RESUMEN
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.
Asunto(s)
Policitemia , Apnea Obstructiva del Sueño , Humanos , Especies Reactivas de Oxígeno , Policitemia/etiología , Hepcidinas , Hipoxia , Apnea Obstructiva del Sueño/complicaciones , InflamaciónRESUMEN
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.
Asunto(s)
Policitemia , Humanos , Policitemia/genética , Hepcidinas/genética , Oxígeno/metabolismo , Mutación , Receptores de Eritropoyetina/genética , Canales Iónicos/genéticaRESUMEN
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.
Asunto(s)
Eritropoyetina , Policitemia , Adulto , Anciano , Animales , Eritropoyesis/genética , Eritropoyetina/genética , Eritropoyetina/farmacología , Mutación con Ganancia de Función , Hepcidinas/genética , Hepcidinas/metabolismo , Hormonas , Humanos , Hierro/metabolismo , Ratones , Policitemia/genética , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/metabolismoRESUMEN
Diamond-Blackfan anemia (DBA) is predominantly underlined by mutations in genes encoding ribosomal proteins (RP); however, its etiology remains unexplained in approximately 25 % of patients. We previously reported a novel heterozygous RPS7 mutation hg38 chr2:g.3,580,153G > T p.V134F in one female patient and two asymptomatic family members, in whom mild anemia and increased erythrocyte adenosine deaminase (eADA) activity were detected. We observed that altered erythrocyte metabolism and oxidative stress which may negatively affect the lifespan of erythrocytes distinguishes the patient from her asymptomatic family members. Pathogenicity of the RPS7 p.V134F mutation was extensively validated including molecular defects in protein translational activity and ribosomal stress activation in the cellular model of this variant.
Asunto(s)
Anemia de Diamond-Blackfan , Proteínas Ribosómicas , Anemia de Diamond-Blackfan/genética , Eritrocitos/metabolismo , Femenino , Humanos , Mutación Missense , Biosíntesis de Proteínas , Proteínas Ribosómicas/genéticaRESUMEN
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.
Asunto(s)
Anemia de Diamond-Blackfan/patología , Daño del ADN , Eritrocitos/patología , Mediadores de Inflamación/metabolismo , Inflamación/patología , Estrés Oxidativo , Adulto , Anemia de Diamond-Blackfan/inmunología , Anemia de Diamond-Blackfan/metabolismo , Animales , Estudios de Casos y Controles , Niño , Eritrocitos/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Pronóstico , Adulto JovenRESUMEN
Diamond-Blackfan anemia (DBA) is a rare congenital erythroid aplasia, underlied by haploinsufficient mutations in genes coding for ribosomal proteins (RP) in approximately 70% of cases. DBA is frequently associated with somatic malformations, endocrine dysfunction and with an increased predisposition to cancer. Here we present clinical and genetic characteristics of 62 patients from 52 families enrolled in the Czech and Slovak DBA Registry. Whole exome sequencing (WES) and array comparative genomic hybridization (aCGH) were employed to identify causative mutations in newly diagnosed patients and in cases with previously unrecognized molecular pathology. RP mutation detection rate was 81% (50/62 patients). This included 8 novel point mutations and 4 large deletions encompassing some of the RP genes. Malignant or predisposing condition developed in 8/62 patients (13%): myelodysplastic syndrome in 3 patients; breast cancer in 2 patients; colorectal cancer plus ocular tumor, diffuse large B-cell lymphoma and multiple myeloma each in one case. These patients exclusively harbored RPL5, RPL11 or RPS19 mutations. Array CGH is beneficial for detection of novel mutations in DBA due to its capacity to detect larger chromosomal aberrations. Despite the importance of genotype-phenotype correlation in DBA, phenotypic differences among family members harboring an identical mutation were observed.
Asunto(s)
Anemia de Diamond-Blackfan/genética , Mutación , Proteínas Ribosómicas/genética , Anemia de Diamond-Blackfan/complicaciones , Anemia de Diamond-Blackfan/epidemiología , Hibridación Genómica Comparativa , República Checa , Familia , Estudios de Asociación Genética , Humanos , Neoplasias/etiología , Sistema de Registros , Eslovaquia , Secuenciación del ExomaRESUMEN
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.
Asunto(s)
Hemoglobinopatías , Talasemia alfa , Talasemia beta , República Checa , Femenino , Hemoglobinopatías/genética , Humanos , Mutación/genética , Embarazo , Talasemia alfa/genética , Talasemia beta/genéticaRESUMEN
AIM: Hepcidin is a central regulator of iron homeostasis. Its production is also influenced by systemic inflammation. The aims of this study were to compare hepcidin levels in paediatric patients newly diagnosed with Crohn's disease (CD) and ulcerative colitis (UC) and to determine the association of hepcidin levels with laboratory and clinical parameters of inflammatory bowel disease (IBD) activity. METHODS: Children with newly diagnosed IBD between January 2012 and September 2016 were enrolled in this comparative cross-sectional study. We analysed levels of serum hepcidin, C-reactive protein, iron, ferritin, soluble transferrin receptors, blood count and faecal calprotectin in all subjects. Serum hepcidin levels were measured by reverse-phase liquid chromatography. The Paediatric Crohn's Disease Activity Index was used to evaluate CD in children, and Paediatric Ulcerative Colitis Activity Index was used for the assessment of UC disease activity. RESULTS: Subjects with CD (n = 53) had significantly higher serum hepcidin levels compared with subjects with UC (n = 23) - 22.6 ng/mL (range 8.5-65.0) versus 6.5 ng/mL (range 2.4-25.8) (P < 0.05). Hepcidin was independently associated with ferritin levels in all IBD patients (P < 0.05). Moreover, there was a significant positive correlation between hepcidin and platelet count (P < 0.05) in children with CD and a negative correlation between hepcidin and faecal calprotectin (P < 0.05) in children with UC. CONCLUSION: Different hepcidin levels between children with newly diagnosed CD and UC suggest the distinct contribution of iron deficiency and/or systemic inflammation to anaemia and may help clinicians choose the best anti-anaemic treatment.
Asunto(s)
Antiinfecciosos/sangre , Hepcidinas/sangre , Enfermedades Inflamatorias del Intestino/diagnóstico , Adolescente , Proteína C-Reactiva/análisis , Niño , Estudios Transversales , Heces/química , Femenino , Ferritinas/sangre , Humanos , Hierro/sangre , Complejo de Antígeno L1 de Leucocito/sangre , MasculinoRESUMEN
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.
Asunto(s)
Sustitución de Aminoácidos , Anemia Hemolítica Congénita no Esferocítica/sangre , Anemia Hemolítica Congénita no Esferocítica/genética , Células Eritroides/metabolismo , Glucosa-6-Fosfato Isomerasa/genética , Hepcidinas/sangre , Mutación , Alelos , Anemia Hemolítica Congénita no Esferocítica/diagnóstico , Biomarcadores , Biopsia , Médula Ósea/patología , Niño , Índices de Eritrocitos , Eritropoyesis/genética , Femenino , Regulación de la Expresión Génica , Genotipo , Glucosa-6-Fosfato Isomerasa/química , Humanos , Hierro/metabolismo , Masculino , Modelos Moleculares , Conformación Proteica , Análisis de Secuencia de ADN , Relación Estructura-ActividadRESUMEN
Thalassemias are a heterogeneous group of red blood cell disorders, considered a major cause of morbidity and mortality among genetic diseases. However, there is still no universally available cure for thalassemias. The underlying basis of thalassemia pathology is the premature apoptotic destruction of erythroblasts causing ineffective erythropoiesis. In ß-thalassemia, ß-globin synthesis is reduced causing α-globin accumulation. Unpaired globin chains, with heme attached to them, accumulate in thalassemic erythroblasts causing oxidative stress and the premature cell death. We hypothesize that in ß-thalassemia heme oxygenase (HO) 1 could play a pathogenic role in the development of anemia and ineffective erythropoiesis. To test this hypothesis, we exploited a mouse model of ß-thalassemia intermedia, Th3/+ We observed that HO inhibition using tin protoporphyrin IX (SnPP) decreased heme-iron recycling in the liver and ameliorated anemia in the Th3/+ mice. SnPP administration led to a decrease in erythropoietin and increase in hepcidin serum levels, changes that were accompanied by an alleviation of ineffective erythropoiesis in Th3/+ mice. Additionally, the bone marrow from Th3/+ mice treated with SnPP exhibited decreased heme catabolism and diminished iron release as well as reduced apoptosis. Our results indicate that the iron released from heme because of HO activity contributes to the pathophysiology of thalassemia. Therefore, new therapies that suppress heme catabolism may be beneficial in ameliorating the anemia and ineffective erythropoiesis in thalassemias.
Asunto(s)
Inhibidores Enzimáticos/uso terapéutico , Hemo-Oxigenasa 1/antagonistas & inhibidores , Sobrecarga de Hierro/tratamiento farmacológico , Metaloporfirinas/uso terapéutico , Protoporfirinas/uso terapéutico , Talasemia beta/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Eritropoyesis/efectos de los fármacos , Eritropoyetina/sangre , Hemo-Oxigenasa 1/análisis , Sobrecarga de Hierro/sangre , Sobrecarga de Hierro/complicaciones , Sobrecarga de Hierro/patología , Hígado/efectos de los fármacos , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Talasemia beta/sangre , Talasemia beta/complicaciones , Talasemia beta/patologíaRESUMEN
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.
Asunto(s)
Mutación de Línea Germinal/genética , Janus Quinasa 2/genética , Megacariocitos/patología , Policitemia/congénito , Adolescente , Adulto , Femenino , Humanos , Masculino , Megacariocitos/metabolismo , Persona de Mediana Edad , Fosforilación , Policitemia/genética , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/metabolismo , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Adulto JovenRESUMEN
BACKGROUND AND AIMS: Erythropoiesis is closely related to iron metabolism in a balanced homeostasis. Analyses of diverse erythroid and iron metabolism disorders have shown that disrupted erythropoiesis negatively affects iron homeostasis and vice versa. The aim of this study was to characterize the relationship between erythropoietic activity and iron homeostasis in pediatric patients with erythrocyte membrane defects and thalassemia traits. METHODS: Selected markers of erythropoietic activity (erythropoietin, soluble transferrin receptor - sTfR and growth differentiation factor 15) and iron status parameters (serum iron, ferritin and hepcidin) were evaluated in pediatric patients with erythrocyte membrane defects and thalassemia traits. RESULTS: The patients with erythrocyte membrane defects and thalassemia traits had altered iron homeostasis due to disturbed erythropoiesis. In comparison with healthy controls, they had a normal to low hepcidin/ferritin ratio and concomitantly elevated sTfR. CONCLUSION: The findings suggest that pediatric patients with erythrocyte membrane defects and thalassemia traits are more susceptible to iron overload than the general population and that the (hepcidin/ferritin)/sTfR ratio can be used to monitor any worsening of the disease.
Asunto(s)
Eliptocitosis Hereditaria/sangre , Membrana Eritrocítica/metabolismo , Eritropoyesis/fisiología , Hierro/metabolismo , Esferocitosis Hereditaria/sangre , Talasemia/metabolismo , Adolescente , Análisis de Varianza , Niño , Preescolar , Hemostasis/fisiología , Hepcidinas/metabolismo , HumanosRESUMEN
UNLABELLED: Mutations of the truncated cytoplasmic domain of human erythropoietin receptor (EPOR) result in gain-of-function of erythropoietin (EPO) signaling and a dominantly inherited polycythemia, primary familial and congenital polycythemia (PFCP). We interrogated the unexplained transient absence of perinatal polycythemia observed in PFCP patients using an animal model of PFCP to examine its erythropoiesis during embryonic, perinatal, and early postnatal periods. In this model, we replaced the murine EpoR gene (mEpoR) with the wild-type human EPOR (wtHEPOR) or mutant human EPOR gene (mtHEPOR) and previously reported that the gain-of-function mtHEPOR mice become polycythemic at 3~6 weeks of age, but not at birth, similar to the phenotype of PFCP patients. In contrast, wtHEPOR mice had sustained anemia. We report that the mtHEPOR fetuses are polycythemic, but their polycythemia is abrogated in the perinatal period and reappears again at 3 weeks after birth. mtHEPOR fetuses have a delayed switch from primitive to definitive erythropoiesis, augmented erythropoietin signaling, and prolonged Stat5 phosphorylation while the wtHEPOR fetuses are anemic. Our study demonstrates the in vivo effect of excessive EPO/EPOR signaling on developmental erythropoiesis switch and describes that fetal polycythemia in this PFCP model is followed by transient correction of polycythemia in perinatal life associated with low Epo levels and increased exposure of erythrocytes' phosphatidylserine. We suggest that neocytolysis contributes to the observed perinatal correction of polycythemia in mtHEPOR newborns as embryos leaving the hypoxic uterus are exposed to normoxia at birth. KEY MESSAGE: Human gain-of-function EPOR (mtHEPOR) causes fetal polycythemia in knock-in mice. Wild-type human EPOR causes fetal anemia in knock-in mouse model. mtHEPOR mice have delayed switch from primitive to definitive erythropoiesis. Polycythemia of mtHEPOR mice is transiently corrected in perinatal life. mtHEPOR newborns have low Epo and increased exposure of erythrocytes' phosphatidylserine.
Asunto(s)
Mutación con Ganancia de Función , Regulación de la Expresión Génica , Hemoglobinas/genética , Receptores de Eritropoyetina/genética , Anemia/sangre , Anemia/genética , Anemia/metabolismo , Animales , Eritrocitos/metabolismo , Células Precursoras Eritroides/metabolismo , Eritropoyesis/genética , Eritropoyetina/metabolismo , Genotipo , Hematócrito , Humanos , Ratones , Ratones Transgénicos , Fosforilación , Policitemia/sangre , Policitemia/genética , Policitemia/metabolismo , Receptores de Eritropoyetina/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de SeñalRESUMEN
UNLABELLED: During prolonged hypoxia, hypoxia-inducible factors (HIFs) mediate an increase in erythropoiesis, leading to an increased red blood cell (RBC) mass and polycythemia. Upon return to normoxia, the increased RBC mass is abruptly overcorrected by the preferential destruction of hypoxia-formed young RBCs, a phenomenon termed neocytolysis. The molecular and biochemical mechanisms involved in neocytolysis are unknown. We developed a murine model of neocytolysis by exposing mice to 12 % oxygen for 10 days followed by return to normoxia. Upon return to normoxia, there was excessive accumulation of reactive oxygen species (ROS) in RBCs from an increased reticulocyte mitochondrial mass correlating with decreased Bnip3L transcripts (Bnip3L mediates reticulocyte mitophagy) and reduced catalase activity. During hypoxia, upregulated miR-21 resulted in low catalase activity in young RBCs. Furthermore, neocytolysis was attenuated by antioxidants and plasma catalase and blunted in mice that had constitutively high expression of HIFs. Among human neonates studied, we report data supporting the existence of neocytolysis during the first week of life. Together, these experiments indicate that the major mechanisms causing neocytolysis involve (1) production of young RBCs with low catalase during hypoxia and (2) lysis of the young RBCs after return to normoxia, mediated by ROS from an increased mitochondrial mass. KEY MESSAGES: We report a mouse model of neocytolysis. Neocytolysis is caused by excessive ROS formation mediated by HIF. ROS is generated from increased mitochondria in reticulocytes. Hypoxia-generated RBCs have low catalase and are preferentially destroyed. Reduced catalase is regulated by increased microRNA-21.
Asunto(s)
Catalasa/metabolismo , Eritrocitos/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Hipoxia/patología , Especies Reactivas de Oxígeno/metabolismo , Animales , Catalasa/genética , Línea Celular , Eritrocitos/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Humanos , Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Mitofagia , Policitemia/genética , Policitemia/metabolismo , Policitemia/patología , Reticulocitos/metabolismo , Reticulocitos/patologíaRESUMEN
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.
Asunto(s)
Anemia Hemolítica Congénita no Esferocítica/genética , Ferritinas/sangre , Hepcidinas/sangre , Hierro/sangre , Mutación , Piruvato Quinasa/deficiencia , Piruvato Quinasa/genética , Errores Innatos del Metabolismo del Piruvato/genética , Adulto , Secuencia de Aminoácidos , Anemia Hemolítica Congénita no Esferocítica/sangre , Niño , Preescolar , Análisis Mutacional de ADN , Eritropoyesis , Femenino , Hepcidinas/biosíntesis , Humanos , Lactante , Recién Nacido , Sobrecarga de Hierro/genética , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Piruvato Quinasa/sangre , Errores Innatos del Metabolismo del Piruvato/sangre , Análisis de Secuencia de ADN , Reacción a la Transfusión , Adulto JovenRESUMEN
Heme is essential for the function of all aerobic cells. However, it can be toxic when it occurs in a non-protein-bound form; cells maintain a fine balance between heme synthesis and catabolism. The only physiological mechanism of heme degradation is by heme oxygenases (HOs). The heme-inducible isoform, HO-1, has been extensively studied in numerous nonerythroid cells, but virtually nothing is known about the expression and potential significance of HO-1 in developing red blood cells. We have demonstrated that HO-1 is present in erythroid cells and that its expression is upregulated during erythroid differentiation. Overexpression of HO-1 in erythroid cells impairs hemoglobin synthesis, whereas HO-1 absence enhances hemoglobinization in cultured erythroid cells. Based on these results, we conclude that HO-1 controls the regulatory heme pool at appropriate levels for any given stage of erythroid differentiation. In summary, our study brings to light the importance of HO-1 expression for erythroid development and expands our knowledge about the fine regulation of hemoglobin synthesis in erythroid cells. Our results indicate that HO-1 plays an important role as a coregulator of the erythroid differentiation process. Moreover, HO-1 expression must be tightly regulated during red blood cell development.
Asunto(s)
Células Eritroides/metabolismo , Hemo-Oxigenasa 1/genética , Hemo/metabolismo , Animales , Células de la Médula Ósea/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Embrión de Mamíferos , Eritropoyesis/genética , Expresión Génica , Hemo-Oxigenasa 1/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
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.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Trastornos Mieloproliferativos/metabolismo , Subunidad p45 del Factor de Transcripción NF-E2/metabolismo , Policitemia/metabolismo , Animales , Hipoxia de la Célula , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Eritropoyetina/metabolismo , Regulación de la Expresión Génica , Granulocitos/citología , Humanos , Leucocitos Mononucleares/citología , Ratones , Ratones Transgénicos , Mutación , Trastornos Mieloproliferativos/genética , Subunidad p45 del Factor de Transcripción NF-E2/genética , Policitemia/genética , Transducción de SeñalRESUMEN
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.
Asunto(s)
Anemia Hipocrómica/genética , Proteínas de Transporte de Catión/genética , Eritrocitos/metabolismo , Estrés Oxidativo , Adenosina Difosfato/sangre , Adenosina Trifosfato/sangre , Anemia Hipocrómica/sangre , Anemia Hipocrómica/patología , Animales , Apoptosis/genética , Proteínas de Transporte de Catión/sangre , Proteínas de Transporte de Catión/deficiencia , Modelos Animales de Enfermedad , Eritrocitos/patología , Eritropoyesis , Glucólisis , Humanos , Ratones , Mutación , Especies Reactivas de Oxígeno/sangreRESUMEN
BACKGROUND: Hypochromic microcytic anemia associated with ineffective erythropoiesis caused by recessive mutations in divalent metal transporter 1 (DMT1) can be improved with high-dose erythropoietin supplementation. The aim of this study was to characterize and compare erythropoiesis in samples from a DMT1-mutant patient before and after treatment with erythropoietin, as well as in a mouse model with a DMT1 mutation, the mk/mk mice. DESIGN AND METHODS: Colony assays were used to compare the in vitro growth of pre-treatment and post-treatment erythroid progenitors in a DMT1-mutant patient. To enable a comparison with human data, high doses of erythropoietin were administered to mk/mk mice. The apoptotic status of erythroblasts, the expression of anti-apoptotic proteins, and the key components of the bone marrow-hepcidin axis were evaluated. RESULTS: Erythropoietin therapy in vivo or the addition of a broad-spectrum caspase inhibitor in vitro significantly improved the growth of human DMT1-mutant erythroid progenitors. A decreased number of apoptotic erythroblasts was detected in the patient's bone marrow after erythropoietin treatment. In mk/mk mice, erythropoietin administration increased activation of signal transducer and activator of transcription 5 (STAT5) and reduced apoptosis in bone marrow and spleen erythroblasts. mk/mk mice propagated on the 129S6/SvEvTac background resembled DMT1-mutant patients in having increased plasma iron but differed by having functional iron deficiency after erythropoietin administration. Co-regulation of hepcidin and growth differentiation factor 15 (GDF15) levels was observed in mk/mk mice but not in the patient. CONCLUSIONS: Erythropoietin inhibits apoptosis of DMT1-mutant erythroid progenitors and differentiating erythroblasts. Ineffective erythropoiesis associated with defective erythroid iron utilization due to DMT1 mutations has specific biological and clinical features.