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1.
Int J Cancer ; 154(9): 1652-1668, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38180088

RESUMO

Patients with myelodysplastic neoplasms (MDS) are classified according to the risk of acute myeloid leukemia transformation. Some lower-risk MDS patients (LR-MDS) progress rapidly despite expected good prognosis. Using diagnostic samples, we aimed to uncover the mechanisms of this accelerated progression at the transcriptome level. RNAseq was performed on CD34+ ribodepleted RNA samples from 53 LR-MDS patients without accelerated progression (stMDS) and 8 who progressed within 20 months (prMDS); 845 genes were differentially expressed (ІlogFCІ > 1, FDR < 0.01) between these groups. stMDS CD34+ cells exhibited transcriptional signatures of actively cycling, megakaryocyte/erythrocyte lineage-primed progenitors, with upregulation of cell cycle checkpoints and stress pathways, which presumably form a tumor-suppressing barrier. Conversely, cell cycle, DNA damage response (DDR) and energy metabolism-related pathways were downregulated in prMDS samples, whereas cell adhesion processes were upregulated. Also, prMDS samples showed high levels of aberrant splicing and global lncRNA expression that may contribute to the attenuation of DDR pathways. We observed overexpression of multiple oncogenes and diminished differentiation in prMDS; the expression of ZEB1 and NEK3, genes not previously associated with MDS prognosis, might serve as potential biomarkers for LR-MDS progression. Our 19-gene DDR signature showed a significant predictive power for LR-MDS progression. In validation samples (stMDS = 3, prMDS = 4), the key markers and signatures retained their significance. Collectively, accelerated progression of LR-MDS appears to be associated with transcriptome patterns of a quiescent-like cell state, reduced lineage differentiation and suppressed DDR, inherent to CD34+ cells. The attenuation of DDR-related gene-expression signature may refine risk assessment in LR-MDS patients.


Assuntos
Síndromes Mielodisplásicas , Neoplasias , Humanos , Transcriptoma , Adesão Celular , Síndromes Mielodisplásicas/genética , Ciclo Celular , Reparo do DNA , Quinases Relacionadas a NIMA/genética , Quinases Relacionadas a NIMA/metabolismo
2.
Br J Haematol ; 202(3): 674-685, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37246471

RESUMO

Congenital erythrocytoses represent a heterogenous group of rare defects of erythropoiesis characterized by elevated erythrocyte mass. We performed molecular-genetic analysis of 21 Czech patients with congenital erythrocytosis and assessed the mutual link between chronic erythrocyte overproduction and iron homoeostasis. Causative mutations in erythropoietin receptor (EPOR), hypoxia-inducible factor 2 alpha (HIF2A) or Von Hippel-Lindau (VHL) genes were detected in nine patients, including a novel p.A421Cfs*4 EPOR and a homozygous intronic c.340+770T>C VHL mutation. The association and possible cooperation of five identified missense germline EPOR or Janus kinase 2 (JAK2) variants with other genetic/non-genetic factors in erythrocytosis manifestation may involve variants of Piezo-type mechanosensitive ion channel component 1 (PIEZO1) or Ten-eleven translocation 2 (TET2), but this requires further research. In two families, hepcidin levels appeared to prevent or promote phenotypic expression of the disease. No major contribution of heterozygous haemochromatosis gene (HFE) mutations to the erythrocytic phenotype or hepcidin levels was observed in our cohort. VHL- and HIF2A-mutant erythrocytosis showed increased erythroferrone and suppressed hepcidin, whereas no overproduction of erythroferrone was detected in other patients regardless of molecular defect, age or therapy. Understanding the interplay between iron metabolism and erythropoiesis in different subgroups of congenital erythrocytosis may improve current treatment options.


Assuntos
Policitemia , Humanos , Policitemia/genética , Hepcidinas/genética , Oxigênio/metabolismo , Mutação , Receptores da Eritropoetina/genética , Canais Iônicos/genética
3.
Am J Hematol ; 98(8): 1265-1276, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37350302

RESUMO

Obstructive sleep apnea (OSA) causes intermittent hypoxia during sleep. Hypoxia predictably initiates an increase in the blood hemoglobin concentration (Hb); yet in our analysis of 527 patients with OSA, >98% did not have an elevated Hb. To understand why patients with OSA do not develop secondary erythrocytosis due to intermittent hypoxia, we first hypothesized that erythrocytosis occurs in these patients, but is masked by a concomitant increase in plasma volume. However, we excluded that explanation by finding that the red cell mass was normal (measured by radionuclide labeling of erythrocytes and carbon monoxide inhalation). We next studied 45 patients with OSA before and after applying continuous positive airway pressure (CPAP). We found accelerated erythropoiesis in these patients (increased erythropoietin and reticulocytosis), but it was offset by neocytolysis (lysis of erythrocytes newly generated in hypoxia upon return to normoxia). Parameters of neocytolysis included increased reactive oxygen species from expanded reticulocytes' mitochondria. The antioxidant catalase was also downregulated in these cells from hypoxia-stimulated microRNA-21. In addition, inflammation-induced hepcidin limited iron availability for erythropoiesis. After CPAP, some of these intermediaries diminished but Hb did not change. We conclude that in OSA, the absence of significant increase in red cell mass is integral to the pathogenesis, and results from hemolysis via neocytolysis combined with inflammation-mediated suppression of erythropoiesis.


Assuntos
Policitemia , Apneia Obstrutiva do Sono , Humanos , Espécies Reativas de Oxigênio , Policitemia/etiologia , Hepcidinas , Hipóxia , Apneia Obstrutiva do Sono/complicações , Inflamação
4.
Int J Mol Sci ; 24(3)2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36768374

RESUMO

The treatment outcome in patients with chronic myeloid leukaemia (CML) in blast crisis (BC) is unsatisfactory despite the use of allogeneic stem cell transplantation (ASCT). Moreover, in some patients ASCT is contraindicated, with limited treatment options. We report the case series of two patients with lymphoid BC CML in whom ASCT was not approachable. The first patient developed BC two months after diagnosis in association with dic(7;9)(p11.2;p11.2) and T315I mutation. Blast crisis with central nervous system leukemic involvement and K611N mutation of the SETD2 gene developed abruptly in the second patient five years after ceasing treatment with nilotinib in major molecular response (MMR) at the patient's request. Both underwent one course of chemotherapy in combination with rituximab and imatinib, followed by dasatinib and interferon α (INFα) treatment in the first and dasatinib alone in the second case. Deep molecular response (DMR; MR 4.0) was achieved within a short time in both cases. It is probable that DMR was caused by a specific immune response to CML cells, described in both agents. The challenging medical condition that prompted these case series, and the subsequent results, suggest a re-visit to the use of a combination of well-known drugs as an area for further investigation.


Assuntos
Crise Blástica , Leucemia Mielogênica Crônica BCR-ABL Positiva , Humanos , Dasatinibe/uso terapêutico , Crise Blástica/tratamento farmacológico , Crise Blástica/genética , Interferon-alfa/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética
5.
Am J Hematol ; 97(10): 1286-1299, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35815815

RESUMO

Iron availability for erythropoiesis is controlled by the iron-regulatory hormone hepcidin. Increased erythropoiesis negatively regulates hepcidin synthesis by erythroferrone (ERFE), a hormone produced by erythroid precursors in response to erythropoietin (EPO). The mechanisms coordinating erythropoietic activity with iron homeostasis in erythrocytosis with low EPO are not well defined as exemplified by dominantly inherited (heterozygous) gain-of-function mutation of human EPO receptor (mtHEPOR) with low EPO characterized by postnatal erythrocytosis. We previously created a mouse model of this mtHEPOR that develops fetal erythrocytosis with a transient perinatal amelioration of erythrocytosis and its reappearance at 3-6 weeks of age. Prenatally and perinatally, mtHEPOR heterozygous and homozygous mice (differing in erythrocytosis severity) had increased Erfe transcripts, reduced hepcidin, and iron deficiency. Epo was transiently normal in the prenatal life; then decreased at postnatal day 7, and remained reduced in adulthood. Postnatally, hepcidin increased in mtHEPOR heterozygotes and homozygotes, accompanied by low Erfe induction and iron accumulation. With aging, the old, especially mtHEPOR homozygotes had a decline of erythropoiesis, myeloid expansion, and local bone marrow inflammatory stress. In addition, mtHEPOR erythrocytes had a reduced lifespan. This, together with reduced iron demand for erythropoiesis, due to its age-related attenuation, likely contributes to increased iron deposition in the aged mtHEPOR mice. In conclusion, the erythroid drive-mediated inhibition of hepcidin production in mtHEPOR mice in the prenatal/perinatal period is postnatally abrogated by increasing iron stores promoting hepcidin synthesis. The differences observed in studied characteristics between mtHEPOR heterozygotes and homozygotes suggest dose-dependent alterations of downstream EPOR stimulation.


Assuntos
Eritropoetina , Policitemia , Adulto , Idoso , Animais , Eritropoese/genética , Eritropoetina/genética , Eritropoetina/farmacologia , Mutação com Ganho de Função , Hepcidinas/genética , Hepcidinas/metabolismo , Hormônios , Humanos , Ferro/metabolismo , Camundongos , Policitemia/genética , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/metabolismo
6.
Apoptosis ; 25(9-10): 674-685, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32638182

RESUMO

Costunolide, a natural sesquiterpene lactone, has multiple pharmacological activities such as neuroprotection or induction of apoptosis and eryptosis. However, the effects of costunolide on pro-survival factors and enzymes in human erythrocytes, e.g. glutathione and glucose-6-phosphate dehydrogenase (G6PDH) respectively, have not been studied yet. Our aim was to determine the mechanisms underlying costunolide-induced eryptosis and to reverse this process. Phosphatidylserine exposure was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry, and intracellular glutathione [GSH]i from high performance liquid chromatography. The oxidized status of intracellular glutathione and enzyme activities were measured by spectrophotometry. Treatment of erythrocytes with costunolide dose-dependently enhanced the percentage of annexin-V-binding cells, decreased the cell volume, depleted [GSH]i and completely inhibited G6PDH activity. The effects of costunolide on annexin-V-binding and cell volume were significantly reversed by pre-treatment of erythrocytes with the specific PKC-α inhibitor chelerythrine. The latter, however, had no effect on costunolide-induced GSH depletion. Costunolide induces eryptosis, depletes [GSH]i and inactivates G6PDH activity. Furthermore, our study reveals an inhibitory effect of chelerythrine on costunolide-induced eryptosis, indicating a relationship between costunolide and PKC-α. In addition, chelerythrine acts independently of the GSH depletion. Understanding the mechanisms of G6PDH inhibition accompanied by GSH depletion should be useful for development of anti-malarial therapeutic strategies or for synthetic lethality-based approaches to escalate oxidative stress in cancer cells for their sensitization to chemotherapy and radiotherapy.


Assuntos
Benzofenantridinas/farmacologia , Inibidores Enzimáticos/farmacologia , Eriptose/genética , Glucosefosfato Desidrogenase/genética , Proteína Quinase C-alfa/genética , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Eriptose/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/patologia , Glucosefosfato Desidrogenase/antagonistas & inibidores , Glutationa/genética , Humanos , Estresse Oxidativo/efeitos dos fármacos , Proteína Quinase C-alfa/antagonistas & inibidores , Espécies Reativas de Oxigênio , Sesquiterpenos/farmacologia
7.
Int J Mol Sci ; 21(24)2020 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-33348919

RESUMO

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.


Assuntos
Anemia de Diamond-Blackfan/patologia , Dano ao DNA , Eritrócitos/patologia , Mediadores da Inflamação/metabolismo , Inflamação/patologia , Estresse Oxidativo , Adulto , Anemia de Diamond-Blackfan/imunologia , Anemia de Diamond-Blackfan/metabolismo , Animais , Estudos de Casos e Controles , Criança , Eritrócitos/metabolismo , Feminino , Seguimentos , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Prognóstico , Adulto Jovem
8.
J Cell Mol Med ; 23(11): 7785-7795, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31517438

RESUMO

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.


Assuntos
Ciclina D1/metabolismo , Dioxigenases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Quelantes de Ferro/farmacologia , Ácidos Cetoglutáricos/farmacologia , Linfoma de Célula do Manto/enzimologia , Aminoácidos Dicarboxílicos/farmacologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA , Desferroxamina/farmacologia , Dioxigenases/metabolismo , Regulação para Baixo/efeitos dos fármacos , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Humanos , Hidroxilação , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Deficiências de Ferro , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
9.
Blood Cells Mol Dis ; 69: 23-29, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28803808

RESUMO

Glucose-6-phosphate isomerase (GPI) deficiency, a genetic disorder responsible for chronic nonspherocytic hemolytic anemia, is the second most common red blood cell glycolytic enzymopathy. We report three patients from two unrelated families of Czech and Slovak origin with macrocytic hemolytic anemia due to GPI deficiency. The first patient had 15% of residual GPI activity resulting from two new heterozygous missense mutations c.478T>C and c.1414C>T leading to substitutions p.(Ser160Pro) and p.(Arg472Cys). Two other patients (siblings) inherited the same c.1414C>T p.(Arg472Cys) mutation in a homozygous constitution and lost approximately 89% of their GPI activity. Erythroid hyperplasia with dysplastic features was observed in the bone marrow of all three patients. Low hepcidin/ferritin ratio and elevated soluble transferrin receptor detected in our GPI-deficient patients suggest disturbed balance between erythropoiesis and iron metabolism contributing to iron overload.


Assuntos
Substituição de Aminoácidos , Anemia Hemolítica Congênita não Esferocítica/sangue , Anemia Hemolítica Congênita não Esferocítica/genética , Células Eritroides/metabolismo , Glucose-6-Fosfato Isomerase/genética , Hepcidinas/sangue , Mutação , Alelos , Anemia Hemolítica Congênita não Esferocítica/diagnóstico , Biomarcadores , Biópsia , Medula Óssea/patologia , Criança , Índices de Eritrócitos , Eritropoese/genética , Feminino , Regulação da Expressão Gênica , Genótipo , Glucose-6-Fosfato Isomerase/química , Humanos , Ferro/metabolismo , Masculino , Modelos Moleculares , Conformação Proteica , Análise de Sequência de DNA , Relação Estrutura-Atividade
10.
Blood ; 128(10): 1418-23, 2016 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-27389715

RESUMO

The role of somatic JAK2 mutations in clonal myeloproliferative neoplasms (MPNs) is well established. Recently, germ line JAK2 mutations were associated with polyclonal hereditary thrombocytosis and triple-negative MPNs. We studied a patient who inherited 2 heterozygous JAK2 mutations, E846D from the mother and R1063H from the father, and exhibited erythrocytosis and megakaryocytic atypia but normal platelet number. Culture of erythroid progenitors from the patient and his parents revealed hypersensitivity to erythropoietin (EPO). Using cellular models, we show that both E846D and R1063H variants lead to constitutive signaling (albeit much weaker than JAK2 V617F), and both weakly hyperactivate JAK2/STAT5 signaling only in the specific context of the EPO receptor (EPOR). JAK2 E846D exhibited slightly stronger effects than JAK2 R1063H and caused prolonged EPO-induced phosphorylation of JAK2/STAT5 via EPOR. We propose that JAK2 E846D predominantly contributes to erythrocytosis, but is not sufficient for the full pathological phenotype to develop. JAK2 R1063H, with very weak effect on JAK2/STAT5 signaling, is necessary to augment JAK2 activity caused by E846D above a threshold level leading to erythrocytosis with megakaryocyte abnormalities. Both mutations were detected in the germ line of rare polycythemia vera, as well as certain leukemia patients, suggesting that they might predispose to hematological malignancy.


Assuntos
Mutação em Linhagem Germinativa/genética , Janus Quinase 2/genética , Megacariócitos/patologia , Policitemia/congênito , Adolescente , Adulto , Feminino , Humanos , Masculino , Megacariócitos/metabolismo , Pessoa de Meia-Idade , Fosforilação , Policitemia/genética , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Adulto Jovem
11.
Vnitr Lek ; 64(5): 476-487, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30193516

RESUMO

This article summarize molecular-genetic basis of hemoglobinopathies, their classification and phenotypic manifestations. The description of individual subgroups is supplemented with a case reports of patients diagnosed in the Czech population. This paper provides an overview of 14 types of α-thalassemic mutations, 34 ß-thalassemic alleles, 4 δß-thalassemic alleles and 22 hemoglobin variants identified in the Czech population in 876 persons from 579 families. In more detail are described hemoglobinopathies, that have been diagnosed and described as novel: ß-thalassemic mutation CD 38/39 (-C); Hb Olomouc; Hb Hana; Hb Hradec Kralove and 18.3 kb deletion downstream of α-globin cluster leading to a new mechanism of α-thalassemia-2. The fact that until the end of 2017 hemoglobinopathies were diagnosed in nearly 900 patients shows that they are not rare in the Czech Republic. This brings increased demands for their diagnostics, including prenatal diagnosis. Key words: hemoglobinopathies - hemoglobinopathy with high affinity to oxygen - sickle cell anemia - thalassemia - thalassemic hemoglobinopathy - unstable hemoglobins.


Assuntos
Hemoglobinopatias , Talassemia alfa , Talassemia beta , República Tcheca , Feminino , Hemoglobinopatias/genética , Humanos , Mutação/genética , Gravidez , Talassemia alfa/genética , Talassemia beta/genética
12.
Hum Mutat ; 37(11): 1153-1156, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27492747

RESUMO

We report an infant with sickle cell disease phenotype by biochemical analysis whose ß-globin gene (HBB) sequencing showed sickle cell mutation (HBBS ) heterozygosity. The proband has a unique head-to-tail duplication of the ß-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 ß-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 ß-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.


Assuntos
Anemia Falciforme/genética , Genes Duplicados , Globinas beta/genética , Região 3'-Flanqueadora , Feminino , Inativação Gênica , Humanos , Lactente , Região de Controle de Locus Gênico , Mutação , Transcrição Gênica
13.
Blood Cells Mol Dis ; 59: 71-6, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27282571

RESUMO

Hexokinase (HK) is a key enzyme of glycolysis, the only metabolic pathway able to provide the red blood cell with ATP. HK deficiency is a very rare hereditary disorder with severe chronic nonspherocytic hemolytic anemia (HNSHA) as a major clinical feature. To date, only 24 patients with HK deficiency have been identified. Here, we report the molecular analysis of six new cases of HK deficiency. A total of six different mutations were detected in HK1, four of them described here for the first time: c.2599C>T p.(His867Tyr), c.1799C>T p.(Thr600Met), c.873-2A>G and c.493-1G>A. The pathogenic nature of the identified missense mutations was confirmed by biochemical and 3-dimensional structural analysis. The effects of the novel splice site mutation c.873-2A>G were studied at the level of pre-mRNA processing, and confirmed at the protein level. All together, these results provide a better insight into the pathogenesis of this rare red cell disorder, and contribute to a better understanding of the genotype-phenotype correlation in HK deficiency.


Assuntos
Eritrócitos/enzimologia , Hexoquinase/deficiência , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética , Hexoquinase/genética , Humanos , Lactente , Masculino , Mutação , Mutação de Sentido Incorreto , Sítios de Splice de RNA/genética , Adulto Jovem
14.
Blood ; 123(3): 391-4, 2014 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-24297870

RESUMO

Overexpression of transcription factors runt-related transcription factor 1 (RUNX1) and nuclear factor, erythroid-derived 2 (NF-E2) was reported in granulocytes of patients with polycythemia vera and other myeloproliferative neoplasms (MPNs). Further, a transgenic mouse overexpressing the NF-E2 transgene was reported to be a model of MPN. We hypothesized that increased transcripts of RUNX1 and NF-E2 might characterize other polycythemic states with primary polycythemic features, that is, those with exaggerated erythropoiesis due to augmented erythropoietin (EPO) sensitivity. We tested the expression of RUNX1 and NF-E2 in polycythemic patients of diverse phenotypes and molecular causes. We report that RUNX1 and NF-E2 overexpression is not specific for MPN; these transcripts were also significantly elevated in polycythemias with augmented hypoxia-inducible factor activity whose erythroid progenitors were hypersensitive to EPO. RUNX1 and NF-E2 overexpression was not detected in patients with EPO receptor (EPOR) gain-of-function, suggesting distinct mechanisms by which erythroid progenitors in polycythemias with defects of hypoxia sensing and EPOR mutations exert their EPO hypersensitivity.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Transtornos Mieloproliferativos/metabolismo , Subunidade p45 do Fator de Transcrição NF-E2/metabolismo , Policitemia/metabolismo , Animais , Hipóxia Celular , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Eritropoetina/metabolismo , Regulação da Expressão Gênica , Granulócitos/citologia , Humanos , Leucócitos Mononucleares/citologia , Camundongos , Camundongos Transgênicos , Mutação , Transtornos Mieloproliferativos/genética , Subunidade p45 do Fator de Transcrição NF-E2/genética , Policitemia/genética , Transdução de Sinais
15.
Hemoglobin ; 40(3): 156-62, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26956563

RESUMO

ß-Thalassemia (ß-thal) is considered rare in Central Europe. As in other malaria-free regions, the presence of ß-thal in Central Europe reflects historical and recent immigration, and demographic changes that have influenced the genetic variability of the current populations living in this area. This study assesses the frequency and spectrum of mutations on the ß-globin gene in Czech and Slovak subjects with clinical symptoms of thalassemia. The results of the initial part of this research were published more than two decades ago; the aim of this study was to update these original reports. During the period from 2002 to 2015, 400 cases from Czech and Slovak hematological centers were analyzed. Twenty-nine ß-thal mutations, identified in 356 heterozygotes from 218 unrelated families, involve five unique mutations including a recently described insertion of a transposable L1 element into the ß-globin gene. One mutation described here is reported for the first time. Most of the mutations were of Mediterranean origin and accounted for 82.0% of cases. All but one case studied were heterozygous carriers, manifesting ß-thal minor, with rare exceptions represented by the rare (ß(0)) codons 46/47 (+G) (HBB: c.142_142dupG) mutation associated with an α-globin gene quadruplication and by dominantly inherited ß-thal with a more severe phenotype. One double heterozygous ß-thal patient was a recent immigrant from Moldavia. The list of δß-thal alleles (26 carriers, 16 families) contains Hb Lepore and two types of δß(0)-thal deletions. In the past, genetic drift and migration as well as recent immigrations were responsible for the introduction of Mediterranean alleles, while several mutations described in single families were of local origin.


Assuntos
Epidemiologia Molecular , Mutação , Talassemia beta/epidemiologia , Povo Asiático/etnologia , Povo Asiático/genética , República Tcheca/epidemiologia , Emigração e Imigração , Frequência do Gene , Deriva Genética , Heterozigoto , Humanos , Linhagem , Eslováquia/epidemiologia , População Branca/etnologia , População Branca/genética , Globinas beta/genética , Talassemia beta/genética
16.
Blood ; 121(19): 3918-24, 2013 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-23538339

RESUMO

Germline von Hippel-Lindau (VHL) gene mutations underlie dominantly inherited familial VHL tumor syndrome comprising a predisposition for renal cell carcinoma, pheochromocytoma/paraganglioma, cerebral hemangioblastoma, and endolymphatic sac tumors. However, recessively inherited congenital polycythemia, exemplified by Chuvash polycythemia, has been associated with 2 separate 3' VHL gene mutations in exon 3. It was proposed that different positions of loss-of-function VHL mutations are associated with VHL syndrome cancer predisposition and only C-terminal domain-encoding VHL mutations would cause polycythemia. However, now we describe a new homozygous VHL exon 2 mutation of the VHL gene:(c.413C>T):P138L, which is associated in the affected homozygote with congenital polycythemia but not in her, or her-heterozygous relatives, with cancer or other VHL syndrome tumors. We show that VHL(P138L) has perturbed interaction with hypoxia-inducible transcription factor (HIF)1α. Further, VHL(P138L) protein has decreased stability in vitro. Similarly to what was reported in Chuvash polycythemia and some other instances of HIFs upregulation, VHL(P138L) erythroid progenitors are hypersensitive to erythropoietin. Interestingly, the level of RUNX1/AML1 and NF-E2 transcripts that are specifically upregulated in acquired polycythemia vera were also upregulated in VHL(P138L) granulocytes.


Assuntos
Mutação , Policitemia/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Adolescente , Éxons/genética , Feminino , Homozigoto , Humanos , Modelos Moleculares , Mutação/fisiologia , Neoplasias/genética , Policitemia/congênito , Estrutura Secundária de Proteína , Proteína Supressora de Tumor Von Hippel-Lindau/química
18.
Br J Haematol ; 165(4): 556-63, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24533562

RESUMO

Pyruvate kinase (PK) deficiency is an iron-loading anaemia characterized by chronic haemolysis, ineffective erythropoiesis and a requirement for blood transfusion in most cases. We studied 11 patients from 10 unrelated families and found nine different disease-causing PKLR mutations. Two of these mutations - the point mutation c.878A>T (p.Asp293Val) and the frameshift deletion c.1553delG (p.(Arg518Leufs*12)) - have not been previously described in the literature. This frameshift deletion was associated with an unusually severe phenotype involving neonatal hyperferritinaemia that is not typical of PK deficiency. No disease-causing mutations in genes associated with haemochromatosis could be found. Inappropriately low levels of hepcidin with respect to iron loading were detected in all PK-deficient patients with increased ferritin, confirming the predominant effect of accelerated erythropoiesis on hepcidin production. Although the levels of a putative hepcidin suppressor, growth differentiation factor-15, were increased in PK-deficient patients, no negative correlation with hepcidin was found. This result indicates the existence of another as-yet unidentified erythroid regulator of hepcidin synthesis in PK deficiency.


Assuntos
Anemia Hemolítica Congênita não Esferocítica/genética , Ferritinas/sangue , Hepcidinas/sangue , Ferro/sangue , Mutação , Piruvato Quinase/deficiência , Piruvato Quinase/genética , Erros Inatos do Metabolismo dos Piruvatos/genética , Adulto , Sequência de Aminoácidos , Anemia Hemolítica Congênita não Esferocítica/sangue , Criança , Pré-Escolar , Análise Mutacional de DNA , Eritropoese , Feminino , Hepcidinas/biossíntese , Humanos , Lactente , Recém-Nascido , Sobrecarga de Ferro/genética , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Piruvato Quinase/sangue , Erros Inatos do Metabolismo dos Piruvatos/sangue , Análise de Sequência de DNA , Reação Transfusional , Adulto Jovem
19.
Cell Physiol Biochem ; 34(6): 2221-31, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25562168

RESUMO

BACKGROUND/AIMS: Deficiency of the divalent metal transporter 1 (DMT1) leads to hypochromic microcytic anemia. We have previously shown that DMT1 deficiency impairs erythroid differentiation and induces apoptosis of erythroid cells. Here we analyzed metabolic processes and survival of mature erythrocytes in order to address potential involvement of erythrocyte defect in the pathophysiology of the disease. METHODS: FACS analysis was used to determine the half-life of erythrocytes (CFSE fluorescence), phosphatidylserine exposure (Annexin V binding), cytosolic Ca(2+) (Fluo3/AM fluorescence) and reactive oxygen species (ROS; DCF fluorescence). Enzyme activities were determined by standard biochemical methods. The concentration of ATP and ADP was measured on HPLC-MS/MS. RESULTS: We observed an accelerated clearance of CFSE-labeled DMT1-mutant erythrocytes from circulating blood when compared to wild-type erythrocytes. In vitro, DMT1-mutant erythrocytes showed significantly increased Annexin V binding after exposure to hyperosmotic shock and glucose depletion. Despite exaggerated anti-oxidative defense, higher ROS levels were present in DMT1-mutant erythrocytes. Accelerated anaerobic glycolysis and reduced ATP/ADP ratio detected in DMT1-mutant erythrocytes indicate enhanced demand for ATP. CONCLUSIONS: We propose that DMT1 deficiency negatively affects metabolism and life span of mature erythrocytes; two other aspects of defective erythropoiesis which contribute to the pathophysiology of the disease.


Assuntos
Anemia Hipocrômica/genética , Proteínas de Transporte de Cátions/genética , Eritrócitos/metabolismo , Estresse Oxidativo , Difosfato de Adenosina/sangue , Trifosfato de Adenosina/sangue , Anemia Hipocrômica/sangue , Anemia Hipocrômica/patologia , Animais , Apoptose/genética , Proteínas de Transporte de Cátions/sangue , Proteínas de Transporte de Cátions/deficiência , Modelos Animais de Doenças , Eritrócitos/patologia , Eritropoese , Glicólise , Humanos , Camundongos , Mutação , Espécies Reativas de Oxigênio/sangue
20.
Hum Mutat ; 34(10): 1361-5, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23878091

RESUMO

We describe the molecular etiology of ß(+)-thalassemia that is caused by the insertion of the full-length transposable element LINE-1 (L1) into the intron-2 of the ß-globin gene (HBB). The transcript level of the affected ß-globin gene was severely reduced. The remaining transcripts consisted of full-length, correctly processed ß-globin mRNA and a minute amount of three aberrantly spliced transcripts with a decreased half-life due to activation of the nonsense-mediated decay pathway. The lower steady-state amount of mRNA produced by the ß-globin(L1) allele also resulted from a reduced rate of transcription and decreased production of full-length ß-globin primary transcripts. The promoter and enhancer sequences of the ß-globin(L1) allele were hypermethylated; however, treatment with a demethylating agent did not restore the impaired transcription. A histone deacetylase inhibitor partially reactivated the ß-globin(L1) transcription despite permanent ß-globin(L1) promoter CpG methylation. This result indicates that the decreased rate of transcription from the ß-globin(L1) allele is associated with an altered chromatin structure. Therefore, the molecular defect caused by intronic L1 insertion in the ß-globin gene represents a novel etiology of ß-thalassemia.


Assuntos
Íntrons , Elementos Nucleotídeos Longos e Dispersos , Mutagênese Insercional , Globinas beta/genética , Talassemia beta/genética , Adulto , Alelos , Processamento Alternativo , Ilhas de CpG , Metilação de DNA , Feminino , Regulação da Expressão Gênica , Ordem dos Genes , Inativação Gênica , Humanos , Regiões Promotoras Genéticas , Estabilidade de RNA , Transcrição Gênica
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