Kazuistika pojednáva o prípade z diabetologickej ambulancie v Moldave nad Bodvou, kde sa 24 ročný muž s MODY-3 diabetom na základe výsledku DNA diagnostiky úspešne previedol z inzulinoterapie na liečbu tabletami sulfonylurey.

The case report discusses a case from a diabetology outpatients-in Moldava nad Bodvou, where a 24 years old man with MODY-3 diabetes mellitus was successfully switched from insulinotherapy to oral therapy by sulphonylurea tablets based on DNA diagnostics.

• MeSH
• Diabetes Mellitus, Type 2 diagnosis   genetics   therapy   MeSH
• Hypoglycemic Agents administration & dosage   MeSH
• Insulin administration & dosage   MeSH
• Humans MeSH
• DNA Mutational Analysis MeSH
• Sulfonylurea Compounds administration & dosage   pharmacology   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Case Reports MeSH

Cíle: zhodnotit senzitivitu a specifitu vyšetření DNA Borrelia burgdorferi (B. burgdorferi) v séru a v synoviální tekutině nemocných s Lymeskou artritidou (LA). Zhodnotit výskyt DNA B. burgdorferi v séru a v synoviální tekutině nemocných s nediferencovanou artritidou (NA). Metody: vyšetřili jsme celkem 102 sér a 37 synoviálních tekutin nemocných s různými revmatologickými diagnózami. První soubor celkem 17 krevních a sérologických vzorků a 9 synoviálních tekutin představovaly klinické vzorky nemocných s definitivní diagnózou LA. Druhý soubor představovaly klinické vzorky nemocných s NA (13 krví, 10 synoviálních tekutin). Třetí soubor obsahoval 72 krevních vzorků a 18 synoviálních tekutin nemocných s jinými definitivními revmatologickými diagnózami. Ve všech vzorcích byla detekována přítomnost DNA B. burgdorferi metodou PCR – polymerázová řetězová reakce a současně byla vyšetřena imunoreaktivita proti B. burgdorferi metodou ELISA – Enzyme linked immunosorbent assay a Western blotem. Přítomnost DNA B. burgdorferi v klinických vzorcích nemocných s LA byla statisticky porovnána s výskytem v druhých dvou souborech a byla zhodnocena senzitivita a specifita tohoto vyšetření pro stanovení diagnózy LA. Výskyt DNA B. burgdorferi ve vzorcích nemocných s NA byl porovnán s výskytem ve třetím souboru a bylo hodnoceno, zda rozdíl mezi soubory dosahuje statistického významu. Výsledky: V souboru 17 nemocných s LA jsme v krvi prokázali DNA B. burgdorferi u 5 nemocných. Senzitivita nálezu v krvi odpovídala 29,41 %. V dostupné synoviální tekutině 9 pacientů tohoto souboru jsme prokázali DNA u 4 nemocných. Senzitivita odpovídala 44,44 % u pacientů s LA nebyly nalezeny rozdíly mezi pozitivními nálezy plasmidové a genomové DNA (48 % a 48 %). Specifita vyšetření byla 40 % v krvi a 65 % v synoviální tekutině. V souboru 13 nemocných s NA jsme prokázali DNA v krvi u 2 nemocných (15,38 %). V 10 dostupných synoviálních tekutinách byla DNA nalezena u 4 nemocných (40 %). Z uvedeného vyplývá, že nebyl přítomen statisticky významný rozdíl mezi nálezy DNA v synoviální tekutině u nemocných s NA a LA (44,44 % proti 40 %).V souboru 72 nemocných s jinými revmatickými chorobami jsme prokázali DNA v krvi u 13 pacientů (18,06 %), z 18 dostupných synoviálních tekutin bylo DNA přítomno u 3 pacientů (16,67 %). Výskyt DNA v synoviální tekutině nemocných s NA byl významně vyšší oproti nemocným s jinými definitivními revmatickými diagnózami (40 % proti 16,67 %). Závěr: vyšetření DNA B. burgdorferi u nemocných s LA vykazovalo v našem souboru nízkou senzitivitu v krvi (29,41 %) a střední senzitivitu v synoviální tekutině (44,44 %). Negativní nález DNA nevylučuje LA. Specifita vyšetření byla v krvi i v synoviální tekutině nízká (40 % v krvi a 65 % v synoviální tekutině). Statisticky nevýznamný rozdíl mezi výskytem DNA v synoviální tekutině nemocných s LA a NA (44,44 % proti 40 %) svědčil pro malý význam tohoto vyšetření v diferenciální diagnóze těchto chorob. Nízká specifita pozitivních nálezů u nemocných s LA oproti kontrolním souborům svědčila rovněž pro malý význam izolovaného nálezu DNA v diferenciální diagnostice LA a jiných revmatických chorob. Nález DNA B. burgdorferi v klinickém vzorku poskytuje pouze doplňující a nikoliv určující diagnostickou informaci pokud je použito výsledků běžné neexperimentální laboratoře.

Aims: to evaluate sensitivity and specificity of Borrelia burgdorferi (B. burgdorferi) DNA detection in sera and synovial fluid from patients with Lyme arthritis (LA). To evaluate presence of B. burgdorferi DNA in serum and synovial fluid from patients with undifferentiated arthritis (UA). Methods: we examined 102 serum and 37 synovial fluid samples from patients with various rheumatic diseases. First group consisted of 17 blood and serum samples and 9 synovial fluid samples from patients with definite diagnosis of LA. In the second group there were 13 blood and 10 synovial fluid samples from patients with UA. Third group consisted of 72 blood samples and 18 synovial fluids from patients with other established rheumatological diagnoses. DNA of B. burgdorferi was analyzed by PCR. Antibodies against B. burgdorferi were examined by ELISA and Western blotting. Sensitivity and specificity of the PCR B. burgdorferi DNA detection for the diagnosis of LA was calculated. Results: We found B. burgdorferi DNA in the blood samples from 5 patients from the group of 17 patients with LA. Sensitivity of this examination was 29.4%. In the synovial fluid we demonstrated DNA in 4 of 9 patients, which corresponds to a sensitivity of 44.4%. There were no differences between positive plasmid and genomic DNA in LA patients (48% and 48%). Specificity of this examination was 40% for blood and 65% for synovial fluid. In the blood from group of 13 patients with UA we found B. burgdorferi DNA in 2 of them (15.4%). In the synovial fluid we demonstrated this DNA in 4 of 10 patients (40%). Abovementioned data showed no statistically significant difference between DNA in synovial fluid from patients with UA and LA (44% vs. 40%). In the blood from the group of 72 patients with other rheumatic diseases we found DNA in 13 patients (18.1%). In the synovial fluid we demonstrated DNA in 3 of 18 patients (16.7%). Presence of the B. burgdorferi DNA in synovial fluid from patients with UA was significantly higher in contrast to patients with other established rheumatic diagnosis (40% vs. 16.7%). Conclusion: the evaluation of B. burgdorferi DNA in patients with LA showed low sensitivity in the blood (29.4%) and medium sensitivity in the synovial fluid (44.4%). Negative DNA finding does not exclude the diagnosis of LA. Specificity of this evaluation in the blood and synovial fluid is low (40% for blood and 65% for synovial fluid). Statistically insignificant difference between the presence of DNA in synovial fluid from patients with LA and UA (44.4% vs. 40%) revealed low value for this evaluation in differential diagnosis of these diseases. Low specificity of positive findings in the patients with LA in contrast to control groups showed also low implication of isolated DNA finding with regard to differential diagnosis of LA and other rheumatic diseases. Presence of B. burgdorferi DNA in the clinical sample represents only additional and not major diagnostic information when the analysis is performed in a general non-experimental laboratory.

• MeSH
• Arthritis diagnosis   etiology   MeSH
• Borrelia burgdorferi genetics   MeSH
• DNA, Bacterial blood   MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Research Support as Topic MeSH
• Humans MeSH
• Lyme Disease diagnosis   MeSH
• Polymerase Chain Reaction MeSH
• Sensitivity and Specificity MeSH
• Synovial Fluid chemistry   MeSH
• Blotting, Western MeSH
• Check Tag
• Humans MeSH

Approximately 13% of the human genome at certain motifs have the potential to form noncanonical (non-B) DNA structures (e.g., G-quadruplexes, cruciforms, and Z-DNA), which regulate many cellular processes but also affect the activity of polymerases and helicases. Because sequencing technologies use these enzymes, they might possess increased errors at non-B structures. To evaluate this, we analyzed error rates, read depth, and base quality of Illumina, Pacific Biosciences (PacBio) HiFi, and Oxford Nanopore Technologies (ONT) sequencing at non-B motifs. All technologies showed altered sequencing success for most non-B motif types, although this could be owing to several factors, including structure formation, biased GC content, and the presence of homopolymers. Single-nucleotide mismatch errors had low biases in HiFi and ONT for all non-B motif types but were increased for G-quadruplexes and Z-DNA in all three technologies. Deletion errors were increased for all non-B types but Z-DNA in Illumina and HiFi, as well as only for G-quadruplexes in ONT. Insertion errors for non-B motifs were highly, moderately, and slightly elevated in Illumina, HiFi, and ONT, respectively. Additionally, we developed a probabilistic approach to determine the number of false positives at non-B motifs depending on sample size and variant frequency, and applied it to publicly available data sets (1000 Genomes, Simons Genome Diversity Project, and gnomAD). We conclude that elevated sequencing errors at non-B DNA motifs should be considered in low-read-depth studies (single-cell, ancient DNA, and pooled-sample population sequencing) and in scoring rare variants. Combining technologies should maximize sequencing accuracy in future studies of non-B DNA.

• MeSH
• DNA genetics   MeSH
• Humans MeSH
• Nanopores * MeSH
• Nucleotide Motifs MeSH
• Sequence Analysis, DNA MeSH
• High-Throughput Nucleotide Sequencing MeSH
• DNA, Z-Form * MeSH
• Base Composition MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, N.I.H., Extramural MeSH

• MeSH
• DNA, Viral isolation & purification   MeSH
• Hepatitis B diagnosis   genetics   blood   MeSH
• Hepatitis C diagnosis   genetics   blood   MeSH
• Microbiological Techniques methods   standards   MeSH
• Quality Control MeSH

Expansions of trinucleotide repeats (TNRs) are associated with genetic disorders such as Friedreich's ataxia. The tumor suppressor p53 is a central regulator of cell fate in response to different types of insults. Sequence and structure-selective modes of DNA recognition are among the main attributes of p53 protein. The focus of this work was analysis of the p53 structure-selective recognition of TNRs associated with human neurodegenerative diseases. Here, we studied binding of full length p53 and several deletion variants to TNRs folded into DNA hairpins or loops. We demonstrate that p53 binds to all studied non-B DNA structures, with a preference for non-B DNA structures formed by pyrimidine (Py) rich strands. Using deletion mutants, we determined the C-terminal DNA binding domain of p53 to be crucial for recognition of such non-B DNA structures. We also observed that p53 in vitro prefers binding to the Py-rich strand over the purine (Pu) rich strand in non-B DNA substrates formed by sequence derived from the first intron of the frataxin gene. The binding of p53 to this region was confirmed using chromatin immunoprecipitation in human Friedreich's ataxia fibroblast and adenocarcinoma cells. Altogether these observations provide further evidence that p53 binds to TNRs' non-B DNA structures.

• MeSH
• DNA chemistry   metabolism   MeSH
• Trinucleotide Repeat Expansion * MeSH
• Gene Expression MeSH
• Friedreich Ataxia genetics   metabolism   MeSH
• Protein Interaction Domains and Motifs MeSH
• Nucleic Acid Conformation * MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 chemistry   metabolism   MeSH
• Pyrimidines MeSH
• Recombinant Proteins MeSH
• Trinucleotide Repeats * MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

DNA conformation may deviate from the classical B-form in ∼13% of the human genome. Non-B DNA regulates many cellular processes; however, its effects on DNA polymerization speed and accuracy have not been investigated genome-wide. Such an inquiry is critical for understanding neurological diseases and cancer genome instability. Here, we present the first simultaneous examination of DNA polymerization kinetics and errors in the human genome sequenced with Single-Molecule Real-Time (SMRT) technology. We show that polymerization speed differs between non-B and B-DNA: It decelerates at G-quadruplexes and fluctuates periodically at disease-causing tandem repeats. Analyzing polymerization kinetics profiles, we predict and validate experimentally non-B DNA formation for a novel motif. We demonstrate that several non-B motifs affect sequencing errors (e.g., G-quadruplexes increase error rates), and that sequencing errors are positively associated with polymerase slowdown. Finally, we show that highly divergent G4 motifs have pronounced polymerization slowdown and high sequencing error rates, suggesting similar mechanisms for sequencing errors and germline mutations.

• MeSH
• DNA chemistry   MeSH
• G-Quadruplexes MeSH
• Genomics * methods   standards   MeSH
• Kinetics MeSH
• Nucleic Acid Conformation * MeSH
• Humans MeSH
• Mutation MeSH
• Nucleotide Motifs MeSH
• DNA Replication MeSH
• Reproducibility of Results MeSH
• Sequence Analysis, DNA * methods   MeSH
• High-Throughput Nucleotide Sequencing * methods   standards   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• DNA, Viral * isolation & purification   MeSH
• Hepatitis B diagnosis   MeSH
• Hepatitis C diagnosis   MeSH
• Quality Control MeSH
• RNA, Viral * isolation & purification   MeSH
• Laboratory Proficiency Testing * MeSH

BACKGROUND: The breast and ovarian cancer susceptibility gene BRCA1 encodes a multifunctional tumor suppressor protein BRCA1, which is involved in regulating cellular processes such as cell cycle, transcription, DNA repair, DNA damage response and chromatin remodeling. BRCA1 protein, located primarily in cell nuclei, interacts with multiple proteins and various DNA targets. It has been demonstrated that BRCA1 protein binds to damaged DNA and plays a role in the transcriptional regulation of downstream target genes. As a key protein in the repair of DNA double-strand breaks, the BRCA1-DNA binding properties, however, have not been reported in detail. RESULTS: In this study, we provided detailed analyses of BRCA1 protein (DNA-binding domain, amino acid residues 444-1057) binding to topologically constrained non-B DNA structures (e.g. cruciform, triplex and quadruplex). Using electrophoretic retardation assay, atomic force microscopy and DNA binding competition assay, we showed the greatest preference of the BRCA1 DNA-binding domain to cruciform structure, followed by DNA quadruplex, with the weakest affinity to double stranded B-DNA and single stranded DNA. While preference of the BRCA1 protein to cruciform structures has been reported previously, our observations demonstrated for the first time a preferential binding of the BRCA1 protein also to triplex and quadruplex DNAs, including its visualization by atomic force microscopy. CONCLUSIONS: Our discovery highlights a direct BRCA1 protein interaction with DNA. When compared to double stranded DNA, such a strong preference of the BRCA1 protein to cruciform and quadruplex structures suggests its importance in biology and may thus shed insight into the role of these interactions in cell regulation and maintenance.

• MeSH
• DNA, B-Form chemistry   metabolism   MeSH
• Nucleic Acid Conformation MeSH
• Humans MeSH
• BRCA1 Protein chemistry   metabolism   MeSH
• Protein Domains MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Leukemie postihuje 2% populace v západní Evropě. Nejčastějším typem je B-CLL, která zahrnuje asi 30% všech případů. I přes značný metodický pokrok ve výzkumu nebyl zjištěný žádný jednoznačný chromozomální nebo protoonkogenový defekt. Vznik nádorů je výsledkem hromadění různých genetických poruch, které regulují specifické buněčné dráhy. Mutace v genu Dok1 a GNB3, které budou analyzovány, spolu s dalšími anomáliemi mohou hrát důležitou roli v iniciaci, progresi nebo léčbě CLL. STAT proteiny mají klíčovouroli v cytokinové signalizaci a následně v proliferaci a apoptóze lymfocytů. Identifikace poruch fosforylace STAT proteinů analyzovaná Western blotem může objasnit úlohu těchto změn ve vývoji CLL. Lymfom plášťové zóny (MCL) je agresivní B-buněčný non-Hodkinský lymfom vznikající z nezralých lymfocytů s translokací t(11;14)(q13;q32). Rozdílná exprese genu c-myc a p16 bude kvantifikována a dána do souvislosti s klinickým obrazem.; In Western countries leukemias affects 2% of the population. CLL is the most common form accounting for about 30% of all cases. Although considerably progress has been made in research no consistent chromosomal or protooncogene defect has been found. Since tumorogenicity is assumed to be a multistep process in which various genetic changes lead to tumor formation,the mutations in Dok1 or GNB3 gene in conjunction with other identified alterations may be important in CLL progression and chemoresistance STATs play central role in cytokine signaling and influence proliferation and apoptosis of lymphocytes. Identification of the defects in STAT phosphorylation by Western blot can clarify relevance of this modification in CLL development. Mantle cell lymphom(MCL) is aggressive B-cell non-Hodkins lymphom arrising from immature lymphocytes with distinctive translocation t(11;14)(q13;q32). C-myc and p16 genes are integrated to the disease development and their expression will be specified.

• MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell diagnosis   MeSH
• Molecular Diagnostic Techniques MeSH
• DNA Mutational Analysis MeSH
• Lymphoma, Non-Hodgkin diagnosis   MeSH
• Polymerase Chain Reaction MeSH
• RNA analysis   MeSH
• STAT Transcription Factors MeSH

• Conspectus
• Biochemie. Molekulární biologie. Biofyzika
• NML Fields
• onkologie
• hematologie a transfuzní lékařství
• biologie
• NML Publication type
• závěrečné zprávy o řešení grantu IGA MZ ČR

• MeSH
• DNA, Viral * isolation & purification   MeSH
• Hepatitis B diagnosis   blood   MeSH
• Hepatitis C diagnosis   blood   MeSH
• Humans MeSH
• Microbiological Techniques methods   standards   MeSH
• RNA, Viral * isolation & purification   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Tables MeSH