Non-invasive prenatal tests for the detection of fetal aneuploidies are predominantly based on the analysis of cell-free DNA (cfDNA) from the plasma of pregnant women by next-generation sequencing. The development of alternative tests for routine genetic laboratories is therefore desirable. Multiplex digital droplet PCR was used to detect 16 amplicons from chromosome 21 and 16 amplicons from chromosome 18 as the reference. Two fluorescently labeled lock nucleic acid probes were used for the detection of reaction products. The required accuracy was achieved by examining 12 chips from each patient using Stilla technology. The plasma cfDNA of 26 pregnant women with euploid pregnancies and 16 plasma samples from pregnancies with trisomy 21 were analyzed to determine the cutoff value for sample classification. The test was validated in a blind study on 30 plasma samples from pregnant patients with a risk for trisomy 21 ranging from 1:4 to 1:801. The results were in complete agreement with the results of the invasive diagnostic procedure (sensitivity, specificity, PPV, and NPV of 100%). Low cost, and speed of analysis make it a potential screening method for implementation into the clinical workflow to support the combined biochemical and ultrasound results indicating a high risk for trisomy 21.

• MeSH
• aneuploidie MeSH
• Downův syndrom * diagnóza   genetika   MeSH
• lidé MeSH
• polymerázová řetězová reakce MeSH
• prenatální diagnóza metody   MeSH
• těhotenství MeSH
• trizomie MeSH
• volné cirkulující nukleové kyseliny * genetika   MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

• Publikační typ
• abstrakt z konference MeSH

Kardiovaskulární onemocnění jsou po komplikacích v těhotenství nejčastější příčinou úmrtí těhotných žen ve vyspělém světě a postihují 1–4 % těhotenství. Během těhotenství nastává v organismu řada fyziologických změn. Mezi ty nejvýznamnější patří hemodynamické změny kardiovaskulárního systému, ke kterým dochází již v časných fázích gravidity a svého vrcholu dosahují kolem 32. týdnu těhotenství. Pro zdravé ženy nepředstavují tyto změny výrazné riziko, ale pro pacientky s již známým či doposud asymptomatickým srdečním onemocněním mohou mít závažný dopad. Diagnostika srdečního selhání v těhotenství bývá ovšem často obtížná, protože klinické projevy kardiální dekompenzace mohou být zaměňovány za obtíže spojené s graviditou a na diagnózu srdečního selhání se ne vždy pomýšlí. Pacientky s již diagnostikovaným srdečním selháním by měly svůj záměr otěhotnět konzultovat se svým kardiologem, který by měl rizika gravidity pro matku i plod zhodnotit a zajistit optimální podmínky pro průběh těhotenství i samotného porodu. Těhotné ženy s kardiovaskulárním onemocněním vždy patří do péče multioborového týmu, jehož nedílnou součástí by měl být i kardiolog. Klinický stav těchto pacientek je nutné velmi pečlivě monitorovat a včasně reagovat na jeho změny odpovídající úpravou farmakoterapie.

Cardiovascular disease is the most common cause of death in pregnant women in developed countries after complications in pregnancy, affecting 1–4% of pregnancies. During pregnancy, several physiological changes occur in the body. Among the most important are the haemodynamic changes in the cardiovascular system, which occur in the early stages of pregnancy and reach their peak around the 32nd week of pregnancy. These changes do not pose a significant risk for healthy women but can seriously impact patients with known or asymptomatic heart disease. However, the diagnosis of heart failure in pregnancy is often difficult because the clinical manifestations of cardiac decompensation may be mistaken for pregnancy‐related difficulties. The diagnosis of heart failure is not always considered. Patients already diagnosed with heart failure should consult their cardiologist about their intention to become pregnant. They should assess the risks of pregnancy for both the mother and the fetus and ensure optimal conditions for the pregnancy and delivery. Pregnant women with cardiovascular disease should always be cared for by a multidisciplinary team, of which the cardiologist should be an integral part. The clinical condition of these patients must be monitored very closely, and changes in their clinical condition must be responded to promptly by appropriate adjustment of drug therapy.

• MeSH
• kardiovaskulární komplikace v těhotenství * farmakoterapie   MeSH
• komplikace těhotenství MeSH
• lidé MeSH
• srdeční selhání * farmakoterapie   MeSH
• těhotenství MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• přehledy MeSH

Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) represents an autoimmunity disease characterized by high mortality. For successful treatment, the detailed knowledge of its complex pathogenesis and the set of biomarkers for differential diagnostics are desired. Analysis of molecular content of small urinary extracellular vesicles (uEV) offers the possibility to find markers in the form of microRNAs (miRNAs) and study the pathways involved in pathogenesis. We used next-generation sequencing in the first preliminary study to detect the miRNAs with altered expression in uEVs of patients with AAV in comparison with age-matched controls. We confirmed the results using single-target quantitative polymerase chain reaction tests on different sets of samples and found five miRNAs (miR-30a-5p, miR-31-3p, miR-99a-5p, miR-106b-5p, miR-182-5p) with highly elevated levels in uEVs of patients. We performed the comparison of their targets with the differentially expressed proteins in uEVs of patients included in the first phase. We realized that upregulated miRNAs and proteins in uEVs in AAV patients target different biological pathways. The only overlap was detected in pathways regulating the actin cytoskeleton assembly and thus potentially affecting the glomerular functions. The associations of upregulated miRNAs with pathways that were neglected as components of complex AAV pathogenesis, e.g., the epidermal growth factor receptor signaling pathway, were found.

• MeSH
• ANCA-asociované vaskulitidy * genetika   MeSH
• biologické markery MeSH
• extracelulární vezikuly * genetika   MeSH
• ledviny MeSH
• lidé MeSH
• mikro RNA * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Cell-free DNA (cfDNA) has recently been used as a non-invasive diagnostic tool for detecting tumour-specific mutations. cfDNA may also be used for monitoring disease progression and treatment response, but so far researchers focused on one or few genes only. A genomic profile may provide better information on patient prognosis compared to single specific mutations. In this hypothesis-generating study, we profiled by whole exome sequencing serial plasma samples from 10 colon cancer (CC) patients collected before and after 5-fluorouracil-based therapy, and one year after diagnosis to determine alterations associated with treatment response. In parallel, genome profiling was also performed in patients' corresponding tumour tissue to ascertain the molecular landscape of resistant tumours. The mutation concordance between cfDNA and tumour tissue DNA was higher in more advanced tumour stages than in the early stages of the disease. In non-responders, a specific mutation profile was observed in tumour tissues (TPSD1 p.Ala92Thr, CPAMD8 p.Arg341Gln, OBP2A p.ArgTyr123CysHis). A pathogenic APC mutation (p.Ser1315Ter) was detected only in cfDNA of one poor responder one year after the diagnosis and after therapy termination. Another poor responder presented a likely pathogenic TP53 mutation (p.Arg110Pro) in cfDNA of all plasma samplings and in tumour tissue. In conclusion, cfDNA could be used for genetic characterisation of CC patients and might be clinically useful for non-invasive therapy response monitoring.

• MeSH
• DNA nádorová * MeSH
• fluoruracil farmakologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace * MeSH
• nádorové biomarkery * MeSH
• nádory tračníku krev   diagnóza   genetika   terapie   MeSH
• prognóza MeSH
• sekvenční analýza DNA MeSH
• senioři MeSH
• staging nádorů MeSH
• stupeň nádoru MeSH
• volné cirkulující nukleové kyseliny * MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND AND OBJECTIVES: The aim of the study was to optimize routine non-invasive prenatal detection of fetal RHD gene from plasma of RhD-negative pregnant women (the median of gestational age was 25 weeks, range 10-38) to detect RhD materno-fetal incompatibility and to avoid the redundant immunoprophylaxis. MATERIALS AND METHODS: Initially only one exon of RHD gene (exon 10) was investigated in 281 plasma samples (144 verified after delivery), in the second phase three RHD exons (5, 7, 10) were analyzed in 246 samples of plasma and maternal genomic DNA (204 verified) by real-time PCR method. Detection of Y-chromosomal sequence DYS-14 and five X-chromosomal insertion/deletion polymorphisms was used to confirm the fetal cfDNA detectability in plasma. Specific polymorphisms in RHD gene were detected by sequence-specific primer PCR in nine samples. RESULTS: When only the RHD exon 10 was tested, 2·8% of verified samples were false positive and 3·5% false negative. With three RHD exons (5, 7, 10) and maternal genomic DNA testing, only one case was false negative (0·5%). Nine samples were inconclusive due to RHD-positive results in maternal genomic DNA. These samples were analyzed for specific mutations in RHD gene. Combination of both methods for fetal cfDNA verification succeeded in 75% of tested group. CONCLUSION: Implementation of analysis of three RHD exons and maternal genomic DNA to routine practice lowers dramatically the ratio of false positive and negative results. This method enables more accurate determination of fetal RHD status with the reduction of unnecessary medical care and RhD immunoprophylaxis.

• MeSH
• DNA MeSH
• genotyp MeSH
• kojenec MeSH
• krevní skupiny - systém Rh-Hr * genetika   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• plod MeSH
• prenatální diagnóza * MeSH
• těhotenství MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

In families with X-linked recessive diseases, foetal sex is determined prenatally by detection of Y-chromosomal sequences in cell-free foetal DNA (cffDNA) in maternal plasma. The same procedure is used to confirm the cffDNA presence during non-invasive prenatal RhD incompatibility testing but there are no generally accepted markers for the detection of cffDNA fraction in female-foetus bearing pregnancies. We present a methodology allowing the detection of paternal X-chromosomal alleles on maternal background and the confirmation of female sex of the foetus by positive amplification signals. Using digital droplet PCR (ddPCR) we examined X-chromosomal INDEL (insertion/deletion) polymorphisms: rs2307932, rs16397, rs16637, rs3048996, rs16680 in buccal swabs of 50 females to obtain the population data. For all INDELs, we determined the limits of detection for each ddPCR assay. We examined the cffDNA from 63 pregnant women bearing Y-chromosome negative foetuses. The analysis with this set of INDELs led to informative results in 66.67% of examined female-foetus bearing pregnancies. Although the population data predicted higher informativity (74%) we provided the proof of principle of this methodology. We successfully applied this methodology in prenatal diagnostics in a family with Wiscott-Aldrich syndrome and in pregnancies tested for the risk of RhD incompatibility.

• MeSH
• analýza určování pohlaví metody   MeSH
• dospělí MeSH
• genetické testování MeSH
• lidé MeSH
• lidské chromozomy X genetika   MeSH
• mutace INDEL * MeSH
• plod chemie   metabolismus   MeSH
• polymerázová řetězová reakce metody   MeSH
• polymorfismus genetický * MeSH
• prenatální diagnóza metody   MeSH
• těhotenství MeSH
• volné cirkulující nukleové kyseliny analýza   genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH

Down syndrome (DS) is one of the most common causes of intellectual disability and new approaches allowing its rapid and effective prenatal detection are being explored. In this study, we investigated the diagnostic potential of plasma microRNAs (miRNAs). This study builds upon our previous study in DS placentas, where seven miRNAs were found to be significantly up-regulated. A total of 70 first-trimester plasma samples from pregnant women were included in the present study (35 samples with DS fetuses; 35 with euploid fetuses). Genome-wide miRNA profiling was performed in the pilot study using Affymetrix GeneChipTM miRNA 4.1 Array Strips (18 samples). Selected miRNAs were then analysed in the validation study using quantitative reverse transcription PCR (RT-qPCR; 52 samples). Based on the current pilot study results (12 miRNAs), our previous research on chorionic villi samples (7 miRNAs) and the literature (4 miRNAs), a group of 23 miRNAs was selected for the validation study. Although the results of the pilot study were promising, the validation study using the more sensitive RT-qPCR technique and a larger group of samples revealed no significant differences in miRNA profiles between the compared groups. Our results suggest that testing of the first-trimester plasma miRNAs is probably not suitable for non-invasive prenatal testing (NIPT). Different results could be theoretically achieved at later gestational ages; however, such a result probably would have limited use in clinical practice.

• MeSH
• celogenomová asociační studie metody   MeSH
• dospělí MeSH
• Downův syndrom genetika   MeSH
• exprese genu genetika   MeSH
• krevní plazma chemie   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• mikro RNA krev   genetika   MeSH
• pilotní projekty MeSH
• plod metabolismus   MeSH
• prenatální diagnóza metody   MeSH
• první trimestr těhotenství krev   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů metody   MeSH
• stanovení celkové genové exprese metody   MeSH
• těhotenství MeSH
• těhotné ženy MeSH
• transkriptom genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Myokarditida je zánětlivé onemocnění srdečního svalu, které může mít řadu infekčních i neinfekčních příčin. V našich podmínkách je etiologie myokarditidy nejčastěji virová. Pokud je myokarditida spojena s dysfunkcí srdečního svalu, označujeme ji jako zánětlivou kardiomyopatii. Mezi příznaky myokarditidy patří bolesti na hrudi imitující akutní koronární syndrom, palpitace, závratě či synkopy a symptomy srdečního selhání. Diagnostika se opírá o klinický obraz a výsledky neinvazivních vyšetřovacích metod, kde v současné době má klíčovou roli vyšetření srdce magnetickou rezonancí. K definitivnímu potvrzení diagnózy je ale nezbytné provedení endomyokardiální biopsie s následným histologickým a imunohistochemickým zhodnocením vzorků myokardu a vyšetřením zaměřeným na detekci případného infekčního agens.

Myocarditis is an inflammatory disease of the heart muscle. It may have a number of infectious and non-infectious causes. In our conditions, the etiology of myocarditis is of viral origin. Should the myocarditis be connected with the dysfunction of the heart muscle, we call it inflammatory cardiomyopathy. The symptoms of myocarditis are as follows: chest pain imitating an acute coronary syndrome, palpitation, dizziness or syncope and symptoms of heart failure. The diagnostics is based on clinical picture and results of non-invasive examination methods, where, currently, the magnetic resonance of heart muscle plays the critical role. However, the application of endomyocardial biopsy followed by histological and immunohistochemical evaluation of myocardial specimens and examination aimed on potential infectious agent detection are crucial for the definitive confirmation of the diagnosis.

• MeSH
• biopsie MeSH
• diagnostické techniky kardiovaskulární MeSH
• kardiomyopatie imunologie   MeSH
• lidé MeSH
• myokarditida * diagnóza   farmakoterapie   patofyziologie   terapie   MeSH
• srdeční selhání MeSH
• Check Tag
• lidé 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
• lidé MeSH
• nádory prsu diagnóza   genetika   MeSH
• polymerázová řetězová reakce * MeSH
• regulace genové exprese u nádorů MeSH
• RNA dlouhá nekódující krev   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH