OBJECTIVE: Array technology in chorionic villus sampling (CVS) - analysis of clinical benefit and a proposal of a more effective 1st trimester genetic testing policy. DESIGN: Retrospective study. SETTING: Gennet, Center of Medical Genetics and Reproductive Medicine, Prague. MATERIAL AND METHODS: Total of 913 CVS were performed at Gennet between 2010-2014. All 913 samples were tested by QF-PCR rapid test for aneuploidy of chromosomes 13, 18, 21, X and Y and karyotyping following standard long term culture. Microarray analysis (Illumina HumanCytoSNP12 v2.1) was performed on 179 samples with normal result from both - QF-PCR and karyotyping. RESULTS: At 229 samples the common chromosomal aneuploidy was detected using rapid QF-PCR (25% from 911 successful rapid tests). Conventional karyotyping revealed 239 unbalanced chromosome aberrations (27% from 897 successful cultivations). 227/239 (95%) positive karyotypes confirmed QF-PCR finding of common aneuploidies. 10 unbalanced chromosome aberrations were not covered by rapid QF-PCR test. Microarray analysis of samples with normal result from both- QF-PCR and karyotyping- revealed 13 clinically relevant chromosome aberrations (7.5%). CONCLUSION: New policy for chorionic villi testing at Gennet was established. Based on evaluation of the results of karyotyping, array and QF-PCR and analysis of published data we decided to replace karyotyping by microarray analysis in all cases of foetuses with normal results from QF-PCR. More effective detection of pathological and clinically relevant chromosome aberrations in examined foetuses is expected.

• Keywords
• CVS, QF-PCR, array., karyotype, prenatal diagnosis,
• MeSH
• Aneuploidy MeSH
• Chromosome Disorders diagnosis   MeSH
• Karyotyping methods   MeSH
• Humans MeSH
• Chorionic Villi Sampling MeSH
• Polymerase Chain Reaction methods   MeSH
• Predictive Value of Tests MeSH
• Prenatal Diagnosis methods   MeSH
• Pregnancy Trimester, First MeSH
• Retrospective Studies MeSH
• Pregnancy MeSH
• Check Tag
• Humans MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Gene expression profiles of 10 children with acute lymphoblastic leukemia (ALL) were detected using cDNA arrays. Total RNAs were isolated from peripheral blood leukocytes of the patients at diagnosis. For detection of expression profiles we used Atlas Human Cancer cDNA Arrays (Clontech) with 588 genes. Although the study revealed variability of gene expression in many genes, we identified a number of genes with the same expression changes (up-regulation: PCNA, ERCC1; down-regulation: jun-B, BCL-2 related protein A1, CRAF-1, PBP) in most examined patients. Our objective was to identify genes that were differentially expressed in ALL and might contribute to development (and characterization) of the disease.

• MeSH
• Precursor Cell Lymphoblastic Leukemia-Lymphoma diagnosis   genetics   MeSH
• Antigens, Neoplasm genetics   MeSH
• Child MeSH
• DNA Primers MeSH
• Down-Regulation MeSH
• Oncogene Proteins, Fusion metabolism   MeSH
• Leukocytes pathology   MeSH
• Humans MeSH
• Adolescent MeSH
• Biomarkers, Tumor metabolism   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Child, Preschool MeSH
• Gene Expression Regulation, Leukemic * MeSH
• RNA, Neoplasm analysis   blood   MeSH
• Oligonucleotide Array Sequence Analysis MeSH
• Gene Expression Profiling * MeSH
• Up-Regulation MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Antigens, Neoplasm MeSH
• DNA Primers MeSH
• Oncogene Proteins, Fusion MeSH
• Biomarkers, Tumor MeSH
• RNA, Neoplasm MeSH

Given the great biological importance and high diversity of temperate Staphylococcus aureus bacteriophages, a method is needed for the description of their genomic structure. Here we have updated a multiplex PCR strategy for the complex characterization of S. aureus phages of the family Siphoviridae. Based on the comparative genomic analysis of the available phage sequences, a multilocus PCR strategy for typing the major modules of the phage genome was designed. The genomic modules were classified on the basis of the genes for integrase (10 types), anti-repressor (five types), replication proteins polA, dnaC and dnaD (four types), dUTPase (four types), portal protein (eight types), tail appendices (four types) and endolysin (four types) corresponding to the integrase locus, lysogeny control region, and modules for DNA replication, transcription regulation, packaging, tail appendices and lysis respectively. The nine PCR assays designed for the above sequences were shown to be capable to identify the bacteriophage gene pool present both in the phage and bacterial genomes and their extensive mosaic structure. The established multiplex PCR-based multilocus diagnostic scheme is convenient for rapid and reliable phage and prophage classification and for the study of bacteriophage evolution.

• MeSH
• DNA, Viral genetics   MeSH
• Genome, Viral * MeSH
• Multilocus Sequence Typing MeSH
• Polymerase Chain Reaction methods   MeSH
• Prophages classification   genetics   MeSH
• Siphoviridae classification   genetics   MeSH
• Comparative Genomic Hybridization MeSH
• Staphylococcus aureus genetics   virology   MeSH
• Viral Proteins genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Viral MeSH
• Viral Proteins MeSH

Long non-coding RNAs (lncRNAs) are defined as RNA molecules longer than 200 nucleotides with poor protein-coding capacity and key functions in regulation of gene expression. Dysregulations of lncRNAs (e.g. HOTAIR and MALAT I) were detected in plasma of breast cancer (BC) patients. Plasma samples are examined as liquid biopsies for purposes of non-invasive diagnostics therefore the research of plasma lncRNAs as potential plasma biomarkers became highly topical. 84 lncRNAs were profiled in 18 plasma samples - 9 BC patients and 9 age-matched healthy - using Human Inflammatory Response & Autoimmunity RT2 lncRNA PCR Array. Total RNA from plasma samples was isolated using miRNeasy Serum/Plasma Kit. Although a pre-amplification recommended for quantification from small starting RNA amounts was used, only 3 lncRNAs (A2ML1-AS1, GAS5 and SNHG5) were detected in all plasma samples. A total of 72 lncRNAs (e.g. HOTAIR or MALAT I) were detected only in some samples and 9 lncRNAs were not detected in any samples. No significant differences were observed in levels of plasma lncRNAs between the BC patients and healthy controls despite the fact that our panel contained also the lncRNAs whose levels were previously reported as significantly different in plasma or cancer tissues (e.g. GAS5, HOTAIR, MALAT I) in BC patients. Detection of lncRNAs in plasma is due to their low concentrations quite difficult as compared with tissues. Our findings suggest that analysis of plasma lncRNAs using this technology is not suitable for use as non-invasive diagnostic tool in BC patients.

• MeSH
• Humans MeSH
• Breast Neoplasms diagnosis   genetics   MeSH
• Polymerase Chain Reaction * MeSH
• Gene Expression Regulation, Neoplastic MeSH
• RNA, Long Noncoding blood   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• RNA, Long Noncoding MeSH

The aim of the study was to define specific genetic profile in Ta and T1 urinary bladder carcinoma patients with and without recurrence by gene expression microarrays. Eleven patients with the time to recurrence shorter than one year (patients with recurrence) and 11 patients with time to recurrence longer than 4 years (patients without recurrence) were enrolled. Data from microarrays were subjected to a panel of statistical analyses to identify bladder cancer recurrence-associated gene signatures. Initial screening using the GeneSpring and Bioconductor software tools revealed a putative set 47 genes differing in gene expression in both groups. After the validation, 33 genes manifested significant differences between both groups. The significant expression was observed in the group of patients without recurrence by 30 genes of which the highest differences were detected by ANXA1, ARHGEF4, FLJ32252, GNE, NINJ1, PRICKLE1, PSAT1, RNASE1, SPTAN1, SYNGR1, TNFSF15, TSPAN1, and WDR34. These genes code for signal transduction, vascular remodeling and vascular endothelial growth inhibition mainly. In the group with recurrence, 3 genes had significant differences, the highest differences were identified by two genes (PLOD2 and WDR72). Loci of genes with significant changes of gene expression were located on characteristic chromosomes for bladder cancer: 7 loci on chromosome 9, 8 loci on chromosomes 1, 2, 3, 12,14,15,16, and 22. We have selected and validated 15 genes that are differentially expressed in superficial bladder cancer. We hope that this cohort of genes will serve as a promising pool of candidate biomarkers for early stage bladder cancer. Our results indicate that it may be possible to identify patients with a low and high risk of disease recurrence at an early stage using a molecular profile.

• MeSH
• Adult MeSH
• Neoplasm Invasiveness MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Middle Aged MeSH
• Humans MeSH
• Neoplasm Recurrence, Local diagnosis   genetics   MeSH
• RNA, Messenger genetics   MeSH
• Biomarkers, Tumor genetics   MeSH
• Urinary Bladder Neoplasms genetics   pathology   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Prognosis MeSH
• Retrospective Studies MeSH
• Risk Factors MeSH
• Oligonucleotide Array Sequence Analysis MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Neoplasm Staging MeSH
• Gene Expression Profiling * MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• RNA, Messenger MeSH
• Biomarkers, Tumor MeSH

γδ T cells are intensively studied because their function in infection, allergy, autoimmune disease, cancer and post-transplant period is not yet fully understood. PCR-based techniques were established to study the γ variable (Vγ) and δ variable (Vδ) gene families. PCR product evaluation is routinely carried out by Southern blot analysis or the third complementarity-determining region spectratyping, but a fast and simple assessment of Vγ and Vδ gene family expression is missing. The aim of our study was to test capillary electrophoresis as a potential method for evaluating the composition of the γδ T-cell population. This report provides optimized PCR conditions for γδ T-cell receptor amplification. Further, it describes the utilization of capillary electrophoresis in the Agilent 2100 Bioanalyzer to evaluate the relative expression of Vγ and Vδ gene families after their amplification. An application of the methodology to peripheral blood mononuclear cell samples from patients during haemato-oncological treatment is shown. The described methodology is fast and simple to operate and is therefore suitable as a first screening of the γδ T-cell population composition in tissues of interest.

• MeSH
• Time Factors MeSH
• Adult MeSH
• Electrophoresis, Capillary methods   MeSH
• Hematologic Neoplasms genetics   pathology   MeSH
• Leukocytes, Mononuclear cytology   metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Multigene Family * MeSH
• Reverse Transcriptase Polymerase Chain Reaction methods   MeSH
• Receptors, Antigen, T-Cell, gamma-delta genetics   metabolism   MeSH
• Oligonucleotide Array Sequence Analysis methods   MeSH
• Case-Control Studies MeSH
• T-Lymphocytes cytology   metabolism   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Receptors, Antigen, T-Cell, gamma-delta MeSH

Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a rapid method for simultaneous detection of multiple molecular markers within a single reaction. MOL-PCR is increasingly employed in microbial detection assays, where its ability to facilitate identification and further characterization via simple analysis is of great benefit and significantly simplifies routine diagnostics. When adapted to microsphere suspension arrays on a MAGPIX reader, MOL-PCR has the potential to outperform standard nucleic acid-based diagnostic assays. This study represents the guideline towards in-house MOL-PCR assay optimization using the example of foodborne pathogens (bacteria and parasites) with an emphasis on the appropriate choice of crucial parameters. The optimized protocol focused on specific sequence detection utilizes the fluorescent reporter BODIPY-TMRX and self-coupled magnetic microspheres and allows for a smooth and brisk workflow which should serve as a guide for the development of MOL-PCR assays intended for pathogen detection.

• MeSH
• Yersinia Infections diagnosis   microbiology   MeSH
• Humans MeSH
• Multiplex Polymerase Chain Reaction methods   MeSH
• Foodborne Diseases diagnosis   microbiology   parasitology   MeSH
• Toxoplasma genetics   isolation & purification   MeSH
• Toxoplasmosis diagnosis   parasitology   MeSH
• Yersinia enterocolitica genetics   isolation & purification   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points.

• Keywords
• BCR-ABL1, CML, LD-PCR, NGS, pyrosequencing, quantitative analysis of mutant subclones,
• MeSH
• Fusion Proteins, bcr-abl chemistry   genetics   MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive genetics   pathology   MeSH
• DNA analysis   genetics   metabolism   MeSH
• Humans MeSH
• Polymerase Chain Reaction * MeSH
• Sequence Analysis, DNA * MeSH
• Comparative Genomic Hybridization MeSH
• High-Throughput Nucleotide Sequencing * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Fusion Proteins, bcr-abl MeSH
• DNA MeSH

Cyanobacteria are well known producers of bioactive metabolites, including harmful substances. The recently discovered "eagle killer" neurotoxin aetokthonotoxin (AETX) is produced by the epiphytic cyanobacterium Aetokthonos hydrillicola growing on invasive water thyme (Hydrilla verticillata). The biosynthetic gene cluster of AETX was previously identified from an Aetokthonos strain isolated from the J. Strom Thurmond Reservoir, Georgia, USA. Here, a PCR protocol for easy detection of AETX-producers in environmental samples of plant-cyanobacterium consortia was designed and tested. Three different loci of the AETX gene cluster were amplified to confirm the genetic potential for AETX production, along with two variable types of rRNA ITS regions to confirm the homogeneity of the producer´s taxonomic identity. In samples of Hydrilla from three Aetokthonos-positive reservoirs and one Aetokthonos-negative lake, the PCR of all four loci provided results congruent with the Aetokthonos presence/absence detected by light and fluorescence microscopy. The production of AETX in the Aetokthonos-positive samples was confirmed using LC-MS. Intriguingly, in J. Strom Thurmond Reservoir, recently Hydrilla free, an Aetokthonos-like cyanobacterium was found growing on American water-willow (Justicia americana). Those specimens were positive for all three aet markers but contained only minute amounts of AETX. The obtained genetic information (ITS rRNA sequence) and morphology of the novel Aetokthonos distinguished it from all the Hydrilla-hosted A. hydrillicola, likely at the species level. Our results suggest that the toxigenic Aetokthonos spp. can colonize a broader array of aquatic plants, however the level of accumulation of the toxin may be driven by host-specific interactions such as the locally hyper-accumulated bromide in Hydrilla.

• Keywords
• Aetokthonos, Cyanotoxin, Hydrilla, Justicia, Vacuolar myelinopathy, rRNA ITS,
• MeSH
• Chromatography, Liquid MeSH
• Mass Spectrometry MeSH
• Lakes * MeSH
• Polymerase Chain Reaction MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• aetokthonotoxin MeSH Browser

The original method for molecular typing of E. coli strains was developed using the polymorphism in chromosomal sequences of bacterial interspersed mosaic elements (BIMEs) detected by multiplex PCR and analysed by AFLP assay. The applicability of the method in the epidemiology of E. coli was tested on a group of 524 strains of human and veterinary origin. In the studied group 18 different genotypes were detected. Significant differences were found in the frequencies of the genotypes among various groups of strains, suggesting the method could be a promising tool in the epidemiology of E. coli.

• MeSH
• Time Factors MeSH
• DNA, Bacterial analysis   genetics   MeSH
• DNA Fingerprinting MeSH
• Electrophoresis, Agar Gel methods   standards   MeSH
• Escherichia coli classification   genetics   MeSH
• Gene Frequency genetics   MeSH
• Genetic Variation genetics   MeSH
• Genotype MeSH
• Escherichia coli Infections epidemiology   microbiology   MeSH
• DNA, Intergenic genetics   MeSH
• Humans MeSH
• Molecular Epidemiology methods   MeSH
• Molecular Sequence Data MeSH
• Polymerase Chain Reaction methods   standards   MeSH
• Polymorphism, Restriction Fragment Length * MeSH
• Interspersed Repetitive Sequences genetics   MeSH
• Oligonucleotide Array Sequence Analysis methods   MeSH
• Cattle MeSH
• Bacterial Typing Techniques methods   standards   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Cattle MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Validation Study MeSH
• Names of Substances
• DNA, Bacterial MeSH
• DNA, Intergenic MeSH