Publications by authors named "Gabriel Minarik"

77 Publications

Uncovering Microbial Composition in Human Breast Cancer Primary Tumour Tissue Using Transcriptomic RNA-seq.

Int J Mol Sci 2021 Aug 22;22(16). Epub 2021 Aug 22.

Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, 813 72 Bratislava, Slovakia.

Recent research studies are showing breast tissues as a place where various species of microorganisms can thrive and cannot be considered sterile, as previously thought. We analysed the microbial composition of primary tumour tissue and normal breast tissue and found differences between them and between multiple breast cancer phenotypes. We sequenced the transcriptome of breast tumours and normal tissues (from cancer-free women) of 23 individuals from Slovakia and used bioinformatics tools to uncover differences in the microbial composition of tissues. To analyse our RNA-seq data (rRNA depleted), we used and tested Kraken2 and Metaphlan3 tools. Kraken2 has shown higher reliability for our data. Additionally, we analysed 91 samples obtained from SRA database, originated in China and submitted by Sichuan University. In breast tissue, the most enriched group were , then and for both datasets, in Slovak samples also , while in Chinese samples were more frequent. We have observed changes in the microbiome between cancerous and healthy tissues and also different phenotypes of diseases, based on the presence of circulating tumour cells and few other markers.
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http://dx.doi.org/10.3390/ijms22169058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8396677PMC
August 2021

Impact of rs243865 and rs3025058 Polymorphisms on Clinical Findings in Alzheimer's Disease Patients.

Mediators Inflamm 2021 19;2021:5573642. Epub 2021 Apr 19.

Institute of Immunology, Faculty of Medicine, Comenius University in Bratislava, Bratislava, Slovakia.

Alzheimer's disease (AD) is a chronic neurodegenerative disease of the central nervous system with higher prevalence in elderly people. Despite numerous research studies, the etiopathogenesis of AD remains unclear. Matrix metalloproteinases (MMPs) are endopeptidases involved in the cleavage of extracellular matrix proteins and basement membrane compounds. In the brain, the pathological role of MMPs includes the disruption of the blood-brain barrier leading to the induction of neuroinflammation. Among various MMPs, MMP-2 and MMP-3 belong to candidate molecules related to AD pathology. In our study, we aimed to evaluate the association of rs243865 and rs3025058 polymorphisms with AD susceptibility and their influence on age at onset and MoCA score in patients from Slovakia. Both MMP gene promoter polymorphisms were genotyped in 171 AD patients and 308 controls by the PCR-RFLP method. No statistically significant differences in the distribution of rs243865 (-1306 C>T) and rs3025058 (-1171 5A>6A) alleles/genotypes were found between AD patients and the control group. However, correlation with clinical findings revealed later age at disease onset in rs243865 CC carriers in the dominant model as compared to T allele carriers (CC vs. CT+TT: 78.44 ± 6.28 vs. 76.36 ± 6.39, = 0.036). The results of rs3025058 analysis revealed that 5A/6A carriers in the overdominant model tended to have earlier age at disease onset as compared to other genotype carriers (5A/6A vs. 5A/5A+6A/6A: 76.61 ± 5.88 vs. 78.57 ± 6.79, = 0.045). In conclusion, our results suggest that rs243865 and rs3025058 promoter polymorphisms may have influence on age at onset in AD patients.
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http://dx.doi.org/10.1155/2021/5573642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8079184PMC
April 2021

Copy Number Variant Detection with Low-Coverage Whole-Genome Sequencing Represents a Viable Alternative to the Conventional Array-CGH.

Diagnostics (Basel) 2021 Apr 15;11(4). Epub 2021 Apr 15.

Geneton s.r.o., 841 04 Bratislava, Slovakia.

Copy number variations (CNVs) represent a type of structural variant involving alterations in the number of copies of specific regions of DNA that can either be deleted or duplicated. CNVs contribute substantially to normal population variability, however, abnormal CNVs cause numerous genetic disorders. At present, several methods for CNV detection are applied, ranging from the conventional cytogenetic analysis, through microarray-based methods (aCGH), to next-generation sequencing (NGS). In this paper, we present GenomeScreen, an NGS-based CNV detection method for low-coverage, whole-genome sequencing. We determined the theoretical limits of its accuracy and obtained confirmation in an extensive in silico study and in real patient samples with known genotypes. In theory, at least 6 M uniquely mapped reads are required to detect a CNV with the length of 100 kilobases (kb) or more with high confidence (Z-score > 7). In practice, the in silico analysis required at least 8 M to obtain >99% accuracy (for 100 kb deviations). We compared GenomeScreen with one of the currently used aCGH methods in diagnostic laboratories, which has mean resolution of 200 kb. GenomeScreen and aCGH both detected 59 deviations, while GenomeScreen furthermore detected 134 other (usually) smaller variations. When compared to aCGH, overall performance of the proposed GenemoScreen tool is comparable or superior in terms of accuracy, turn-around time, and cost-effectiveness, thus providing reasonable benefits, particularly in a prenatal diagnosis setting.
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http://dx.doi.org/10.3390/diagnostics11040708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8071346PMC
April 2021

5'URR regulatory polymorphisms are associated with the risk of developing gliomas.

Int J Neurosci 2021 Sep 28:1-10. Epub 2021 Sep 28.

Institute of Immunology, Faculty of Medicine, Comenius University in Bratislava, Bratislava, Slovakia.

Background: Human leukocyte antigen G (HLA-G) belongs to non-classical MHC class I molecules that is involved in the suppression of immune response. As HLA-G plays important role in the maintenance of fetal tolerance, its overexpression has been associated with tumor progression. For the regulation of HLA-G levels, genetic variants within the 5' upstream regulatory region (5'URR) are of crucial importance. Our study aimed to analyze the association between 16 5'URR variants, sHLA-G level and clinical variables in glioma patients.

Methods: We investigated 59 patients with gliomas (mean age 54.70 ± 15.10 years) and 131 healthy controls (mean age 41.45 ± 9.75 years). Patient's blood was obtained on the day of surgical treatment. The 5'URR polymorphisms were typed by direct sequencing and the plasma level of sHLA-G assessed by ELISA.

Results: Haploblock within 5'URR consisting of -762T, -716G, -689G, -666T, -633A, followed by -486C and -201A alleles were significantly more frequent in patients with gliomas than in the controls ( < 0.05). No correlation of 5'URR variants with sHLA-G plasma level was found. Analysis of 5'URR variants with main clinical variables in patients with grade IV gliomas revealed that haploblock carriers of -762CT, -716TG, -689AG, -666GT, -633GA, -486AC, -477GC, -201GA followed by -369AC carriers tend to have lower age at onset as compared to other genotype carriers ( = 0.04).

Conclusion: Our results suggest genetic association of 5'URR variants with risk of developing gliomas and possible contribution of HLA-G to disease pathology.
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http://dx.doi.org/10.1080/00207454.2021.1922401DOI Listing
September 2021

Prognostic role of matrix metalloproteinase 9 in early breast cancer.

Oncol Lett 2021 Feb 30;21(2):78. Epub 2020 Nov 30.

Translational Research Unit, Faculty of Medicine, Comenius University, 833 10 Bratislava, Slovakia.

MMP9 is involved in extracellular matrix degradation during various physiological and pathological conditions, including tumorigenesis. The present study aimed to assess the prognostic role of intratumoral MMP9 and to determine its association with circulating tumor cells (CTCs) in patients with early breast cancer. A total of 318 patients with primary breast cancer (PBC) were enrolled into the present study. Specimens were subjected to immunohistochemistry analysis, using the MMP9 monoclonal antibody. MMP9 expression was scored using a weighted histoscore (WH). The results demonstrated that the mean WH ± SEM for MMP9 expression was significantly higher in breast tumor cells compared with tumor associated stromas (132.0±5.2 vs. 50.8±3.7; P<0.00001). Furthermore, a positive association was observed between MMP9 expression, the hormone positive status and proliferation index of analysed breast cancer tumour cells. Notably, the prognostic role of MMP9 was not observed in tumor cells [hazard ratio (HR) =0.96; 95% confidence interval (CI), 0.58-1.59; P=0.864] or tumor associated stroma (HR=1.29; 95% CI, 0.60-2.78; P=0.547). Subgroup analysis demonstrated that patients that were HR negative or triple negative, with low MMP9 expression in tumor cells and stroma had a significantly improved disease-free survival than patients with high MMP9 expression. Taken together, the results of the present study demonstrated that high MMP9 expression in PBC was associated with favorable tumor characteristics. However, the prognostic value of MMP9 was limited to only the HR negative and CTC epithelial-to-mesenchymal transition positive subgroups. Thus, analyzing MMP9 tumor expression may help identify patients with increased risk of disease recurrence in these subgroups.
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http://dx.doi.org/10.3892/ol.2020.12339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7723168PMC
February 2021

Monosomy 3 Influences Epithelial-Mesenchymal Transition Gene Expression in Uveal Melanoma Patients; Consequences for Liquid Biopsy.

Int J Mol Sci 2020 Dec 17;21(24). Epub 2020 Dec 17.

Department of Molecular Oncology, Cancer Research Institute, Biomedical Research Center, Slovak Academy of Sciences, Dubravska Cesta 9, 845 05 Bratislava, Slovakia.

Despite outstanding advances in diagnosis and the treatment of primary uveal melanoma (UM), nearly 50% of UM patients develop metastases via hematogenous dissemination, driven by the epithelial-mesenchymal transition (EMT). Despite the failure in UM to date, a liquid biopsy may offer a feasible non-invasive approach for monitoring metastatic disease progression and addressing protracted dormancy. To detect circulating tumor cells (CTCs) in UM patients, we evaluated the mRNA expression of EMT-associated transcription factors in CD45-depleted blood fraction, using qRT-PCR. ddPCR was employed to assess UM-specific , , , and mutations in plasma DNA. Moreover, microarray analysis was performed on total RNA isolated from tumor tissues to estimate the prognostic value of EMT-associated gene expression. In total, 42 primary UM and 11 metastatic patients were enrolled. All CD45-depleted samples were negative for CTC when compared to the peripheral blood fraction of 60 healthy controls. Tumor-specific mutations were detected in the plasma of 21.4% patients, merely, in 9.4% of primary UM, while 54.5% in metastatic patients. Unsupervised hierarchical clustering of differentially expressed EMT genes showed significant differences between monosomy 3 and disomy 3 tumors. Newly identified genes can serve as non-invasive prognostic biomarkers that can support therapeutic decisions.
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http://dx.doi.org/10.3390/ijms21249651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7767066PMC
December 2020

Increased Stromal Infiltrating Lymphocytes Are Associated with the Risk of Disease Progression in Mesenchymal Circulating Tumor Cell-Positive Primary Breast Cancer Patients.

Int J Mol Sci 2020 Dec 12;21(24). Epub 2020 Dec 12.

2nd Department of Oncology, Faculty of Medicine, Comenius University and National Cancer Institute, Klenova 1, 833 10 Bratislava, Slovakia.

Circulating tumor cells (CTCs) and the immune infiltration of tumors are closely related to clinical outcomes. This study aimed to verify the influence of stromal lymphocyte infiltration and the immune context of tumor microenvironment on the hematogenous spread and prognosis of 282 chemotherapy naïve primary BC patients. To detect the presence of mesenchymal CTCs, RNA extracted from CD45-depleted peripheral blood was interrogated for the expression of mesenchymal gene transcripts. Tumor-infiltrating lymphocytes (TILs) were detected in the stromal areas by immunohistochemistry, using CD3, CD8, and CD45RO antibodies. The concentrations of 51 plasma cytokines were measured by multiplex bead arrays. TILs infiltration in mesenchymal CTC-positive patients significantly decreased their progression-free survival (HR = 4.88, 95% CI 2.30-10.37, < 0.001 for CD3; HR = 6.17, 95% CI 2.75-13.80, < 0.001 for CD8; HR = 6.93, 95% CI 2.86-16.81, < 0.001 for CD45RO). Moreover, the combination of elevated plasma concentrations of transforming growth factor beta-3 (cut-off 662 pg/mL), decreased monocyte chemotactic protein-3 (cut-off 52.5 pg/mL) and interleukin-15 (cut-off 17.1 pg/mL) significantly increased the risk of disease recurrence (HR = 4.838, 95% CI 2.048-11.427, < 0.001). Our results suggest a strong impact of the immune tumor microenvironment on BC progression, especially through influencing the dissemination and survival of more aggressive, mesenchymal CTC subtypes.
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http://dx.doi.org/10.3390/ijms21249460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763628PMC
December 2020

Comparative analysis of monozoic fish tapeworms Caryophyllaeus laticeps (Pallas, 1781) and recently described Caryophyllaeus chondrostomi Barčák, Oros, Hanzelová, Scholz, 2017, using microsatellite markers.

Parasitol Res 2020 Dec 1;119(12):3995-4004. Epub 2020 Oct 1.

Biology Centre CAS, Institute of Parasitology, and Faculty of Science, University of South Bohemia, Branišovská 31, 370 05, České Budějovice, Czech Republic.

The monozoic tapeworm Caryophyllaeus laticeps has been characterized by five markedly different morphotypes largely corresponding to different fish hosts. Recently, the most distinct morphotype 4 from the common nase Chondrostoma nasus was studied in more details resulting in description of a new species Caryophyllaeus chondrostomi. The molecular study based on mitochondrial cox1 and ribosomal lsrDNA did not reveal any interspecific differences between C. laticeps and C. chondrostomi and did not provide any molecular support for recognition of these two species. In the current study, six polymorphic microsatellite markers were applied in order to detect molecular differences between the two species and to provide molecular evidence of validity of C. chondrostomi. While all six microsatellite loci were amplified in different geographic populations of C. laticeps, only two of them provided the amplification product in C. chondrostomi. Results on the Bayesian analysis assigned C. chondrostomi and all geographic populations of C. laticeps to distinct clusters. Neither any close relationships among C. laticeps populations nor specific position of C. chondrostomi were revealed. Contrary, the results of the principal coordinate analysis revealed striking genetic separation of C. chondrostomi with no overlaps with any of the C. laticeps population or morphotype. Caryophyllaeus chondrostomi very probably underwent morphological divergence as a result of ongoing speciation, but this process has not yet been accompanied by sufficient genetic divergence. In this particular case, microsatellites were proved to be better molecular discriminative markers than rDNA and mtDNA.
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http://dx.doi.org/10.1007/s00436-020-06898-8DOI Listing
December 2020

Plasma Nucleosomes in Primary Breast Cancer.

Cancers (Basel) 2020 Sep 10;12(9). Epub 2020 Sep 10.

Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, 81372 Bratislava, Slovakia.

When cells die, nucleosomes composed of DNA and histone proteins enter the extracellular space and end eventually in the circulation. In plasma, they might serve as a nonspecific marker of cell death, potentially useful for noninvasive monitoring of tumor dynamics. The aim of this study was to analyze circulating nucleosomes in relation to patient/tumor characteristics and prognosis in primary breast cancer. This study included 92 patients with breast cancer treated with surgery for whom plasma isolated was available in the biobank. Plasma nucleosomes were detected in samples taken in the morning on the day of surgery using Cell Death Detection ELISA kit with anti-histone and anti-DNA antibodies. Circulating nucleosomes were positively associated with the systemic inflammatory index (SII), but not with other patient/tumor characteristics. Patients with high SII in comparison to low SII had higher circulating nucleosomes (by 59%, = 0.02). Nucleosomes correlated with plasma plasminogen activator inhibitor-1, IL-15, IL-16, IL-18, and hepatocyte growth factor. Patients with lower nucleosomes had significantly better disease-free survival (HR = 0.46, = 0.05). In a multivariate analysis, nucleosomes, hormone receptor status, HER2 status, lymph node involvement, and tumor grade were independent predictors of disease-free survival. Our data suggest that plasma nucleosomes in primary breast cancer are associated with systemic inflammation and might have a prognostic value. The underlying mechanisms require further studies.
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http://dx.doi.org/10.3390/cancers12092587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563724PMC
September 2020

Gut Microbiota Diversity in Lean Athletes Is Associated with Positive Energy Balance.

Ann Nutr Metab 2020 7;76(4):242-250. Epub 2020 Sep 7.

Comenius University Science Park, Comenius University in Bratislava, Bratislava, Slovakia.

Introduction: In contrast to obesity, little is known about the human lean phenotype associated with gut microbiota composition.

Objective: We aimed to investigate whether the bacterial composition of lean athletes with a positive energy balance differs from the equal-calorie food group.

Methods: Twenty-four male participants were included in this cross-sectional study: lean athletes with a positive energy balance (LA, n 12) and control group athletes (CTRLs, n 12). Nutritional data, resting and total energy expenditure, and body composition were determined. DNA was extracted from stool samples and subjected to 16S rRNA gene analysis.

Results: We found 7 differentially abundant bacterial taxa between the LA and CTRL groups. Of those, 5 were significantly less abundant and 2 were enriched in the LA group. The following categories significantly associated with the community structure were identified: body fat parameters, BMI, energy intake and expenditure, oxygen consumption, and respiratory exchange ratio.

Conclusions: Although we are far from a detailed interpretation of lean human body maintenance, the primary findings of our study suggest that gut microbial composition may be a factor influencing the regulation of weight gain in lean athletes with a positive energy balance.
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http://dx.doi.org/10.1159/000509833DOI Listing
August 2021

Non-invasive prenatal testing (NIPT) by low coverage genomic sequencing: Detection limits of screened chromosomal microdeletions.

PLoS One 2020 26;15(8):e0238245. Epub 2020 Aug 26.

Geneton Ltd., Bratislava, Slovakia.

To study the detection limits of chromosomal microaberrations in non-invasive prenatal testing with aim for five target microdeletion syndromes, including DiGeorge, Prader-Willi/Angelman, 1p36, Cri-Du-Chat, and Wolf-Hirschhorn syndromes. We used known cases of pathogenic deletions from ISCA database to specifically define regions critical for the target syndromes. Our approach to detect microdeletions, from whole genome sequencing data, is based on sample normalization and read counting for individual bins. We performed both an in-silico study using artificially created data sets and a laboratory test on mixed DNA samples, with known microdeletions, to assess the sensitivity of prediction for varying fetal fractions, deletion lengths, and sequencing read counts. The in-silico study showed sensitivity of 79.3% for 10% fetal fraction with 20M read count, which further increased to 98.4% if we searched only for deletions longer than 3Mb. The test on laboratory-prepared mixed samples was in agreement with in-silico results, while we were able to correctly detect 24 out of 29 control samples. Our results suggest that it is possible to incorporate microaberration detection into basic NIPT as part of the offered screening/diagnostics procedure, however, accuracy and reliability depends on several specific factors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0238245PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449492PMC
October 2020

Validation of Copy Number Variants Detection from Pregnant Plasma Using Low-Pass Whole-Genome Sequencing in Noninvasive Prenatal Testing-Like Settings.

Diagnostics (Basel) 2020 Aug 8;10(8). Epub 2020 Aug 8.

Trisomy Test s.r.o., Ilkovičova 8, 841 04 Bratislava, Slovakia.

Detection of copy number variants as an integral part of noninvasive prenatal testing is increasingly used in clinical practice worldwide. We performed validation on plasma samples from 34 pregnant women with known aberrations using cell-free DNA sequencing to evaluate the sensitivity for copy number variants (CNV) detection using an in-house CNV fraction-based detection algorithm. The sensitivity for CNVs smaller than 3 megabases (Mb), larger than 3Mb, and overall was 78.57%, 100%, and 90.6%, respectively. Regarding the fetal fraction, detection sensitivity in the group with a fetal fraction of less than 10% was 57.14%, whereas there was 100% sensitivity in the group with fetal fraction exceeding 10%. The assay is also capable of indicating whether the origin of an aberration is exclusively fetal or fetomaternal/maternal. This validation demonstrated that a CNV fraction-based algorithm was applicable and feasible in clinical settings as a supplement to testing for common trisomies 21, 18, and 13.
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http://dx.doi.org/10.3390/diagnostics10080569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460070PMC
August 2020

Development of 14 Microsatellite Markers for Zoonotic Tapeworm (Cestoda: Diphyllobothriidea).

Genes (Basel) 2020 07 12;11(7). Epub 2020 Jul 12.

Institute of Parasitology, Slovak Academy of Sciences, Hlinkova 3, 04001 Košice, Slovakia.

is one of the causative agents of the fish-borne zoonosis diphyllobothriosis. Polymorphic microsatellite markers were originally developed for future genetic studies using microsatellite library screening and next-generation sequencing (NGS). Out of 128 microsatellite candidates selected after NGS analysis, 126 yielded PCR products of the expected size. A declared repetitive motif was confirmed in 92 loci by Sanger sequencing. The level of polymorphism was tested by fragment analysis. Statistical tests for observed and expected heterozygosities and deviations from Hardy-Weinberg equilibrium revealed 14 polymorphic microsatellite loci suitable for studies on the finer genetic structure of global populations of .
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http://dx.doi.org/10.3390/genes11070782DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397143PMC
July 2020

Inflammation-Based Scores Increase the Prognostic Value of Circulating Tumor Cells in Primary Breast Cancer.

Cancers (Basel) 2020 May 1;12(5). Epub 2020 May 1.

2nd Department of Oncology, Faculty of Medicine, Comenius University and National Cancer Institute, Klenova 1, 833 10 Bratislava, Slovakia.

A correlation between circulating tumor cells (CTCs) and monocytes in metastatic breast cancer (BC), where CTCs and monocyte-to-lymphocyte ratio (MLR) were predictors of overall survival (OS), was recently shown. Herein, we aimed to assess the association between CTCs and the complete blood count (CBC)-derived inflammation-based scores in 284 primary BC patients. CTCs were determined in CD45-depleted peripheral blood mononuclear cells by real time-PCR. This method allowed us to detect a subset of CTCs with an epithelial-to-mesenchymal transition phenotype (CTC EMT), previously associated with inferior outcomes in primary BC. In the present study, CTC EMT positivity (hazard ratio (HR) = 2.4; 95% CI 1.20-4.66, = 0.013) and elevated neutrophil-to-lymphocyte ratio (NLR) (HR = 2.20; 95% CI 1.07-4.55; = 0.033) were associated with shorter progression-free survival (PFS) in primary BC patients. Multivariate analysis showed that CTC EMT-positive patients with NLR ≥ 3 had 8.6 times increased risk of disease recurrence (95% CI 2.35-31.48, = 0.001) compared with CTC EMT-negative patients with NLR < 3. Similarly, disease recurrence was 13.14 times more likely in CTC EMT-positive patients with MLR ≥ 0.34 (95% CI 4.35-39.67, < 0.001). Given its low methodological and financial demands, the CBC-derived inflammation-based score determination could, after broader validation, significantly improve the prognostication of BC patients.
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http://dx.doi.org/10.3390/cancers12051134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281016PMC
May 2020

Spinal muscular atrophy caused by a novel Alu-mediated deletion of exons 2a-5 in SMN1 undetectable with routine genetic testing.

Mol Genet Genomic Med 2020 07 26;8(7):e1238. Epub 2020 Apr 26.

Research Unit for Rare Diseases, Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University, Prague, Czech Republic.

Background: Spinal muscular atrophy (SMA) is an inherited neuromuscular disease affecting 1 in 8,000 newborns. The majority of patients carry bi-allelic variants in the survival of motor neuron 1 gene (SMN1). SMN1 is located in a duplicated region on chromosome 5q13 that contains Alu elements and is predisposed to genomic rearrangements. Due to the genomic complexity of the SMN region and genetic heterogeneity, approximately 50% of SMA patients remain without genetic diagnosis that is a prerequisite for genetic treatments. In this work we describe the diagnostic odyssey of one SMA patient in whom routine diagnostics identified only a maternal heterozygous SMN1Δ(7-8) deletion.

Methods: We characterized SMN transcripts, assessed SMN protein content in peripheral blood mononuclear cells (PBMC), estimated SMN genes dosage, and mapped genomic rearrangement in the SMN region.

Results: We identified an Alu-mediated deletion encompassing exons 2a-5 of SMN1 on the paternal allele and a complete deletion of SMN1 on the maternal allele as the cause of SMA in this patient.

Conclusion: Alu-mediated rearrangements in SMN1 can escape routine diagnostic testing. Parallel analysis of SMN gene dosage, SMN transcripts, and total SMN protein levels in PBMC can identify genomic rearrangements and should be considered in genetically undefined SMA cases.
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http://dx.doi.org/10.1002/mgg3.1238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336725PMC
July 2020

Circulating tumor cells and breast cancer-specific mutations in primary breast cancer.

Mol Clin Oncol 2020 Jun 1;12(6):565-573. Epub 2020 Apr 1.

Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Mlynska dolina, 842 15 Bratislava, Slovakia.

Circulating tumor cells (CTCs) play a pivotal role in tumor dissemination and progression, and are considered to be a critical part of the metastatic cascade. The aim of the present research article was to examine breast cancer-specific mutations in primary breast cancer (PBC) using targeted resequencing. A total of 78 patients with PBC were enrolled into this translational study. Reverse transcription-quantitative PCR assay for the expression of epithelial markers () or epithelial-to-mesenchymal transition (EMT)-related genes (, , and ) was applied for identification of CTCs prior to surgery. Total DNA was isolated from fresh frozen primary tumors. Sequencing was performed by Agilent SureSelect target enrichment and Illumina paired-end sequencing on the MiSeq platform. The most commonly affected genes were (mutated in 21 tumors; 26.9%), followed by (mutated in 16 tumors; 20.5%) and (mutated in 7 tumors, n=2 and n=5; 9.0%). In our cohort, a significantly higher proportion of patients with epithelial CTCs harbored mutations in the genes in the tumor tissue. There were no mutations in specific genes associated with CTCs with the EMT phenotype. To the best of our knowledge, this study is the first to report a correlation between the presence of epithelial CTCs in the peripheral blood and mutations of the genes in primary tumor tissue.
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http://dx.doi.org/10.3892/mco.2020.2026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7179389PMC
June 2020

Association of CD33 rs3865444:C˃A polymorphism with a reduced risk of late-onset Alzheimer's disease in Slovaks is limited to subjects carrying the APOE ε4 allele.

Int J Immunogenet 2020 Oct 24;47(5):397-405. Epub 2020 Apr 24.

Institute of Immunology, Faculty of Medicine, Comenius University in Bratislava, Bratislava, Slovakia.

CD33 rs3865444:C>A single nucleotide polymorphism (SNP) has been previously associated with the risk of late-onset Alzheimer's disease (LOAD); however, the results have been inconsistent across different populations. CD33 is a transmembrane receptor that plays an important role in AD pathogenesis by inhibiting amyloid β42 uptake by microglial cells. In this study, we aimed to validate the association between rs3865444 and LOAD risk in the Slovak population and to evaluate whether it was affected by the carrier status of the major LOAD risk allele apolipoprotein (APOE) ε4. CD33 rs3865444 and APOE variants were genotyped in 206 LOAD patients and 487 control subjects using the polymerase chain reaction-restriction fragment length polymorphism method and direct sequencing, respectively. Logistic regression analysis revealed a significant association of rs3865444 A allele with a reduced LOAD risk that was only present in APOE ε4 allele carriers (AA + CA versus CC: p = .0085; OR = 0.45; 95% CI = 0.25-0.82). On the other hand, no such association was found in subjects without the APOE ε4 (p = .75; OR = 0.93; 95% CI = 0.61-1.42). Moreover, regression analysis detected a significant interaction between CD33 rs3865444 A and APOE ε4 alleles (p = .021 for APOE ε4 allele dosage and p = .051 for APOE ε4 carriage status), with synergy factor (SF) value of 0.49 indicating an antagonistic effect between the two alleles in LOAD risk. In conclusion, our results suggest that CD33 rs3865444:C˃A substitution may reduce the risk of LOAD in Slovaks by antagonizing the effect conferred by the major susceptibility allele APOE ε4.
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http://dx.doi.org/10.1111/iji.12489DOI Listing
October 2020

Fatal neonatal nephrocutaneous syndrome in 18 Roma children with EGFR deficiency.

J Dermatol 2020 Jun 6;47(6):663-668. Epub 2020 Apr 6.

Department of Pediatrics and Adolescent Medicine, General University Hospital, First Medical Faculty, Charles University, Prague, Czech Republic.

Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein with tyrosine-kinase signaling activity, involved in many cellular functions including cell growth and differentiation. Germ line loss-of-function mutations in EGFR lead to a severe neonatal skin disorder (Online Mendelian Inheritance in Man #131550). We report 18 premature Roma children from 16 families with birthweights ranging 440-1470 g and multisystem diseases due to the homozygous mutation c.1283G˃A (p.Gly428Asp) in EGFR. They presented with thin, translucent, fragile skin (14/15), skin desquamation (10/17), ichthyosis (9/17), recurrent skin infections and sepsis (9/12), nephromegaly (10/16) and congenital heart defects (7/17). Their prognosis was poor, and all died before the age of 6 months except one 13-year-old boy with a severe skin disorder, dentinogenesis imperfecta, Fanconi-like syndrome and secondary hyperaldosteronism. Management of ion and water imbalances and extremely demanding skin care may improve the unfavorable outcome of such patients.
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http://dx.doi.org/10.1111/1346-8138.15317DOI Listing
June 2020

Result of Prospective Validation of the Trisomy Test for the Detection of Chromosomal Trisomies.

Diagnostics (Basel) 2019 Oct 2;9(4). Epub 2019 Oct 2.

Trisomy Test Ltd., Ilkovičova 8, 841 04 Bratislava, Slovakia.

Noninvasive prenatal testing (NIPT) is one of the most common prenatal screening tests used worldwide. Trisomy Test belongs to NIPT tests based on low-coverage whole-genome sequencing. In our prospective study, 7279 samples of pregnant women collected during approximately two years were analyzed. In this cohort, 117 positive cases for trisomies 21, 18, and 13 were reported. An in-house designed bioinformatic pipeline and proprietary biostatistical approach was used for the detection of trisomies. The pooled sensitivity and specificity of our test reached 99.12% and 99.94%, respectively. The proportion of repeatedly uninformative results after repeated blood draws was 1.11%. Based on the presented results, we can confirm that the Trisomy Test is fully comparable with other commercial NIPT tests available worldwide.
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http://dx.doi.org/10.3390/diagnostics9040138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6963324PMC
October 2019

Non-invasive prenatal testing as a valuable source of population specific allelic frequencies.

J Biotechnol 2019 Jun 1;299:72-78. Epub 2019 May 1.

Geneton Ltd., Bratislava, Slovakia; Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia; Comenius University Science Park, Bratislava, Slovakia. Electronic address:

Low-coverage massively parallel genome sequencing for non-invasive prenatal testing (NIPT) of common aneuploidies is one of the most rapidly adopted and relatively low-cost DNA tests. Since aggregation of reads from a large number of samples allows overcoming the problems of extremely low coverage of individual samples, we describe the possible re-use of the data generated during NIPT testing for genome scale population specific frequency determination of small DNA variants, requiring no additional costs except of those for the NIPT test itself. We applied our method to a data set comprising of 1501 original NIPT test results and evaluated the findings on different levels, from in silico population frequency comparisons up to wet lab validation analyses using a gold-standard method based on Sanger sequencing. The revealed high reliability of variant calling and allelic frequency determinations suggest that these NIPT data could serve as valuable alternatives to large scale population studies even for smaller countries around the world.
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http://dx.doi.org/10.1016/j.jbiotec.2019.04.026DOI Listing
June 2019

Circulating Tumor Cells With Epithelial-to-mesenchymal Transition Phenotypes Associated With Inferior Outcomes in Primary Breast Cancer.

Anticancer Res 2019 Apr;39(4):1829-1837

2nd Department of Oncology, Faculty of Medicine, Comenius University, Bratislava, Slovak Republic.

Background/aim: Circulating tumor cells (CTCs) comprise a heterogeneous population of cancer cells with different clinical and biological value. The aim of this study was to evaluate the prognostic value of CTCs with an epithelial-mesenchymal transition (EMT) phenotype in primary breast cancer (PBC) patients.

Patients And Methods: This study included 427 primary breast cancer patients. RNA extracted from CD45-depleted peripheral blood mononuclear cell (PBMCs) was evaluated for the expression of EMT transcription factors (TWIST1, SNAIL1, SLUG, ZEB1) by quantitative real time polymerase chain reaction (qRT-PCR).

Results: In total, CTC EMT was detected in 77 (18.0%) patients. Patients without detectable CTC EMT in peripheral blood had significantly longer disease-free survival than patients with detectable CTC EMT. The prognostic value of CTC EMT was demonstrated in all subgroups of patients.

Conclusion: CTCs with an EMT phenotype have a prognostic value in primary breast cancer.
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http://dx.doi.org/10.21873/anticanres.13290DOI Listing
April 2019

A disintegrin and metalloprotease 23 hypermethylation predicts decreased disease-free survival in low-risk breast cancer patients.

Cancer Sci 2019 May 18;110(5):1695-1704. Epub 2019 Mar 18.

Cancer Research Institute, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia.

A Disintegrin And Metalloprotease 23 (ADAM23), a member of the ADAM family, is involved in neuronal differentiation and cancer. ADAM23 is considered a possible tumor suppressor gene and is frequently downregulated in various types of malignancies. Its epigenetic silencing through promoter hypermethylation was observed in breast cancer (BC). In the present study, we evaluated the prognostic significance of ADAM23 promoter methylation for hematogenous spread and disease-free survival (DFS). Pyrosequencing was used to quantify ADAM23 methylation in tumors of 203 BC patients. Presence of circulating tumor cells (CTC) in their peripheral blood was detected by quantitative RT-PCR. Expression of epithelial (KRT19) or mesenchymal (epithelial-mesenchymal transition [EMT]-inducing transcription factors TWIST1, SNAI1, SLUG and ZEB1) mRNA transcripts was examined in CD45-depleted peripheral blood mononuclear cells. ADAM23 methylation was significantly lower in tumors of patients with the mesenchymal CTC (P = .006). It positively correlated with Ki-67 proliferation, especially in mesenchymal CTC-negative patients (P = .001). In low-risk patients, characterized by low Ki-67 and mesenchymal CTC absence, ADAM23 hypermethylation was an independent predictor of DFS (P = .006). Our results indicate that ADAM23 is likely involved in BC progression and dissemination of mesenchymal CTC. ADAM23 methylation has the potential to function as a novel prognostic marker and therapeutic target.
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http://dx.doi.org/10.1111/cas.13985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6500989PMC
May 2019

Combining count- and length-based z-scores leads to improved predictions in non-invasive prenatal testing.

Bioinformatics 2019 04;35(8):1284-1291

Bratislava, Geneton s.r.o, Bratislava, Slovakia.

Motivation: Non-invasive prenatal testing or NIPT is currently among the top researched topic in obstetric care. While the performance of the current state-of-the-art NIPT solutions achieve high sensitivity and specificity, they still struggle with a considerable number of samples that cannot be concluded with certainty. Such uninformative results are often subject to repeated blood sampling and re-analysis, usually after two weeks, and this period may cause a stress to the future mothers as well as increase the overall cost of the test.

Results: We propose a supplementary method to traditional z-scores to reduce the number of such uninformative calls. The method is based on a novel analysis of the length profile of circulating cell free DNA which compares the change in such profiles when random-based and length-based elimination of some fragments is performed. The proposed method is not as accurate as the standard z-score; however, our results suggest that combination of these two independent methods correctly resolves a substantial portion of healthy samples with an uninformative result. Additionally, we discuss how the proposed method can be used to identify maternal aberrations, thus reducing the risk of false positive and false negative calls.

Availability And Implementation: The open-source code of the proposed methods, together with test data, is freely available for non-commercial users at github web page https://github.com/jbudis/lambda.

Supplementary Information: Supplementary materials are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty806DOI Listing
April 2019

Decreased methylation in the SNAI2 and ADAM23 genes associated with de-differentiation and haematogenous dissemination in breast cancers.

BMC Cancer 2018 Sep 6;18(1):875. Epub 2018 Sep 6.

Department of Genetics, Cancer Research Institute, Biomedical Research Center, Slovak Academy of Sciences, v.v.i., Dubravska cesta 9, 845 05, Bratislava, Slovak Republic.

Background: In breast cancer (BC), deregulation of DNA methylation leads to aberrant expressions and functions of key regulatory genes. In our study, we investigated the relationship between the methylation profiles of genes associated with cancer invasivity and clinico-pathological parameters. In detail, we studied differences in the methylation levels between BC patients with haematogenous and lymphogenous cancer dissemination.

Methods: We analysed samples of primary tumours (PTs), lymph node metastases (LNMs) and peripheral blood cells (PBCs) from 59 patients with sporadic disseminated BC. Evaluation of the DNA methylation levels of six genes related to invasivity, ADAM23, uPA, CXCL12, TWIST1, SNAI1 and SNAI2, was performed by pyrosequencing.

Results: Among the cancer-specific methylated genes, we found lower methylation levels of the SNAI2 gene in histologic grade 3 tumours (OR = 0.61; 95% CI, 0.39-0.97; P = 0.038) than in fully or moderately differentiated cancers. We also evaluated the methylation profiles in patients with different cancer cell dissemination statuses (positivity for circulating tumour cells (CTCs) and/or LNMs). We detected the significant association between reduced DNA methylation of ADAM23 in PTs and presence of CTCs in the peripheral blood of patients (OR = 0.45; 95% CI, 0.23-0.90; P = 0.023).

Conclusion: The relationships between the decreased methylation levels of the SNAI2 and ADAM23 genes and cancer de-differentiation and haematogenous dissemination, respectively, indicate novel functions of those genes in the invasive processes. After experimental validation of the association between the lower values of SNAI2 and ADAM23 methylation and clinical features of aggressive BCs, these methylation profiles could improve the management of metastatic disease.
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http://dx.doi.org/10.1186/s12885-018-4783-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6127923PMC
September 2018

Development of microsatellite loci in zoonotic tapeworm Dibothriocephalus latus (Linnaeus, 1758), Lühe, 1899 (syn. Diphyllobothrium latum) using microsatellite library screening.

Mol Biochem Parasitol 2018 10 18;225:1-3. Epub 2018 Aug 18.

Biology Centre CAS, Institute of Parasitology, Branišovská 31, 37005 České Budějovice, Czech Republic; Faculty of Science, University of South Bohemia, Branišovská 1160/31, 37005 České Budějovice, Czech Republic.

The broad fish tapeworm Dibothriocephalus latus is a causative agent of human food-borne disease called diphyllobothriosis. Medical importance, scattered geographical distribution and unknown origin of D. latus in Europe and North America make this species to be an interesting model for population genetics. Microsatellite markers were originally designed by library screening using NGS approach and validated as tools for future studies on population genetics of D. latus. Out of 122 candidates selected after NGS analysis, 110 yielded PCR products of the expected size, and in 78 of them, a declared repetitive motif was confirmed by Sanger sequencing. After the fragment analysis, six loci were proved to be polymorphic and tested for observed (Ho) and expected (He) heterozygosity, and deviations from Hardy-Weinberg equilibrium (HWE). They promise future application in studies on genetic interrelationships, origin and migratory routes of this medically important emerging tapeworm.
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http://dx.doi.org/10.1016/j.molbiopara.2018.08.003DOI Listing
October 2018

Association between genetic variability of neuronal nitric oxide synthase and sensorimotor gating in humans.

Nitric Oxide 2018 11 8;80:32-36. Epub 2018 Aug 8.

Department of Behavioural Neuroscience, Institute of Normal and Pathological Physiology, Centre of Experimental Medicine, Slovak Academy of Sciences, Bratislava, Slovakia; Social, Cognitive and Affective Neuroscience Unit, Department of Basic Psychological Research and Research Methods, Faculty of Psychology, University of Vienna, Vienna, Austria. Electronic address:

Research increasingly suggests that nitric oxide (NO) plays a role in the pathogenesis of schizophrenia. One important line of evidence comes from genetic studies, which have repeatedly detected an association between the neuronal isoform of nitric oxide synthase (nNOS or NOS1) and schizophrenia. However, the pathogenetic pathways linking nNOS, NO, and the disorder remain poorly understood. A deficit in sensorimotor gating is considered to importantly contribute to core schizophrenia symptoms such as psychotic disorganization and thought disturbance. We selected three candidate nNOS polymorphisms (Ex1f-VNTR, rs6490121 and rs41279104), associated with schizophrenia and cognition in previous studies, and tested their association with the efficiency of sensorimotor gating in healthy human adults. We found that risk variants of Ex1f-VNTR and rs6490121 (but not rs41279104) were associated with a weaker prepulse inhibition (PPI) of the acoustic startle reflex, a standard measure of sensorimotor gating. Furthermore, the effect of presence of risk variants in Ex1f-VNTR and rs6490121 was additive: PPI linearly decreased with increasing number of risk alleles, being highest in participants with no risk allele, while lowest in individuals who carry three risk alleles. Our findings indicate that NO is involved in the regulation of sensorimotor gating, and highlight one possible pathogenetic mechanism for NO playing a role in the development of schizophrenia psychosis.
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http://dx.doi.org/10.1016/j.niox.2018.08.002DOI Listing
November 2018

A Novel Association of Polymorphism in the Gene Encoding the VLA-4 4 Subunit with Increased Risk of Alzheimer's Disease.

Mediators Inflamm 2018 27;2018:7623823. Epub 2018 Mar 27.

Institute of Immunology, Faculty of Medicine, Comenius University in Bratislava, Bratislava, Slovakia.

Alzheimer's disease (AD) is the most prevalent cause of dementia in elderly people worldwide. Many studies support the hypothesis that the inflammation of the CNS contributes to the neurodegeneration and disease progression. The integrin molecule 41, also known as very late antigen 4 (VLA-4), belongs to adhesion molecules that activate the inflammatory process through the migration of immune cells into the CNS. Therefore, the objective of our study was to analyze the association between two polymorphisms located in the gene encoding the 4 subunit of VLA-4 and the risk of AD. 104 late-onset AD patients and 206 control subjects from Slovakia were genotyped for gene SNP polymorphism rs113276800 (-269C/A) and rs1143676 (+3061A/G). The same study cohorts were also genotyped for the -4, which is a known genetic factor associated with increased risk of AD developing. polymorphism analysis revealed significantly higher frequency of the +3061AG carriers in AD group compared to the controls ( ≤ 0.05). Following the -4 stratification of study groups, the association remained significant only in -4 noncarriers. Our study suggests a novel association of +3061A/G polymorphism with AD and its possible contribution to the disease pathology.
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http://dx.doi.org/10.1155/2018/7623823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892238PMC
October 2018

Draft Genome Sequencing of an Acinetobacter ursingii Isolate from Healthy Human Skin, Carrying Multidrug Resistance Genes.

Genome Announc 2018 May 10;6(19). Epub 2018 May 10.

Comenius University Science Park, Bratislava, Slovakia.

In this paper, we report the data from whole-genome shotgun sequencing of an isolate from healthy human skin of the forearm. The bacterial genome includes 3,473 genes and carries beta-lactamase resistance genes as well as resistance genes for several heavy metals.
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http://dx.doi.org/10.1128/genomeA.00394-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5946048PMC
May 2018

Draft Genome Sequence of Escherichia coli KL53.

Genome Announc 2018 Mar 29;6(13). Epub 2018 Mar 29.

Faculty of Natural Sciences, Department of Molecular Biology in Bratislava, Comenius University in Bratislava, Bratislava, Slovakia.

Here, we report the draft genome sequence of a clinical isolate of the uropathogenic strain KL53. A total of 5,083,632 bp was assembled into 170 contigs containing 89 RNAs and 5,034 protein-coding genes. Remarkable is the presence of the tellurite resistance () operon on a plasmid.
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http://dx.doi.org/10.1128/genomeA.00220-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5876489PMC
March 2018

Is the DNA of placental origin packaged in exosomes isolated from plasma and serum of pregnant women?

Clin Chem Lab Med 2018 05;56(6):e150-e153

Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, Slovakia.

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http://dx.doi.org/10.1515/cclm-2017-0560DOI Listing
May 2018
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