BACKGROUND & AIMS: Lymphedema cholestasis syndrome 1 or Aagenaes syndrome is a condition characterized by neonatal cholestasis, lymphedema, and giant cell hepatitis. The genetic background of this autosomal recessive disease was unknown up to now. METHODS: A total of 26 patients with Aagenaes syndrome and 17 parents were investigated with whole-genome sequencing and/or Sanger sequencing. PCR and western blot analyses were used to assess levels of mRNA and protein, respectively. CRISPR/Cas9 was used to generate the variant in HEK293T cells. Light microscopy, transmission electron microscopy and immunohistochemistry for biliary transport proteins were performed in liver biopsies. RESULTS: One specific variant (c.-98G>T) in the 5'-untranslated region of Unc-45 myosin chaperone A (UNC45A) was identified in all tested patients with Aagenaes syndrome. Nineteen were homozygous for the c.-98G>T variant and seven were compound heterozygous for the variant in the 5'-untranslated region and an exonic loss-of-function variant in UNC45A. Patients with Aagenaes syndrome exhibited lower expression of UNC45A mRNA and protein than controls, and this was reproduced in a CRISPR/Cas9-created cell model. Liver biopsies from the neonatal period demonstrated cholestasis, paucity of bile ducts and pronounced formation of multinucleated giant cells. Immunohistochemistry revealed mislocalization of the hepatobiliary transport proteins BSEP (bile salt export pump) and MRP2 (multidrug resistance-associated protein 2). CONCLUSIONS: c.-98G>T in the 5'-untranslated region of UNC45A is the causative genetic variant in Aagenaes syndrome. IMPACT AND IMPLICATIONS: The genetic background of Aagenaes syndrome, a disease presenting with cholestasis and lymphedema in childhood, was unknown until now. A variant in the 5'-untranslated region of the Unc-45 myosin chaperone A (UNC45A) was identified in all tested patients with Aagenaes syndrome, providing evidence of the genetic background of the disease. Identification of the genetic background provides a tool for diagnosis of patients with Aagenaes syndrome before lymphedema is evident.
- MeSH
- 5' nepřekládaná oblast genetika MeSH
- cholestáza * genetika MeSH
- HEK293 buňky MeSH
- intracelulární signální peptidy a proteiny * genetika MeSH
- lidé MeSH
- lymfedém * diagnóza genetika metabolismus MeSH
- myosiny genetika metabolismus MeSH
- novorozenec MeSH
- transportní proteiny genetika MeSH
- Check Tag
- lidé MeSH
- novorozenec MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- akutní lymfatická leukemie farmakoterapie genetika patologie MeSH
- akutní nemoc MeSH
- buněčný rodokmen genetika MeSH
- genová přestavba MeSH
- histonlysin-N-methyltransferasa genetika MeSH
- indukční chemoterapie metody MeSH
- kineziny genetika MeSH
- lidé MeSH
- lidské chromozomy, pár 11 genetika MeSH
- lidské chromozomy, pár 6 genetika MeSH
- malování chromozomů metody MeSH
- mladiství MeSH
- myeloidní leukemie genetika patologie MeSH
- myosiny genetika MeSH
- protoonkogenní protein MLL genetika MeSH
- translokace genetická * MeSH
- Check Tag
- lidé MeSH
- mladiství MeSH
- ženské pohlaví MeSH
- Publikační typ
- dopisy MeSH
- kazuistiky MeSH
Non-syndromic autosomal recessive hearing loss is an extremely heterogeneous disease caused by mutations in more than 80 genes. We examined Czech patients with early/prelingual non-syndromic, presumably genetic hearing loss (NSHL) without known cause after GJB2 gene testing. Four hundred and twenty-one unrelated patients were examined for STRC gene deletions with quantitative comparative fluorescent PCR (QCF PCR), 197 unrelated patients with next-generation sequencing by custom-designed NSHL gene panels and 19 patients with whole-exome sequencing (WES). Combining all methods, we discovered the cause of the disease in 54 patients. The most frequent type of NSHL was DFNB16 (STRC), which was detected in 22 patients, almost half of the clarified patients. Other biallelic pathogenic mutations were detected in the genes: MYO15A, LOXHD1, TMPRSS3 (each gene was responsible for five clarified patients, CDH23 (four clarified patients), OTOG and OTOF (each gene was responsible for two clarified patients). Other genes (AIFM1, CABP2, DIAPH1, PTPRQ, RDX, SLC26A4, TBC1D24, TECTA, TMC1) that explained the cause of hearing impairment were further detected in only one patient for each gene. STRC gene mutations, mainly deletions remain the most frequent NSHL cause after mutations in the GJB2.
- MeSH
- dítě MeSH
- dospělí MeSH
- genetická predispozice k nemoci MeSH
- hluchota embryologie genetika patologie MeSH
- kadheriny genetika MeSH
- konexin 26 genetika MeSH
- lidé MeSH
- membránové glykoproteiny genetika MeSH
- membránové proteiny genetika MeSH
- mezibuněčné signální peptidy a proteiny genetika MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mutace genetika MeSH
- myosiny genetika MeSH
- nádorové proteiny genetika MeSH
- nedoslýchavost epidemiologie genetika patologie MeSH
- sekvenování exomu MeSH
- serinové endopeptidasy genetika MeSH
- transportní proteiny genetika MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
A girl with profound congenital deafness and balance problems was found at 3.5 years of age to be a carrier of two novel compound heterozygous mutations in MYO7A that were predicted to be disease-causing. She also carried one known pathogenic mutation and one rare variant in USH2A. Fundus examination performed at 4.75 years revealed one small peripheral pigment deposit in the right eye, indicating probable retinal degeneration. Spectral domain optical coherence tomography (SD-OCT) showed a loss of photoreceptors throughout the macular area, except for the foveolar region, clearly confirming a diagnosis of Usher syndrome type 1. This case demonstrates that SD-OCT may be easily used in young children to confirm retinal disease, quantify the extent of retinal damage, and monitor disease progression.
- MeSH
- extracelulární matrix - proteiny genetika MeSH
- lidé MeSH
- missense mutace MeSH
- myosiny genetika MeSH
- nemoci retiny diagnóza diagnostické zobrazování MeSH
- optická koherentní tomografie metody MeSH
- předškolní dítě MeSH
- Usherovy syndromy diagnóza genetika MeSH
- Check Tag
- lidé MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
Purpose: Characterization of colorectal cancer transcriptome by high-throughput techniques has enabled the discovery of several differentially expressed genes involving previously unreported miRNA abnormalities. Here, we followed a systematic approach on a global scale to identify miRNAs as clinical outcome predictors and further validated them in the clinical and experimental setting.Experimental Design:Genome-wide miRNA sequencing data of 228 colorectal cancer patients from The Cancer Genome Atlas dataset were analyzed as a screening cohort to identify miRNAs significantly associated with survival according to stringent prespecified criteria. A panel of six miRNAs was further validated for their prognostic utility in a large independent validation cohort (n= 332).In situhybridization and functional experiments in a panel of colorectal cancer cell lines and xenografts further clarified the role of clinical relevant miRNAs.Results:Six miRNAs (miR-92b-3p, miR-188-3p, miR-221-5p, miR-331-3p, miR-425-3p, and miR-497-5p) were identified as strong predictors of survival in the screening cohort. High miR-188-3p expression proves to be an independent prognostic factor [screening cohort: HR = 4.137; 95% confidence interval (CI), 1.568-10.917;P= 0.004; validation cohort: HR = 1.538; 95% CI, 1.107-2.137;P= 0.010, respectively]. Forced miR-188-3p expression increased migratory behavior of colorectal cancer cellsin vitroand metastases formationin vivo(P< 0.05). The promigratory role of miR-188-3p is mediated by direct interaction with MLLT4, a novel identified player involved in colorectal cancer cell migration.Conclusions:miR-188-3p is a novel independent prognostic factor in colorectal cancer patients, which can be partly explained by its effect on MLLT4 expression and migration of cancer cells.Clin Cancer Res; 23(5); 1323-33. ©2016 AACR.
- MeSH
- genom lidský MeSH
- HCT116 buňky MeSH
- karcinogeneze genetika MeSH
- kineziny genetika MeSH
- kolorektální nádory genetika patologie MeSH
- lidé MeSH
- mikro RNA genetika izolace a purifikace MeSH
- myosiny genetika MeSH
- myši MeSH
- nádorové biomarkery genetika MeSH
- prognóza MeSH
- transkriptom genetika MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- xenogenní modely - testy antitumorózní aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The differentiation of pluripotent embryonic stem (ES) cells into various lineages in vitro represents an important tool for studying the mechanisms underlying mammalian embryogenesis. It is a key technique in studies evaluating the molecular mechanisms of cardiomyogenesis and heart development and also in embryotoxicology. Herein, modest modifications of the basic protocol for ES cell differentiation into cardiomyocytes were evaluated in order to increase the yield and differentiation status of developed cardiomyocytes. Primarily, the data show that ES cell cultivation in the form of non-adherent embryoid bodies (EBs) for 5 days compared to 8 days significantly improved cardiomyogenic differentiation. This is illustrated by the appearance of beating foci in the adherent EBs layer at earlier phases of differentiation from day 10 up to day 16 and by the significantly higher expression of genes characteristic of cardiomyogenic differentiation (sarcomeric alpha actinin, myosin heavy chain alpha and beta, myosin light chain 2 and 7, and transcriptional factor Nkx2.5) in EBs cultivated under non-adherent conditions for 5 days. The ratio of cardiomyocytes per other cells was also potentiated in EBs cultivated in non-adherent conditions for only 5 days followed by cultivation in adherent serum-free culture conditions. Nevertheless, the alteration in the percentage of beating foci among these two tested cultivation conditions vanished at later phases and also did not affect the total number of cardiomyocytes determined as myosin heavy chain positive cells at the end of the differentiation process on day 20. Thus, although these modifications of the conditions of ES cells differentiation may intensify cardiomyocyte differentiation, the final count of cardiomyocytes might not change. Thus, serum depletion was identified as a key factor that intensified cardiomyogenesis. Further, the treatment of EBs with N-acetylcysteine, a reactive oxygen species scavenger, did not affect the observed increase in cardiomyogenesis under serum depleted conditions. Interestingly, a mild induction of the ventricular-like phenotype of cardiomyocytes was observed in 5-day-old EBs compared to 8-day-old EBs. Overall, these findings bring crucial information on the mechanisms of ES cells differentiation into cardiomyocytes and on the establishment of efficient protocols for the cardiomyogenic differentiation of ES cells. Further, the importance of determining the absolute number of formed cardiomyocyte-like cells per seeded pluripotent cells in contrast to the simple quantification of the ratios of cells is highlighted.
- MeSH
- acetylcystein aplikace a dávkování MeSH
- aktinin genetika MeSH
- embryonální kmenové buňky cytologie MeSH
- homeoboxový protein Nkx-2.5 genetika MeSH
- kardiomyocyty cytologie MeSH
- kultivační média bez séra * MeSH
- kultivované buňky MeSH
- myosiny genetika MeSH
- myši MeSH
- techniky in vitro MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
AIM: To analyze the genesis of hypertrophic cardiomyopathy on a large cohort of patients from molecular genetics point of view and perform the functional analysis of the 3D molecular model of defective myosin-7 protein in silico. METHODS: The study enrolled 153 patients with diagnosed hypertrophic cardiomyopathy from different parts of the Czech Republic. DNA samples were analyzed for mutations in exons 21 and 22 of the MYH7 gene, which have been associated with high mutation clustering. The 3D model of human myosin-7 was built using the x-ray structure of nucleotide-free scallop myosin S1 as the structural template. We performed de novo structure prediction of mutant and wild type peptides spanning the 769-788 amino acids region of the myosin-7 protein. RESULTS: The Arg870His and Asp778Val amino acid alterations were found in 2 unrelated patients with a severe form of hypertrophic cardiomyopathy. The Asp778Val variation was chosen for subsequent 3D molecular modeling in silico. The mutation of the Asp by Val not only changes the character of the interaction pattern with other amino acids or ions but Val, being a small hydrophobic amino acid, can also completely change the stability of the region. CONCLUSION: Mutation location in the MYH7 gene and changes in amino acid composition may have a crucial negative impact on the outcome of the disease in patients with hypertrophic cardiomyopathy. In addition, a mutation that changes the charge of the amino acid is more likely to affect protein function than a conservative mutation.
- MeSH
- databáze genetické MeSH
- DNA analýza MeSH
- dospělí MeSH
- hodnocení rizik MeSH
- hypertrofická kardiomyopatie diagnóza genetika patologie MeSH
- kohortové studie MeSH
- lidé MeSH
- mladý dospělý MeSH
- molekulární modely MeSH
- mutace MeSH
- myosiny genetika MeSH
- počítačová simulace MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
The role of bone morphogenetic proteins (BMPs) in vasculogenesis is still not well understood, despite many recent developments in this area of research. In this review, we discuss the most recent studies that identify new critical mechanisms through which BMP signaling acts with a focus on angiogenesis. RECENT FINDINGS: New evidence brought to light over the last few years suggests that BMP-binding proteins, formerly thought of as antagonists, can also increase BMP activity under certain conditions. It has also recently been determined that components of the extracellular matrix are involved in the BMP signaling pathways that regulate angiogenesis. Through the BMP pathway, myosin-X and cyclooxygenase 2 serve as target genes that have been determined to play a role in blood vessel formation. BMPs also conduct Smad-independent signaling and crosstalk with other pathways. Finally, BMPs have been shown to play an antiangiogenic role in specific settings. SUMMARY: Better understanding of the BMP signaling pathway and its regulators can have potentially great effects on therapeutic strategies from cardiovascular disease to cancer.
- MeSH
- cyklooxygenasa 2 genetika metabolismus MeSH
- endoteliální buňky fyziologie MeSH
- endoteliální růstové faktory fyziologie MeSH
- extracelulární matrix metabolismus MeSH
- fyziologická neovaskularizace MeSH
- kostní morfogenetické proteiny fyziologie MeSH
- lidé MeSH
- myosiny genetika metabolismus MeSH
- proteiny Smad fyziologie MeSH
- signální transdukce fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Fibre type determination requires a large series of differently stained muscle sections. The manual identification of individual fibres through the series is tedious and time consuming. This paper presents a software that enables (i) adjusting the position of individual fibres through a series of differently stained sections (image registration) and identification of individual fibres through the series as well as (ii) muscle fibre classification and (iii) quantitative analysis. The data output of the system is the following: numerical and areal proportions of fibre types, fibre type size and optical density (grey level) of the final reaction product in every fibre. The muscle fibre type can be determined stepwise, based on one set of stained sections while further, newly stained sections can be added to the already defined muscle fibre profile. Several advantages of the presented software application in skeletal muscle research are presented. The system is semiquantitative, flexible, and user friendly.
- MeSH
- hybridizace in situ MeSH
- imunohistochemie MeSH
- kosterní svalová vlákna cytologie klasifikace MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- musculus masseter cytologie MeSH
- myosiny genetika metabolismus MeSH
- počítačové zpracování obrazu metody MeSH
- protein - isoformy genetika metabolismus MeSH
- reprodukovatelnost výsledků MeSH
- software MeSH
- těžké řetězce myosinu genetika metabolismus MeSH
- uživatelské rozhraní počítače MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- MeSH
- bodová mutace MeSH
- dyneiny genetika MeSH
- inzerční mutageneze MeSH
- lidé MeSH
- missense mutace MeSH
- místa sestřihu RNA genetika MeSH
- molekulární sekvence - údaje MeSH
- mutace * MeSH
- mutační analýza DNA MeSH
- myosin VIIa MeSH
- myosiny genetika MeSH
- nesmyslný kodon MeSH
- polymorfismus konformace jednovláknové DNA MeSH
- rodokmen MeSH
- sekvence aminokyselin MeSH
- sekvenční delece MeSH
- sekvenční homologie aminokyselin MeSH
- sekvenční seřazení MeSH
- tandemové repetitivní sekvence MeSH
- Usherovy syndromy etnologie genetika MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Geografické názvy
- Česká republika MeSH
- Itálie MeSH
- Maroko MeSH
- Španělsko MeSH
- Turecko MeSH
