This project aims to establish genetic background, etiology and pathogenesis of new cases of thalassemias, enzymopathies, erythrocyte membrane defects associated with hemolytic anemia, congenital dyserythropoietic anemia as well as some congenital defects of the iron transport pathway in erythroid cells and congenital polycythemias. Based on experiences gained during previous grant periods we estimate that in approximately two hundreds of pediatric patients hereditary defects of erythropoiesis will be confirmed. We propose several approaches (CRISPR/Cas9 editing, induced pluripotent stem cells, zebrafish and mouse model) to unravel the molecular-genetic mechanisms contributing to defective erythropoiesis. Results of our work will improve understanding of the molecular-genetic factors contributing to the pathogenesis of disordered erythropoiesis, and will help to elucidate the interconnections of disbalanced erythropoiesis and iron metabolism and the role of the hypoxia signaling pathway proteins, which emerge as important targets for therapeutic interventions in tumors.

Navrhovaný projekt je zaměřen na studium genetického podkladu, etiologie a patogeneze nových případů talasémií, erytroenzymopatií, defektů membrány erytrocytů vedoucích k hemolytickým anémiím, vrozených dyserytropoetických anémií, vrozených poruch transportu železa v erytroidních buňkách a vrozených polycytémií. Na základě zkušeností získaných v průběhu předchozích grantových období odhadujeme, že přibližně u dvou set dětských pacientů budou potvrzeny vrozené defekty erytropoézy. V rámci projektu budeme využívat několika moderních přístupů (CRISPR/Cas9 editace, indukované pluripotentní kmenové buňky, myší a rybí modely), které nám umožní odhalit molekulárně-genetické mechanismy přispívající k defektní erytropoéze. Výsledky naší práce pomohou pochopit molekulárně genetické faktory přispívající k patologické erytropoéze, objasnit propojení procesu nevyvážené erytropoézy s metabolismem železa a úlohu proteinů signalizačních cest hypoxie, jednoho z důležitých cílů pro terapeutické intervence u nádorů.

• Klíčová slova
• zebrafish, hemolytická anémie, kongenitální polycytémie, HIF dráha, Danio rerio, hemolytic anemia, congenital polycythemia, HIF pathway,

• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Polycythemia vera (PV) is a clonal disorder arising from the acquired somatic mutations of the JAK2 gene, including JAK2V617F or several others in exon 12. A 38-year-old female had a stroke at age 32 and found to have elevated hemoglobin, normal leukocytes, normal platelets, and tested negative for JAK2V617F and exon 12 mutations. Next generation sequencing revealed a novel mutation: JAK2R715T in the pseudokinase domain (JH2) at 47.5%. Its presence in her nail DNA confirmed a germline origin. Her mother and her son similarly had erythrocytosis and a JAK2R715T mutation. Computer modeling indicated gain-of-function JAK2 activity. The propositus and her mother had polyclonal myelopoiesis, ruling out another somatic mutation-derived clonal hematopoiesis. Some erythroid progenitors of all three generations grew without erythropoietin, a hallmark of PV. The in vitro reporter assay confirmed increased activity of the JAK2R715T kinase. Similar to PV, the JAK2R715T native cells have increased STAT5 phosphorylation, augmented transcripts of prothrombotic and inflammatory genes, and decreased KLF2 transcripts. The propositus was not controlled by hydroxyurea, and JAK2 inhibitors were not tolerated; however, Ropeginterferon-alfa-2b (Ropeg-IFN-α) induced a remission. Ropeg-IFN-α treatment also reduced JAK2 activity in the propositus, her mother and JAK2V617F PV subjects. We report dominantly inherited erythrocytosis secondary to a novel germline JAK2R715T gain-of-function mutation with many but not all comparable molecular features to JAK2V617F PV. We also document a previously unreported inhibitory mechanism of JAK2 signaling by Ropeg-IFN-α.

• MeSH
• aktivační mutace MeSH
• dospělí MeSH
• interferon alfa terapeutické užití   MeSH
• Janus kinasa 2 * genetika   MeSH
• lidé MeSH
• polycytemie * genetika   farmakoterapie   MeSH
• polycythaemia vera genetika   farmakoterapie   MeSH
• rodokmen MeSH
• zárodečné mutace * MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH

Molecular pathophysiology of Diamond-Blackfan anemia (DBA) involves disrupted erythroid-lineage proliferation, differentiation and apoptosis; with the activation of p53 considered as a key component. Recently, oxidative stress was proposed to play an important role in DBA pathophysiology as well. CRISPR/Cas9-created Rpl5- and Rps19-deficient murine erythroleukemia (MEL) cells and DBA patients' samples were used to evaluate proinflammatory cytokines, oxidative stress, DNA damage and DNA damage response. We demonstrated that the antioxidant defense capacity of Rp-mutant cells is insufficient to meet the greater reactive oxygen species (ROS) production which leads to oxidative DNA damage, cellular senescence and activation of DNA damage response signaling in the developing erythroblasts and altered characteristics of mature erythrocytes. We also showed that the disturbed balance between ROS formation and antioxidant defense is accompanied by the upregulation of proinflammatory cytokines. Finally, the alterations detected in the membrane of DBA erythrocytes may cause their enhanced recognition and destruction by reticuloendothelial macrophages, especially during infections. We propose that the extent of oxidative stress and the ability to activate antioxidant defense systems may contribute to high heterogeneity of clinical symptoms and response to therapy observed in DBA patients.

• MeSH
• Diamondova-Blackfanova anemie imunologie   metabolismus   patologie   MeSH
• dítě MeSH
• dospělí MeSH
• erytrocyty metabolismus   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mediátory zánětu metabolismus   MeSH
• mladý dospělý MeSH
• myši MeSH
• následné studie MeSH
• oxidační stres * MeSH
• poškození DNA * MeSH
• prognóza MeSH
• studie případů a kontrol MeSH
• zánět imunologie   metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Myeloproliferative neoplasms (MPN) are genetically very complex and heterogeneous diseases in which the acquisition of a somatic driver mutation triggers three main myeloid cytokine receptors, and phenotypically expresses as polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF). The course of the diseases may be influenced by germline predispositions, modifying mutations, their order of acquisition and environmental factors such as aging and inflammation. Deciphering these contributory elements, their mutual interrelationships, and their contribution to MPN pathogenesis brings important insights into the diseases. Animal models (mainly mouse and zebrafish) have already significantly contributed to understanding the role of several acquired and germline mutations in MPN oncogenic signaling. Novel technologies such as induced pluripotent stem cells (iPSCs) and precise genome editing (using CRISPR/Cas9) contribute to the emerging understanding of MPN pathogenesis and clonal architecture, and form a convenient platform for evaluating drug efficacy. In this overview, the genetic landscape of MPN is briefly described, with an attempt to cover the main discoveries of the last 15 years. Mouse and zebrafish models of the driver mutations are discussed and followed by a review of recent progress in modeling MPN with patient-derived iPSCs and CRISPR/Cas9 gene editing.

• MeSH
• dánio pruhované MeSH
• esenciální trombocytemie genetika   MeSH
• fenotyp MeSH
• indukované pluripotentní kmenové buňky metabolismus   MeSH
• Janus kinasa 2 genetika   MeSH
• kalretikulin genetika   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• mutace MeSH
• myeloproliferativní poruchy genetika   patofyziologie   MeSH
• myši MeSH
• nádory genetika   MeSH
• polycythaemia vera genetika   MeSH
• primární myelofibróza genetika   MeSH
• receptory thrombopoetinu genetika   MeSH
• signální transdukce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

The patients with mantle cell lymphoma (MCL) have translocation t(11;14) associated with cyclin D1 overexpression. We observed that iron (an essential cofactor of dioxygenases including prolyl hydroxylases [PHDs]) depletion by deferoxamine blocked MCL cells' proliferation, increased expression of DNA damage marker γH2AX, induced cell cycle arrest and decreased cyclin D1 level. Treatment of MCL cell lines with dimethyloxalylglycine, which blocks dioxygenases involving PHDs by competing with their substrate 2-oxoglutarate, leads to their decreased proliferation and the decrease of cyclin D1 level. We then postulated that loss of EGLN2/PHD1 in MCL cells may lead to down-regulation of cyclin D1 by blocking the degradation of FOXO3A, a cyclin D1 suppressor. However, the CRISPR/Cas9-based loss-of-function of EGLN2/PHD1 did not affect cyclin D1 expression and the loss of FOXO3A did not restore cyclin D1 levels after iron chelation. These data suggest that expression of cyclin D1 in MCL is not controlled by ENGL2/PHD1-FOXO3A pathway and that chelation- and 2-oxoglutarate competition-mediated down-regulation of cyclin D1 in MCL cells is driven by yet unknown mechanism involving iron- and 2-oxoglutarate-dependent dioxygenases other than PHD1. These data support further exploration of the use of iron chelation and 2-oxoglutarate-dependent dioxygenase inhibitors as a novel therapy of MCL.

• MeSH
• aminokyseliny dikarboxylové farmakologie   MeSH
• chelátory železa farmakologie   MeSH
• cyklin D1 metabolismus   MeSH
• deferoxamin farmakologie   MeSH
• deficit železa MeSH
• dioxygenasy antagonisté a inhibitory   metabolismus   MeSH
• down regulace účinky léků   MeSH
• hydroxylace MeSH
• hypoxie buňky účinky léků   MeSH
• inhibitory enzymů farmakologie   MeSH
• kyseliny ketoglutarové farmakologie   MeSH
• lidé MeSH
• lymfom z plášťových buněk enzymologie   MeSH
• messenger RNA genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• poškození DNA MeSH
• prolyl-4-hydroxylasy HIF metabolismus   MeSH
• protein FOXO3 genetika   metabolismus   MeSH
• železo MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• Janus kinasa 2 * genetika   metabolismus   MeSH
• lidé MeSH
• missense mutace * MeSH
• myeloproliferativní poruchy * enzymologie   genetika   patologie   MeSH
• signální transdukce genetika   MeSH
• substituce aminokyselin MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH

T-cell factor 4 (TCF4), together with β-catenin coactivator, functions as the major transcriptional mediator of the canonical wingless/integrated (Wnt) signaling pathway in the intestinal epithelium. The pathway activity is essential for both intestinal homeostasis and tumorigenesis. To date, several mouse models and cellular systems have been used to analyze TCF4 function. However, some findings were conflicting, especially those that were related to the defects observed in the mouse gastrointestinal tract after Tcf4 gene deletion, or to a potential tumor suppressive role of the gene in intestinal cancer cells or tumors. Here, we present the results obtained using a newly generated conditional Tcf4 allele that allows inactivation of all potential Tcf4 isoforms in the mouse tissue or small intestinal and colon organoids. We also employed the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to disrupt the TCF4 gene in human cells. We showed that in adult mice, epithelial expression of Tcf4 is indispensable for cell proliferation and tumor initiation. However, in human cells, the TCF4 role is redundant with the related T-cell factor 1 (TCF1) and lymphoid enhancer-binding factor 1 (LEF1) transcription factors.

• Publikační typ
• časopisecké články MeSH

Tibetans existed in high altitude for ~25 thousand years and have evolutionary selected unique haplotypes assumed to be beneficial to hypoxic adaptation. EGLN1/PHD2 and EPAS1/HIF-2α, both crucial components of hypoxia sensing, are the two best-established loci contributing to high altitude adaptation. The co-adapted Tibetan-specific haplotype encoding for PHD2:p.[D4E/C127S] promotes increased HIF degradation under hypoxic conditions. The Tibetan-specific 200 kb EPAS1 haplotype introgressed from an archaic human population related to Denisovans which underwent evolutionary decay; however, the functional variant(s) responsible for high-altitude adaptation at EPAS1/HIF-2α have not yet been identified. Since HIF modulates the behavior of cancer cells, we hypothesized that these Tibetan selected genomic variants may modify cancer risk predisposition. Here, we ascertained the frequencies of EGLN1D4E/C127S and EGLN1C127S variants and ten EPAS1/HIF-2α variants in lung cancer patients and controls in Nepal, whose population consists of people with Indo-Aryan origin and Tibetan-related Mongoloid origin. We observed a significant association between the selected Tibetan EGLN1/PHD2 haplotype and lung cancer (p=0.0012 for D4E, p=0.0002 for C127S), corresponding to a two-fold increase in lung cancer risk. We also observed a two-fold or greater increased risk for two of the ten EPAS1/HIF-2α variants, although the association was not significant after correcting for multiple comparisons (p=0.12). Although these data cannot address the role of these genetic variants on lung cancer initiation or progression, we conclude that some selected Tibetan variants are strongly associated with a modified risk of lung cancer.

• MeSH
• aklimatizace MeSH
• faktor 1 indukovatelný hypoxií - podjednotka alfa genetika   metabolismus   MeSH
• hypoxie genetika   metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádory plic genetika   metabolismus   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Tibet MeSH

• MeSH
• fenylalanin genetika   MeSH
• Janus kinasa 2 genetika   MeSH
• jednonukleotidový polymorfismus MeSH
• kohortové studie MeSH
• kultivované buňky MeSH
• lidé MeSH
• missense mutace * MeSH
• mutační analýza DNA MeSH
• polycythaemia vera genetika   patologie   MeSH
• substituce aminokyselin MeSH
• valin genetika   MeSH
• zárodečné mutace * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

We report an infant with sickle cell disease phenotype by biochemical analysis whose β-globin gene (HBB) sequencing showed sickle cell mutation (HBBS ) heterozygosity. The proband has a unique head-to-tail duplication of the β-globin gene cluster having wild-type (HBBA ) and HBBS alleles inherited from her father; constituting her HBBS /HBBS -HBBA genotype. Further analyses revealed that proband's duplicated β-globin gene cluster (∼650 kb) encompassing HBBA does not include the immediate upstream locus control region (LCR) or 3' DNase I hypersensitivity (HS) element. The LCR interacts with β-globin gene cluster involving long range DNA interactions mediated by various transcription factors to drive the regulation of globin genes expression. However, a low level of HBBA transcript was clearly detected by digital PCR. In this patient, the observed transcription from the duplicated, distally displaced HBBA cluster demonstrates that the loss of LCR and flanking 3'HS sites do not lead to complete silencing of HBB transcription.

• MeSH
• 3' přiléhající oblast DNA MeSH
• beta-globiny genetika   MeSH
• duplicitní geny * MeSH
• genetická transkripce MeSH
• kojenec MeSH
• lidé MeSH
• mutace MeSH
• regulační oblast lokusu (genu) MeSH
• srpkovitá anemie genetika   MeSH
• umlčování genů MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH