse transcriptases (RTs) are a standard tool in both fundamental studies and diagnostics. RTs should possess elevated temperature optimum, high thermal stability, processivity and tolerance to contaminants. Here, we constructed a set of chimeric RTs, based on the combination of the Moloney murine leukaemia virus (M-MuLV) RT and either of two DNA-binding domains: the DNA-binding domain of the DNA ligase from Pyrococcus abyssi or the DNA-binding Sto7d protein from Sulfolobus tokodaii. The processivity and efficiency of cDNA synthesis of the chimeric RT with Sto7d at the C-end are increased several fold. The attachment of Sto7d enhances the tolerance of M-MuLV RT to the most common amplification inhibitors: NaCl, urea, guanidinium chloride, formamide, components of human whole blood and human blood plasma. Thus, fusing M-MuLV RT with an additional domain results in more robust and efficient RTs.

Spectrum of TP53 Mutations in BRCA1/2 Associated High-Grade Serous Ovarian Cancer.

Objective: Mutations in TP53 lead to loss of function (LOF) or gain of function (GOF) of the corresponding protein p53 and produce a different effect on the tumor. Our goal was to determine the spectrum of somatic TP53 variants in BRCA1/2 associated high-grade serous ovarian cancer (HGSOC). Methods: The population under study comprised of HGSOCs with pathogenic variants in BRCA1 (n = 78) or BRCA2 (n = 21). Only chemo-naive and platinum-sensitive patients were included in this study. The case group of the IARC database (n = 1249) with HGSOC not stratified by BRCA status was used as a reference. A custom NGS panel was used for sequencing TP53 and mutational hot-spots of other genes, and p53 expression was evaluated by immunohistochemistry for 68 cases of HGSOCs. Results: Somatic TP53 variants (95) or inhibition of wild-type p53 expression (3) were observed in 98 cases. The sample with normal p53 had CDKNA1 variants. The frequency of truncating variants was significantly higher than in the reference cohort (30.3 vs. 21.0%, p = 0.01). Most of the samples (41/68) demonstrated low (or absent) expression of p53, and 17 samples overexpressed p53. LOH was typical for TP53 nonsense variants (14/15). In total, 68/95 samples were LOH positive and showed LOH in all tumorous cells, thus indicating the driver effect of TP53 mutations. Three specimens had KRAS, BAX, APC, and CTNNB1 subclones variants. Conclusion: High frequency of TP53 truncating variants, the low expression of mutant p53, and low incidence of oncogene mutations show potential GOF properties of p53 to be poorly represented in BRCA1/2 associated HGSOC.

Walking pathways with positive feedback loops reveal DNA methylation biomarkers of colorectal cancer.

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.

Computational master-regulator search reveals mTOR and PI3K pathways responsible for low sensitivity of NCI-H292 and A427 lung cancer cell lines to cytotoxic action of p53 activator Nutlin-3.

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.

Derivatives of Bst-like Gss-polymerase with improved processivity and inhibitor tolerance.

At the moment, one of the actual trends in medical diagnostics is a development of methods for practical applications such as point-of-care testing, POCT or research tools, for example, whole genome amplification, WGA. All the techniques are based on using of specific DNA polymerases having strand displacement activity, high synthetic processivity, fidelity and, most significantly, tolerance to contaminants, appearing from analysed biological samples or collected under purification procedures. Here, we have designed a set of fusion enzymes based on catalytic domain of DNA polymerase I from Geobacillus sp. 777 with DNA-binding domain of DNA ligase Pyrococcus abyssi and Sto7d protein from Sulfolobus tokodaii, analogue of Sso7d. Designed chimeric DNA polymerases DBD-Gss, Sto-Gss and Gss-Sto exhibited the same level of thermal stability, thermal transferase activity and fidelity as native Gss; however, the processivity was increased up to 3-fold, leading to about 4-fold of DNA product in WGA which is much more exiting. The attachment of DNA-binding proteins enhanced the inhibitor tolerance of chimeric polymerases in loop-mediated isothermal amplification to several of the most common DNA sample contaminants-urea and whole blood, heparin, ethylenediaminetetraacetic acid, NaCl, ethanol. Therefore, chimeric Bst-like Gss-polymerase will be promising tool for both WGA and POCT due to increased processivity and inhibitor tolerance.

Highly Sensitive and Reliable Detection of EGFR Exon 19 Deletions by Droplet Digital Polymerase Chain Reaction.

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.

Comparison of fluorescent intercalating dyes for quantitative loop-mediated isothermal amplification (qLAMP).

Real-time or quantitative loop-mediated isothermal amplification (qLAMP) is a promising technique for the accurate detection of pathogens in organisms and the environment. Here we present a comparative study of the performance of six fluorescent intercalating dyes-SYTO-9, SYTO-13, SYTO-82, SYBR Green I, SYBR Gold, EvaGreen-in three different qLAMP model systems. SYTO-9 and SYTO-82, which had the best results, were used for additional enzyme and template titration studies. SYTO-82 demonstrated the best combination of time-to-threshold (Tt) and signal-to-noise ratio (SNR).

Large Fragment of DNA Polymerase I from Geobacillus sp. 777: Cloning and Comparison with DNA Polymerases I in Practical Applications.

A truncated gene of DNA polymerase I from the thermophilic bacteria Geobacillus sp. 777 encoding a large fragment of enzyme (LF Gss pol) was cloned and sequenced. The resulting sequence is 1776-bp long and encodes a 592 aa protein with a predicted molecular mass of 69.8 kDa. Enzyme was overexpressed in E. coli, purified by metal-chelate chromatography, and biochemically characterized. The specific activity of LF Gss pol is 104,000 U/mg (one unit of enzyme was defined as the amount of enzyme that incorporated 10 nmol of dNTP into acid insoluble material in 30 min at 65 °C). The properties of LF Gss pol were compared to commercially available large fragments of DNA polymerase I from G. stearothermophilus (LF Bst pol) and Bacillus smithii (LF Bsm pol). Studied enzymes showed maximum activity at similar pH and concentrations of monovalent/divalent ions, whereas LF Gss pol and LF Bst pol were more thermostable than LF Bsm pol. LF Gss pol is more resistant to enzyme inhibitors (SYBR Green I, heparin, ethanol, urea, blood plasma) in comparison with LF Bst pol and LF Bsm pol. LF Gss pol is also suitable for loop-mediated isothermal amplification and whole genome amplification of human genomic DNA.

A replication study examining association of rs6983267, rs10090154, and rs1447295 common single nucleotide polymorphisms in 8q24 region with prostate cancer in Siberians.

INTRODUCTION: Multiple genetic studies have confirmed association of 8q24 variants with susceptibility to prostate cancer (CaP). However, the risk conferred in men living in Russia is unknown. MATERIALS AND METHODS: In this work we studied the association of rs6983267, rs10090154, and rs1447295 single nucleotide polymorphisms (SNPs) with a risk of CaP development in men of Caucasoid descent living in the Siberian region of Russia. Three 8q24 SNPs were genotyped by real-time polymerase chain reaction in histologically confirmed CaP "cases" (n = 392) and clinically evaluated "controls" (n = 344). To evaluate the SNP effects on CaP susceptibility, odds ratio (OR) and confidence interval (CI) 95% were calculated. Allele and genotype frequencies in the groups were compared using logistic regression; differences were considered statistically significant if P<0.05. RESULTS: We showed statistically significant association of the A allele of rs1447295 (OR [CI 95%] = 1.96 [1.37-2.81], P<0.0001) and the T allele of rs10090154 (OR [CI 95%] = 2.14 [1.41-3.26], P<0.0001) with CaP. The T-A rs10090154 to rs1447295 haplotype was also associated with CaP (OR [CI 95%] = 2.47 [1.59-3.85], P<0.0001). There was no significant association with the T allele of rs6983267: OR [CI 95%] = 0.9 [0.73-1.11], P> 0.05. CONCLUSION: Thus, our investigation confirms the role of chromosomal region 8q24 in the development of CaP in the Russian population.

Downregulation of serotonergic gene expression in the Raphe nuclei of the midbrain under chronic social defeat stress in male mice.

There is ample experimental evidence supporting the hypothesis that the brain serotonergic system is involved in the control of chronic social defeat stress (CSDS), depression, and anxiety. The study aimed to analyze mRNA levels of the serotonergic genes in the raphe nuclei of midbrain that may be associated with chronic social defeats consistently shown by male mice in special experimental settings. The serotonergic genes were the Tph2, Sert, Maoa, and Htr1a. The Bdnf and Creb genes were also studied. The experimental groups were composed of male mice with experience of defeats in 21 daily encounters and male mice with the same track record of defeats followed by a no-defeat period without agonistic interactions (relative rest for 14 days). It has been shown that mRNA levels of the Tph2, Maoa, Sert, Htr1a, Bdnf, and Creb genes in the raphe nuclei of defeated mice are decreased as compared with the controls. The expression of the serotonergic genes as well as the Creb gene is not restored to the control level after the 2 weeks of relative rest. mRNA levels of Bdnf gene are not recovered to the control levels, although some upregulation was observed in rested losers. CSDS experience inducing the development of mixed anxiety/depression-like state in male mice downregulates the expression of serotonergic genes associated with the synthesis, inactivation, and reception of serotonin. The Bdnf and Creb genes in the midbrain raphe nuclei are also downregulated under CSDS. Period of relative rest is not enough for most serotonergic genes to recover expression to the control levels.

Reprogramming somatic cells by fusion with embryonic stem cells does not cause silencing of the Dlk1-Dio3 region in mice.

AIM: To examine the imprinted Dlk1-Dio3 locus in pluripotent embryonic stem (ES) cell/fibroblast hybrid cells. METHODS: Gtl2, Rian, and Mirg mRNA expression in mouse pluripotent ES cell/fibroblast hybrid cells was examined by real-time reverse transcription-polymerase chain reaction. Pyrosequencing and bisulfate sequencing were used to determine the DNA methylation level of the Dlk1-Dio3 locus imprinting control region. RESULTS: The selected hybrid clones had a near-tetraploid karyotype and were highly pluripotent judging from their capacity to generate chimeric embryos and adult chimeras. Our data clearly demonstrate that Gtl2, Rian, and Mirg, which are imprinted genes within the Dlk1-Dio3 locus, are active in all examined ES cell/fibroblast hybrid clones. In spite of interclonal variability, the expression of the imprinted genes is comparable to that of ES cells and fibroblasts. Quantitative analysis of the DNA methylation status of the intergenic differentially methylated region (IG DMR) within the Dlk1-Dio3 locus by pyrosequencing and bisulfite sequencing clearly showed that the DNA methylation status of the imprinted region in the tested hybrid clones was comparable to that of both ES cells and fibroblasts. CONCLUSION: Reprogramming process in a hybrid cell system is achieved without marked alteration of the imprinted Dlk1-Dio3 locus.

Snca and Bdnf gene expression in the VTA and raphe nuclei of midbrain in chronically victorious and defeated male mice.

BACKGROUND: Alpha-synuclein (α-Syn) is a small neuronal protein that has been found to be expressed throughout the brain. It has been shown that α-Syn regulates the homeostasis of monoamine neurotransmitters and is involved in various degenerative and affective disorders. There is indication that α-Syn may regulate expression of the brain-derived neurotropic factor (BDNF) which plays an important role in the mood disorders. METHODOLOGY/PRINCIPAL FINDINGS: The study aimed to analyze the mRNA levels of Snca and Bdnf genes in the ventral tegmental area (VTA) and raphe nuclei of the midbrain in male mice that had each won or defeated 20 encounters (20-time winners and 20-time losers, respectively) in daily agonistic interactions. Groups of animals that had the same winning and losing track record followed by a no-fight period for 14 days (no-fighting winners and no-fighting losers) were also studied. Snca mRNA levels were increased in the raphe nuclei in the 20-time losers and in the VTA of the 20-time winners. After no-fight period Snca mRNA levels decreased in both groups. Snca mRNA levels were similar to the control level in the VTA of the 20-time losers and in the raphe nuclei of the 20-time winners. However Snca gene expression increased in these areas in the no-fighting winners and no-fighting losers in comparison with respective mRNA levels in animals before no-fight period. Bdnf mRNA levels increased in VTA of 20-time winners. Significant positive correlations were found between the mRNA levels of Snca and Bdnf genes in the raphe nuclei. CONCLUSIONS/SIGNIFICANCE: Social experience affects Snca gene expression depending on brain areas and functional activity of monoaminergic systems in chronically victorious or defeated mice. These findings may be useful for understanding the mechanisms of forming different alpha-synucleinopathies.

Molecular implications of repeated aggression: Th, Dat1, Snca and Bdnf gene expression in the VTA of victorious male mice.

BACKGROUND: It is generally recognized that recurrent aggression can be the result of various psychiatric disorders. The aim of our study was to analyze the mRNA levels, in the ventral tegmental area (VTA) of the midbrain, of the genes that may possibly be associated with aggression consistently shown by male mice in special experimental settings. METHODOLOGY/PRINCIPAL FINDINGS: The genes were Th, Dat1, Snca and Bdnf; the male mice were a group of animals that had each won 20 daily encounters in succession and a group of animals that had the same winning track record followed by a no-fight period for 14 days. Increased Th, Dat1 and Snca mRNA levels were in the fresh-from-the-fight group as compared to the controls. Increased Th and Dat1 mRNA levels were in the no-fight winners as compared to the controls. Significant positive correlations were found between the level of aggression and Th and Snca mRNA levels. CONCLUSIONS: Repeated positive fighting experience enhances the expression of the Th, Dat1 and Snca genes, which are associated with brain dopaminergic systems. The expression of the Th and Dat1 genes stays enhanced for a long time.