Cíl studie: Syndrom Noonanové (NS), jedna z nejčastějších RASopatií, má odhadovanou incidenci 1 z 1000 až 2500 živě narozených jedinců. V prenatálním období lze u postižených plodů s tímto syndromem pozorovat zvýšené šíjové projasnění, hygroma colli, hydrops plodu, vrozené srdeční vady, vady ledvin, lymfatické vaky nebo větší množství plodové vody. U plodů s těmito nálezy byla při nálezu normálního karyotypu a po vyloučení mikrodelečních/mikroduplikačních syndromů doplněna prenatální diagnostika i o vyšetření vybraných genů pro RASopatie. Cílem studie bylo zhodnotit klinický přínos masivně paralelního sekvenování (MPS, massively parallel sequencing) DNA plodů suspektních pro NS, tj. diagnostickou výtěžnost na jedné straně a podíl detekovaných variant nejasného významu na straně druhé. Typ studie: Klinicky diagnostická. Název a sídlo pracoviště: Centrum prenatální diagnostiky, Brno, s.r.o.; Cytogenetická laboratoř Brno, s.r.o. Metodika: Analyzovány byly vzorky plodových vod, resp. choriových klků. Z nich izolovaná DNA byla po zjištění normálního karyotypu a array-CGH (aCGH) postoupena k analýze genů asociovaných s RASopatiemi metodou MPS. Výsledky: Ve sledovaném dvouletém období byl NS potvrzen celkem u 10 z 95 vyšetřovaných plodů. To představuje 10,5% diagnostickou výtěžnost metody. Celkem u 10 plodů byla detekována varianta DNA nejasného významu (VOUS, variants of unknown significance), následná segregační analýza pomohla objasnit její význam u šesti plodů. Závěr: MPS umožňuje rychlou molekulárně-genetickou diagnostiku RASopatií již v prenatálním období. Tato metoda tak přispívá k objasnění nejen fenotypových projevů u již narozených jedinců, ale také ultrazvukových nálezů u plodů s normálním karyotypem i aCGH.

Objective: Noonan syndrome (NS), one of the most common RASopathies, has an estimated incidence of 1 in 1,0002,500 live births. In the prenatal period increased nuchal translucency, hygroma colli, hydrops fetus, congenital heart disease, kidney defects, larger amount of amniotic fluid can be observed in affected fetuses with this syndrome. In the fetuses with normal karyotype and no microdeletion/microduplication syndromes the examination of selected genes for RASopathies was added. The aim of the study was to evaluate the clinical benefit of massively parallel sequencing (MPS) of susceptible fetal DNA for NS, i.e., the diagnostic yield on the one hand and the proportion of detected variants of unknown significance (VOUS) on the other. Design: Clinically diagnostic. Setting: Centrum prenatální diagnostiky, Brno, s.r.o; Cytogenetická laboratoř Brno, s.r.o. Methods: Samples of amniotic fluid or chorionic villus were analyzed. Selected genes associated with RASopathies were analyzed in case of the negative result of karyotype and array-CGH. A panel of twenty genes was investigated by MPS. Results: In the two-years period, Noonan syndrome was detected in 10 from 95 investigated fetuses. This represents a 10.5% diagnostic efficiency of the method. DNA variants of unknown significance were detected in 10 fetuses. A segregation analysis helped to clarify their meaning in six fetuses. Conclusion: MPS allows fast molecular-genetic diagnosis of RASopathies already in the prenatal period. This method contributes to the clarification not only of phenotypic manifestations in already born individuals but also of ultrasound findings in fetuses with both normal karyotype and aCGH.

• Klíčová slova
• cystický hygrom,
• MeSH
• hydrops fetalis MeSH
• komplikace těhotenství MeSH
• lidé MeSH
• měření nuchální translucence metody   MeSH
• Noonanové syndrom * diagnóza   MeSH
• prenatální diagnóza * metody   MeSH
• retrospektivní studie MeSH
• těhotenství MeSH
• vrozené srdeční vady diagnóza   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH

The inevitable accumulation of chromosomal abnormalities in human embryonic stem cells (hESCs) during in vitro expansion represents a considerable obstacle for cell replacement therapies. To determine the source of chromosomal abnormalities, we examined hESCs maintained in culture for over 55 months for defects in telomere maintenance and DNA repair. Although prolonged culture affected neither telomerase activity nor nonhomologous end joining, the efficiency of base excision repair (BER) was significantly decreased and correlated with reduced expression of apurinic/apyrimidinic endonuclease 1 (APE1), the major nuclease required for BER. Interestingly, the expression of other BER enzymes was unchanged. Addition of human recombinant APE1 protein to nuclear extracts from late passage hESCs increased BER efficiency to the level typical of early passage hESCs. The link between BER and double-strand breaks (DSB) was demonstrated by decreased DSB release after downregulation of APE1 in early passage hESCs via siRNA. Correspondingly lower APE1 level in late passage hESC resulted in slower and less intensive but long lasting DSB release upon ionizing radiation (IR). Downregulation of APE1 in early passage hESCs also led to approximately 30% decrease in γ-H2AX signaling following IR, similar to that in late passage hESCs. We suggest that downregulation of APE1 significantly contributes to the failure of BER during long-term culture of hESCs, and further that BER failure is one of the factors affecting the genomic instability of hESCs by altering BER-dependent DSB release and cell cycle/checkpoint signaling.

• MeSH
• buněčné linie MeSH
• DNA-lyasa (apurinová nebo apyrimidinová) genetika   metabolismus   MeSH
• dvouřetězcové zlomy DNA MeSH
• embryonální kmenové buňky enzymologie   metabolismus   MeSH
• imunohistochemie MeSH
• karyotypizace MeSH
• lidé MeSH
• oprava DNA genetika   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Telomere repeats are added onto chromosome ends by telomerase, consisting of two main core components: a catalytic protein subunit (telomerase reverse trancriptase, TERT), and an RNA subunit (telomerase RNA, TR). Here, we report for the first time evidence that HMGB1 (a chromatin-associated protein in mammals, acting as a DNA chaperone in transcription, replication, recombination, and repair) can modulate cellular activity of mammalian telomerase. Knockout of the HMGB1 gene (HMGB1 KO) in mouse embryonic fibroblasts (MEFs) results in chromosomal abnormalities, enhanced colocalization of γ-H2AX foci at telomeres, and a moderate shortening of telomere lengths. HMGB1 KO MEFs also exhibit significantly (>5-fold) lower telomerase activity than the wild-type MEFs. Correspondingly, enhanced telomerase activity is observed upon overexpression of HMGB1 in MEFs. HMGB1 physically interacts with both TERT and TR, as well as with active telomerase complex in vitro. However, direct interaction of HMGB1 with telomerase is most likely not accountable for the observed higher telomerase activity in HMGB1-containing cells, as revealed from the inability of purified HMGB1 protein to stimulate telomerase activity in vitro. While no transcriptional silencing of TERT is observed in HMGB1 KO MEFs, levels of TR are diminished (~3-fold), providing possible explanation for the observed lower telomerase activity in HMGB1 KO cells. Interestingly, knockout of the HMGB2 gene elevates telomerase activity (~3-fold) in MEFs, suggesting that the two closely related proteins of the HMGB family, HMGB1 and HMGB2, have opposite effects on telomerase activity in the cell. The ability of HMGB1 to modulate cellular activity of telomerase and to maintain telomere integrity can help to understand some aspects of the protein involvement in chromosome stability and cancer.

• MeSH
• buněčné linie MeSH
• chromozomální aberace MeSH
• down regulace MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• fragmentace DNA MeSH
• genový knockout MeSH
• histony genetika   metabolismus   MeSH
• hybridizace in situ fluorescenční MeSH
• myši MeSH
• poškození DNA MeSH
• protein HMGB1 genetika   metabolismus   MeSH
• protein HMGB2 genetika   metabolismus   MeSH
• replikace DNA MeSH
• RNA genetika   metabolismus   MeSH
• telomerasa genetika   metabolismus   MeSH
• telomery metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Analysis of plants bearing a T-DNA insertion is a potent tool of modern molecular biology, providing valuable information about the function and involvement of genes in metabolic pathways. A collection of 12 Arabidopsis thaliana lines with T-DNA insertions in the gene coding for the catalytic subunit of telomerase (AtTERT) and in adjacent regions was screened for telomerase activity [telomere repeat amplification protocol (TRAP) assay], telomere length (terminal restriction fragments), and AtTERT transcription (quantitative reverse transcription-PCR). Lines with the insertion located upstream of the start codon displayed unchanged telomere stability and telomerase activity, defining a putative minimal AtTERT promoter and the presence of a regulatory element linked to increased transcription in the line SALK_048471. Lines bearing a T-DNA insertion inside the protein-coding region showed telomere shortening and lack of telomerase activity. Transcription in most of these lines was unchanged upstream of the T-DNA insertion, while it was notably decreased downstream. The expression profile varied markedly in mutant lines harbouring insertions at the 5' end of AtTERT which showed increased transcription and abolished tissue specificity. Moreover, the line FLAG_385G01 (T-DNA insertion inside intron 1) revealed the presence of a highly abundant downstream transcript with normal splicing but without active telomerase. The role of regulatory elements found along the AtTERT gene is discussed in respect to natural telomerase expression and putative intron-mediated enhancement.

• MeSH
• Arabidopsis genetika   MeSH
• DNA bakterií genetika   MeSH
• genotyp MeSH
• inzerční mutageneze MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proteiny huseníčku genetika   MeSH
• regulace genové exprese u rostlin MeSH
• regulační oblasti nukleových kyselin genetika   MeSH
• telomerasa genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• abstrakt z konference MeSH

BACKGROUND: Telomerase activity is associated with many malignancies, including head and neck cancer. The use of telomerase activity as a diagnostic and prognostic marker of head and neck cancer development was examined and compared with standard histological analysis. PATIENTS AND METHODS: Telomerase activity was determined using quantitative dual-colour real-time TRAP (telomeric repeat amplification protocol). In each of 58 patients, a sample of tumour tissue, adjacent mucosa and normal muscle was collected. RESULTS: Telomerase activation was observed in 88% of tumour tissues and 34% of tumour-adjacent mucosa samples. No telomerase activity was detected in normal muscle tissues. Telomerase activity correlated with tumour grade, with an average of 4.6 telomerase units (T.U.) in well-differentiated, 8.3 T.U. in moderately-differentiated and 20 T.U. in poorly differentiated tumours. Relapse occurred in 13 patients and no telomerase activity was detected in 3 recurrent tumours. CONCLUSION: Telomerase activity may be used as an objective parameter inversely related to tumour differentiation. Prognosis in telomerase-negative tumours is worse than that of the telomerase-positive group.

• MeSH
• kouření genetika   metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorové biomarkery metabolismus   MeSH
• nádory hlavy a krku enzymologie   genetika   MeSH
• prognóza MeSH
• senioři MeSH
• telomerasa metabolismus   MeSH
• telomery metabolismus   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• práce podpořená grantem MeSH

Telomerase assay in multiple myeloma is new perspective prognostic factor in multiple myeloma The use od hTERT as tumor antigen is promising new treatment modality and could circumvent limitation of current vaccination protocols in multiple myeloma in which low immunogenic antigens and consequently minimal responses are reported.

Vyhodnocení telomerázové aktivity a měření telomer by mohlo patřit k novým perspektivním prognostickým faktorům u mnohočetného myelomu. Možnost využít hTERT jako nádorového antigenu by výrazným způsobem obohatila vakcinační strategii u mnohočetného myelomu, kde je doposud limitací nízká účinnost, respektive nepřítomnost silného antigenu.

• MeSH
• mnohočetný myelom MeSH
• prognóza MeSH
• telomerasa MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• alergologie a imunologie
• onkologie
• hematologie a transfuzní lékařství
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

• Publikační typ
• abstrakt z konference MeSH