RESUMO
Diamond-Blackfan anemia (DBA) is a rare genetic disorder in which patients present a scarcity of erythroid precursors in an otherwise normocellular bone marrow. Most, but not all, patients carry mutations in ribosomal proteins such as RPS19, suggesting that compromised mRNA translation and ribosomal stress are pathogenic mechanisms causing depletion of erythroid precursors. To gain further insight to disease mechanisms in DBA, we performed a custom short hairpin RNA (shRNA) based screen against 750 genes hypothesized to affect DBA pathophysiology. Among the hits were two shRNAs against the erythroid specific heme-regulated eIF2α kinase (HRI), which is a negative regulator of mRNA translation. This study shows that shRNA-mediated HRI silencing or loss of one HRI allele improves expansion of Rps19-deficient erythroid precursors, as well as improves the anemic phenotype in Rps19-deficient animals. We found that Rps19-deficient erythroblasts have elevated levels of unbound intracellular heme, which is normalized by HRI heterozygosity. Additionally, targeting elevated heme levels by treating cells with the heme scavenger alpha-1-microglobulin (A1M), increased proliferation of Rps19-deficient erythroid precursors and decreased heme levels in a disease-specific manner. HRI heterozygosity, but not A1M treatment, also decreased the elevated p53 activity observed in Rps19-deficient cells, indicating that p53 activation is caused by ribosomal stress and aberrant mRNA translation and not heme overload in Rps19-deficiency. Together, these findings suggest that targeting elevated heme levels is a promising new treatment strategy for DBA.
Assuntos
alfa-Globulinas/uso terapêutico , Anemia de Diamond-Blackfan/terapia , Heme/análise , Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Deleção de Genes , Inativação Gênica , Terapia Genética , Heme/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/uso terapêutico , Proteínas Ribossômicas/genéticaRESUMO
Diamond-Blackfan anemia is a congenital bone marrow failure syndrome characterized by red blood cell (RBC) aplasia with varied malformations in infants. Elevated activity of adenosine deaminase (ADA) has been considered as a useful biomarker of Diamond-Blackfan anemia, and ADA assay has been shown to be more sensitive than genetic diagnosis. Approximately, 80% of the examined patients showed elevated ADA activity, whereas genetic tests of ribosome subunit genes identified mutations in approximately 60% of the patients. We previously reported that reduced glutathione (GSH) levels in RBCs may serve as a biomarker of Diamond-Blackfan anemia. In this study, to confirm the universality of our data, we extended the analysis to seven RBC enzymes and GSH of 14 patients with Diamond-Blackfan anemia and performed a cross-analysis study using enzyme activity assay and recently reported proteome data. Statistical analysis revealed that both data exhibited high similarity, upregulation in the hexokinase and pentose-phosphate pathway, and downregulation in glycolytic enzymes such as phosphofructokinase and pyruvate kinase, in the RBCs obtained from the subjects with Diamond-Blackfan anemia. The only discrepancy between enzyme activity and proteome data was observed in glucose-6-phosphate dehydrogenase (G6PD), as increased G6PD activity showed no relation with the significant elevation in protein levels. These results suggest that our enzymatic activity data of Diamond-Blackfan anemia are universal and that the enzymatic activation of G6PD via a hitherto-unveiled mechanism is another metabolic feature of RBCs of Diamond-Blackfan anemia.
Assuntos
Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/enzimologia , Eritrócitos/enzimologia , Adolescente , Aminoidrolases/sangue , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , Pré-Escolar , Regulação para Baixo , Glucosefosfato Desidrogenase/sangue , Glutationa/sangue , Glicólise , Humanos , Lactente , Japão , Via de Pentose Fosfato , Regulação para CimaRESUMO
BACKGROUND AND OBJECTIVES: The status of red blood cell alloimmunization in patients with constitutional anemias including hemoglobinopathies is not known in Norway. The study objective was to investigate the impact of a strategy based on phenotype-matching for C, c, E, e, K, Jka, Jkb, Fya, Fyb, S and s on alloimmunization. MATERIALS AND METHODS: We reviewed transfusions of 40 patients retrospectively using the computerized blood bank management system and medical records; including diagnosis, age at start of transfusion therapy, gender, number and age of packed red blood cell units transfused, follow-up time, phenotypes of the donors and patients, antigen-negative patients exposed to antigen-positive packed red blood cell units, transfusion reactions and alloantibody specificities. RESULTS: Forty patients received 5402 packed red blood cell units. Alloimmunization frequency was 20 % for the whole group, being 7%, 25 % and 30 % in patients with sickle cell disease (n = 14), thalassemia (n = 16) and other conditions (n = 10), respectively. The alloantibodies detected were anti-E, -c, -C, -Cw, -K, -Jka and -Lua. CONCLUSION: Good communication between the clinicians and the transfusion services is essential for successful management of patients with constitutional anemias. Providing full phenotype-matched units was not always possible. Extended pheno-/genotyping before the first transfusion and providing antigen-negative units for antigen-negative patients for at least C, c, E and K in every red cell transfusion would probably have reduced the alloimmunization rate. Non-phenotype-matched transfusions seem to be the main reason for alloimmunization. Finding markers for identifying responders prone to alloimmunization will ensure targeted transfusion strategies.
Assuntos
Anemia de Diamond-Blackfan/terapia , Anemia Falciforme/terapia , Antígenos de Grupos Sanguíneos/imunologia , Anemia de Fanconi/terapia , Isoanticorpos/sangue , Talassemia/terapia , Adolescente , Adulto , Anemia de Diamond-Blackfan/sangue , Anemia Falciforme/sangue , Transfusão de Sangue , Criança , Transfusão de Eritrócitos , Eritrócitos/imunologia , Anemia de Fanconi/sangue , Feminino , Genótipo , Humanos , Masculino , Noruega/epidemiologia , Fenótipo , Estudos Retrospectivos , Talassemia/sangue , Reação Transfusional , Adulto JovemRESUMO
The diagnostic evaluation of Diamond Blackfan Anaemia (DBA), an inherited bone marrow failure syndrome characterised by erythroid hypoplasia, is challenging because of a broad phenotypic variability and the lack of functional screening tests. In this study, we explored the potential of untargeted metabolomics to diagnose DBA. In dried blood spot samples from 18 DBA patients and 40 healthy controls, a total of 1752 unique metabolite features were identified. This metabolic fingerprint was incorporated into a machine-learning algorithm, and a binary classification model was constructed using a training set. The model showed high performance characteristics (average accuracy 91·9%), and correct prediction of class was observed for all controls (n = 12) and all but one patient (n = 4/5) from the validation or 'test' set (accuracy 94%). Importantly, in patients with congenital dyserythropoietic anaemia (CDA) - an erythroid disorder with overlapping features - we observed a distinct metabolic profile, indicating the disease specificity of the DBA fingerprint and underlining its diagnostic potential. Furthermore, when exploring phenotypic heterogeneity, DBA treatment subgroups yielded discrete differences in metabolic profiles, which could hold future potential in understanding therapy responses. Our data demonstrate that untargeted metabolomics in dried blood spots is a promising new diagnostic tool for DBA.
Assuntos
Anemia de Diamond-Blackfan , Teste em Amostras de Sangue Seco , Metabolômica , Adolescente , Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/diagnóstico , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Valor Preditivo dos TestesRESUMO
BACKGROUND: Diamond-Blackfan anemia is a rare inherited bone marrow failure disease. Typical findings include hypoplastic macrocytic anemia, congenital anomalies, and a predisposition to cancer. The molecular basis of the disease is heterozygous mutations of ribosomal proteins without a strict correlation between genotype and phenotype. OBSERVATION: We present 2 cases of Diamond-Blackfan anemia diagnosed during infancy with interesting clinical, molecular, and family characteristics. CONCLUSIONS: A thorough evaluation of all family members is imperative to identify possible 'silent carriers' who are those with no physical stigmata and minor or absent hematologic manifestations. New mutations could add in the map of the disease.
Assuntos
Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/diagnóstico , Anemia de Diamond-Blackfan/terapia , Transfusão de Sangue , Feminino , Humanos , Lactente , Unidades de Terapia Intensiva , Masculino , Resultado do TratamentoRESUMO
Diamond-Blackfan anemia (DBA) is a rare, inherited bone marrow failure syndrome, characterized by red blood cell aplasia, developmental abnormalities, and enhanced risk of malignancy. However, the underlying pathogenesis of DBA is yet to be understood. Recently, mutations in the gene encoding ribosomal protein (RP) L18 were identified in DBA patients. RPL18 is a crucial component of the ribosomal large subunit but its role in hematopoiesis remains unknown. To genetically model the ribosomal defect identified in DBA, we generated a rpl18 mutant line in zebrafish, using CRISPR/Cas9 system. Molecular characterization of this mutant line demonstrated that Rpl18 deficiency mirrored the erythroid defects of DBA, namely a lack of mature red blood cells. Rpl18 deficiency caused an increase in p53 activation and JAK2-STAT3 activity. Furthermore, we found inhibitors of JAK2 or STAT3 phosphorylation could rescue anemia in rpl18 mutants. Our research provides a new in vivo model of Rpl18 deficiency and suggests involvement of signal pathway of JAK2-STAT3 in the DBA pathogenesis.
Assuntos
Anemia de Diamond-Blackfan/enzimologia , Células Eritroides/enzimologia , Eritropoese , Janus Quinase 2/metabolismo , Proteínas Ribossômicas/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/tratamento farmacológico , Anemia de Diamond-Blackfan/genética , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Células Eritroides/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Janus Quinase 2/antagonistas & inibidores , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Ribossômicas/genética , Fator de Transcrição STAT3/antagonistas & inibidores , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/antagonistas & inibidoresRESUMO
OBJECTIVE: Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive autoinflammatory disorder associated with ADA2 mutations. We aimed to investigate the characteristics and ADA2 enzyme activities of patients with DADA2 compared to non-DADA2 patients. METHODS: This is a descriptive study of 24 patients with DADA2 who were admitted to the Adult and Pediatric Rheumatology, Pediatric Haematology, and Pediatric Immunology Departments of Hacettepe University. All ADA2 exons were screened by Sanger sequencing. Serum ADA2 enzyme activity was measured by modified spectrophotometric method. RESULTS: Twenty-four patients with DADA2 were included: 14 with polyarteritis nodosa (PAN)-like phenotype (Group 1); 9 with Diamond-Blackfan anemia (DBA)-like features, and 1 with immunodeficiency (Group 2). Fourteen PAN-like DADA2 patients did not have the typical thrombocytosis seen in classic PAN. Inflammatory attacks were evident only in Group 1 patients. Serum ADA2 activity was low in all patients with DADA2 except one, who was tested after hematopoietic stem cell transplantation. There was no significant difference in ADA2 activities between PAN-like and DBA-like patients. In DADA2 patients with one ADA2 mutation, serum ADA2 activities were as low as those of patients with homozygote DADA2. ADA2 activities were normal in non-DADA2 patients. ADA2 mutations were affecting the dimerization domain in Group 1 patients and the catalytic domain in Group 2 patients. CONCLUSION: We suggest assessing ADA2 activity along with genetic analysis because there are patients with one ADA2 mutation and absent enzyme activity. Our data suggest a possible genotype-phenotype correlation in which dimerization domain mutations are associated with PAN-like phenotype, and catalytic domain mutations are associated with hematological manifestations.
Assuntos
Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Agamaglobulinemia/enzimologia , Anemia de Diamond-Blackfan/enzimologia , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Fenótipo , Poliarterite Nodosa/enzimologia , Imunodeficiência Combinada Severa/enzimologia , Adenosina Desaminase/sangue , Adenosina Desaminase/química , Adolescente , Adulto , Agamaglobulinemia/sangue , Anemia de Diamond-Blackfan/sangue , Domínio Catalítico/genética , Criança , Pré-Escolar , Estudos de Coortes , Dimerização , Éxons , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Transplante de Células-Tronco Hematopoéticas , Homozigoto , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Peptídeos e Proteínas de Sinalização Intercelular/química , Masculino , Pessoa de Meia-Idade , Mutação , Poliarterite Nodosa/sangue , Imunodeficiência Combinada Severa/sangue , Adulto JovemRESUMO
Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia arising from ribosomal protein (RP) defects. Affected patients present with neonatal anemia, occasional dysmorphism, and cancer predisposition. An anemic newborn was diagnosed with DBA due to RPL5 mutation (c.473_474del, p.K158SfsX26). Refractory anemia required regular transfusions and iron chelation therapy. Pancytopenia occurred at age 16 years. Bone-marrow studies showed myelodysplasia, erythroblastosis, and clonal evolution of del(20)(q11.2q13.3). Severe anemia required transfusions. Del(20q), including the L3MBTL1 gene, is reported to be relevant to the hematological phenotype of Shwachman-Diamond syndrome. A combined defect of RPL5 and L3MBTL1 may contribute to the aberrant erythropoiesis in the present case.
Assuntos
Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/complicações , Deleção Cromossômica , Cromossomos Humanos Par 20/genética , Evolução Clonal/genética , Eritroblastose Fetal/sangue , Eritroblastose Fetal/etiologia , Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/etiologia , Síndromes Mielodisplásicas/genética , Adolescente , Anemia de Diamond-Blackfan/terapia , Transfusão de Sangue , Proteínas Cromossômicas não Histona/genética , Progressão da Doença , Eritroblastose Fetal/genética , Eritropoese/genética , Feminino , Transplante de Células-Tronco Hematopoéticas , Humanos , Síndromes Mielodisplásicas/terapia , Pancitopenia/etiologia , Proteínas Repressoras , Proteínas Ribossômicas/genética , Índice de Gravidade de Doença , Proteínas Supressoras de TumorRESUMO
Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by erythroid hypoplasia, usually without perturbation of other hematopoietic lineages. Approximately 65% of DBA patients with autosomal dominant inheritance have heterozygous mutations or deletions in ribosomal protein (RP) genes while <1% of patients with X-linked inheritance have been identified with mutations in the transcription factor GATA1 Erythroid cells from patients with DBA have not been well characterized, and the mechanisms underlying the erythroid specific effects of either RP or GATA1 associated DBA remain unclear. We have developed an ex vivo culture system to expand peripheral blood CD34+ progenitor cells from patients with DBA and differentiate them into erythroid cells. Cells from patients with RP or GATA1 mutations showed decreased proliferation and delayed erythroid differentiation in comparison with controls. RNA transcript analyses of erythroid cells from controls and patients with RP or GATA1 mutations showed distinctive differences, with upregulation of heme biosynthesis genes prominently in RP-mediated DBA and failure to upregulate components of the translational apparatus in GATA1-mediated DBA. Our data show that dysregulation of translation is a common feature of DBA caused by both RP and GATA1 mutations. This trial was registered at www.clinicaltrials.gov as #NCT00106015.
Assuntos
Anemia de Diamond-Blackfan/genética , Adolescente , Adulto , Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/metabolismo , Estudos de Casos e Controles , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Criança , Pré-Escolar , Células Eritroides/metabolismo , Células Eritroides/patologia , Eritropoese/genética , Feminino , Fator de Transcrição GATA1/genética , Genes Dominantes , Genes Ligados ao Cromossomo X , Humanos , Masculino , Modelos Genéticos , Mutação , Proteínas Ribossômicas/genética , Transcriptoma , Adulto JovemRESUMO
Diamond-Blackfan anemia (DBA) is a rare congenital disorder characterized by pure erythrocyte aplasia, and approximately 70% of patients carry mutations in the genes encoding ribosomal proteins (RP). Here, we report the case of a male infant with DBA who presented with anemic crisis (hemoglobin [Hb] concentration 1.5 g/dL) at 58 days after birth. On admission, the infant was pale and had tachypnea, but recovered with intensive care, including red blood cell transfusions, and prednisolone. Based on the clinical diagnosis of DBA, the father of the infant had cyclosporine-A-dependent anemia. On analysis of RP genes when the infant was 6 months old, both the infant and the father, but not the mother, were found to harbor a mutation of RPS19 (c.167G > C, p. R56P). Therefore, genetic background search and early neonatal health check-ups are recommended for families with a history of inherited bone marrow failure syndromes.
Assuntos
Anemia de Diamond-Blackfan/genética , DNA de Neoplasias/genética , Mutação de Sentido Incorreto , Proteínas Ribossômicas/genética , Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/diagnóstico , Análise Mutacional de DNA , Humanos , Lactente , Masculino , Proteínas Ribossômicas/metabolismoRESUMO
Diamond-Blackfan Anemia (DBA) is a rare heterogeneous genetic disease characterized by severe anemia, reduction or absence of erythroid progenitors, and pro-apoptoptic hematopoiesis, which culminates in bone marrow failure. The disease generally manifests in infancy, as craniofacial, cardiac, genitourinary, and upper limb congenital anomalies. Therapy with corticoids is the treatment of choice, while blood transfusion is adopted during diagnosis and as a chronic approach if the patient does not respond to corticoids. This case report describes DBA in a patient that presented with lesions on the oral mucosa caused by secondary neutropenia. The stomatologist plays an important role in a transdisciplinary team and must remain attentive to the general health conditions of patients, since some oral lesions may be associated with systemic events.
Assuntos
Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/complicações , Neutropenia/diagnóstico por imagem , Corticosteroides/uso terapêutico , Negro ou Afro-Americano , Anemia de Diamond-Blackfan/diagnóstico , Anemia de Diamond-Blackfan/etnologia , Apoptose , Transfusão de Sangue , Brasil , Comorbidade , Feminino , Hematopoese/fisiologia , Hemodinâmica , Hospitalização , Humanos , Inflamação , Mucosa Bucal/patologia , Neutropenia/complicações , Adulto JovemRESUMO
Diamond Blackfan Anemia (DBA) is a rare, congenital erythrocyte aplasia that is usually caused by haploinsufficiency of ribosomal proteins due to diverse mutations in one of several ribosomal genes. A striking feature of this disease is that a range of different mutations in ribosomal proteins results in similar disease phenotypes primarily characterized by erythrocyte abnormalities and macrocytic anemia, while most other cell types in the body are minimally affected. Previously, we analyzed the erythrocyte membrane proteomes of several DBA patients and identified several proteins that are not typically associated with this cell type and that suggested inflammatory mechanisms contribute to the pathogenesis of DBA. In this study, we evaluated the erythrocyte cytosolic proteome of DBA patients through in-depth analysis of hemoglobin-depleted erythrocyte cytosols. Simple, reproducible, hemoglobin depletion using nickel columns enabled in-depth analysis of over 1000 cytosolic erythrocyte proteins with only moderate total analysis time per proteome. Label-free quantitation and statistical analysis identified 29 proteins with significantly altered abundance levels in DBA patients compared to matched healthy control donors. Proteins that were significantly increased in DBA erythrocyte cytoplasms included three proteasome subunit beta proteins that make up the immunoproteasome and proteins induced by interferon-γ such as n-myc interactor and interferon-induced 35 kDa protein [NMI and IFI35 respectively]. Pathway analysis confirmed the presence of an inflammatory signature in erythrocytes of DBA patients and predicted key upstream regulators including mitogen activated kinase 1, interferon-γ, tumor suppressor p53, and tumor necrosis factor. These results show that erythrocytes in DBA patients are intrinsically different from those in healthy controls which may be due to an inflammatory response resulting from the inherent molecular defect of ribosomal protein haploinsufficiency or changes in the bone marrow microenvironment that leads to red cell aplasia in DBA patients.
Assuntos
Anemia de Diamond-Blackfan/sangue , Citosol/metabolismo , Eritrócitos/metabolismo , Proteoma/metabolismo , Adolescente , Adulto , Feminino , Humanos , Inflamação/sangue , MasculinoRESUMO
Diamond-Blackfan Anaemia (DBA) is a rare inherited anaemia caused by heterozygous mutations in one of 13 ribosomal protein genes. Erythroid progenitors (BFU-E and CFU-E) in bone marrow (BM) show a proapoptotic phenotype. Suspicion of DBA is reached after exclusion of other forms of BM failure syndromes. To improve DBA diagnosis, which is confirmed by mutation analysis, we tested a new approach based on the study of extracellular vesicles (EVs) isolated from plasma by differential centrifugations and analysed by flow cytometry. We chose CD34, CD71 and CD235a markers to study erythroid EVs. We characterised the EVs immunophentoypic profiles of 13 DBA patients, 22 healthy controls and 16 patients with other haematological diseases. Among the three EVs clusters we found, only the CD34+/CD71low population showed statistically significant differences between DBA patients and controls (p< 0.05). The absence of this cluster is in agreement with the low levels of BFU-E found in DBA patients. The assessment of ROC curves demonstrated the potential diagnostic value of this population. We suggest that this assay may be useful to improve DBA diagnosis as a quicker and less invasive alternative to BM BFU-E culture analysis.
Assuntos
Anemia de Diamond-Blackfan/diagnóstico , Células Precursoras Eritroides/metabolismo , Vesículas Extracelulares/metabolismo , Imunofenotipagem/métodos , Adolescente , Adulto , Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/genética , Antígenos CD/sangue , Antígenos CD34/sangue , Biomarcadores/sangue , Criança , Pré-Escolar , Ensaio de Unidades Formadoras de Colônias , Feminino , Citometria de Fluxo , Glicoforinas/análise , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Curva ROC , Receptores da Transferrina/sangue , Adulto JovemRESUMO
Defects in ribosome biogenesis are associated with a group of diseases called the ribosomopathies, of which Diamond-Blackfan anemia (DBA) is the most studied. Ribosomes are composed of ribosomal proteins (RPs) and ribosomal RNA (rRNA). RPs and multiple other factors are necessary for the processing of pre-rRNA, the assembly of ribosomal subunits, their export to the cytoplasm and for the final assembly of subunits into a ribosome. Haploinsufficiency of certain RPs causes DBA, whereas mutations in other factors cause various other ribosomopathies. Despite the general nature of their underlying defects, the clinical manifestations of ribosomopathies differ. In DBA, for example, red blood cell pathology is especially evident. In addition, individuals with DBA often have malformations of limbs, the face and various organs, and also have an increased risk of cancer. Common features shared among human DBA and animal models have emerged, such as small body size, eye defects, duplication or overgrowth of ectoderm-derived structures, and hematopoietic defects. Phenotypes of ribosomopathies are mediated both by p53-dependent and -independent pathways. The current challenge is to identify differences in response to ribosomal stress that lead to specific tissue defects in various ribosomopathies. Here, we review recent findings in this field, with a particular focus on animal models, and discuss how, in some cases, the different phenotypes of ribosomopathies might arise from differences in the spatiotemporal expression of the affected genes.
Assuntos
Anemia de Diamond-Blackfan/fisiopatologia , Ribossomos/ultraestrutura , Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/genética , Animais , Ciclo Celular , Proliferação de Células , Modelos Animais de Doenças , Eritrócitos/patologia , Eritropoese , Hematopoese , Humanos , Imunidade Inata , Mutação , Neoplasias/metabolismo , Fenótipo , RNA Ribossômico/análise , Proteínas Ribossômicas/genética , Ribossomos/patologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Diamond-Blackfan anaemia (DBA) is a rare congenital disease causing severe anaemia and progressive bone marrow failure. The majority of patients carry mutations in ribosomal proteins, which leads to depletion of erythroid progenitors in the bone marrow. As many as 40% of all DBA patients receive glucocorticoids to alleviate their anaemia. However, despite their use in DBA treatment for more than half a century, the therapeutic mechanisms of glucocorticoids remain largely unknown. Therefore we sought to study disease specific effects of glucocorticoid treatment using a ribosomal protein s19 (Rps19) deficient mouse model of DBA. This study determines for the first time that a mouse model of DBA can respond to glucocorticoid treatment, similar to DBA patients. Our results demonstrate that glucocorticoid treatment reduces apoptosis, rescues erythroid progenitor depletion and premature differentiation of erythroid cells. Furthermore, glucocorticoids prevent Trp53 activation in Rps19-deficient cells- in a disease-specific manner. Dissecting the therapeutic mechanisms behind glucocorticoid treatment of DBA provides indispensible insight into DBA pathogenesis. Identifying mechanisms important for DBA treatment also enables development of more disease-specific treatments of DBA.
Assuntos
Anemia de Diamond-Blackfan/tratamento farmacológico , Eritropoese/efeitos dos fármacos , Prednisolona/uso terapêutico , Proteínas Ribossômicas/deficiência , Proteína Supressora de Tumor p53/fisiologia , Adolescente , Anemia de Diamond-Blackfan/sangue , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/genética , Dexametasona/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Células Precursoras Eritroides/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Prednisolona/farmacologia , Quimera por Radiação , Proteínas Ribossômicas/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Regulação para Cima/efeitos dos fármacos , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/genéticaRESUMO
Diamond-Blackfan Anemia (DBA) is a bone marrow failure disorder characterized by low red blood cell count. Mutations in ribosomal protein genes have been identified in approximately half of all DBA cases. Corticosteriod therapy and bone marrow transplantation are common treatment options for patients; however, significant risks and complications are associated with these treatment options. Therefore, novel therapeutic approaches are needed for treating DBA. Sotatercept (ACE-011, and its murine ortholog RAP-011) acts as an activin receptor type IIA ligand trap, increasing hemoglobin and hematocrit in pharmacologic models, in healthy volunteers, and in patients with ß-thalassemia, by expanding late-stage erythroblasts through a mechanism distinct from erythropoietin. Here, we evaluated the effects of RAP-011 in zebrafish models of RPL11 ribosome deficiency. Treatment with RAP-011 dramatically restored hemoglobin levels caused by ribosome stress. In zebrafish embryos, RAP-011 likely stimulates erythropoietic activity by sequestering lefty1 from erythroid cells. These findings identify lefty1 as a signaling component in the development of erythroid cells and rationalize the use of sotatercept in DBA patients.
Assuntos
Anemia de Diamond-Blackfan/tratamento farmacológico , Eritropoese/efeitos dos fármacos , Fatores de Determinação Direita-Esquerda/antagonistas & inibidores , Proteínas Recombinantes de Fusão/uso terapêutico , Proteínas de Peixe-Zebra/antagonistas & inibidores , Receptores de Activinas Tipo II/antagonistas & inibidores , Receptores de Activinas Tipo II/sangue , Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/genética , Animais , Modelos Animais de Doenças , Eritropoese/genética , Técnicas de Silenciamento de Genes , Genes p53 , Humanos , Fatores de Determinação Direita-Esquerda/sangue , Fatores de Determinação Direita-Esquerda/genética , Ligantes , Proteínas Ribossômicas/sangue , Proteínas Ribossômicas/deficiência , Proteínas Ribossômicas/genética , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra , Proteínas de Peixe-Zebra/sangue , Proteínas de Peixe-Zebra/genética , Talassemia beta/sangue , Talassemia beta/tratamento farmacológicoRESUMO
Diamond-Blackfan anemia (DBA) is a disorder characterized by a selective defect in erythropoiesis. Delineation of the precise defect is hampered by a lack of markers that define cells giving rise to erythroid burst- and erythroid colony-forming unit (BFU-E and CFU-E) colonies, the clonogenic assays that quantify early and late erythroid progenitor (EEP and LEP) potential, respectively. By combining flow cytometry, cell-sorting, and single-cell clonogenic assays, we identified Lin(-)CD34(+)CD38(+)CD45RA(-)CD123(-)CD71(+)CD41a(-)CD105(-)CD36(-) bone marrow cells as EEP giving rise to BFU-E, and Lin(-)CD34(+/-)CD38(+)CD45RA(-)CD123(-)CD71(+)CD41a(-)CD105(+)CD36(+) cells as LEP giving rise to CFU-E, in a hierarchical fashion. We then applied these definitions to DBA and identified that, compared with controls, frequency, and clonogenicity of DBA, EEP and LEP are significantly decreased in transfusion-dependent but restored in corticosteroid-responsive patients. Thus, both quantitative and qualitative defects in erythroid progenitor (EP) contribute to defective erythropoiesis in DBA. Prospective isolation of defined EPs will facilitate more incisive study of normal and aberrant erythropoiesis.