RESUMEN
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.
Asunto(s)
Anemia de Diamond-Blackfan/sangre , Anemia de Diamond-Blackfan/enzimología , Eritrocitos/enzimología , Adolescente , Aminohidrolasas/sangre , Biomarcadores/sangre , Estudios de Casos y Controles , Niño , Preescolar , Regulación hacia Abajo , Glucosafosfato Deshidrogenasa/sangre , Glutatión/sangre , Glucólisis , Humanos , Lactante , Japón , Vía de Pentosa Fosfato , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Anemia de Diamond-Blackfan/enzimología , Células Eritroides/enzimología , Eritropoyesis , Janus Quinasa 2/metabolismo , Proteínas Ribosómicas/metabolismo , Factor de Transcripción STAT3/metabolismo , Proteínas de Pez Cebra/metabolismo , Anemia de Diamond-Blackfan/sangre , Anemia de Diamond-Blackfan/tratamiento farmacológico , Anemia de Diamond-Blackfan/genética , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Células Eritroides/efectos de los fármacos , Eritropoyesis/efectos de los fármacos , Janus Quinasa 2/antagonistas & inhibidores , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Ribosómicas/genética , Factor de Transcripción STAT3/antagonistas & inhibidores , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Adenosina Desaminasa/deficiencia , Adenosina Desaminasa/genética , Agammaglobulinemia/enzimología , Anemia de Diamond-Blackfan/enzimología , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Péptidos y Proteínas de Señalización Intercelular/genética , Fenotipo , Poliarteritis Nudosa/enzimología , Inmunodeficiencia Combinada Grave/enzimología , Adenosina Desaminasa/sangre , Adenosina Desaminasa/química , Adolescente , Adulto , Agammaglobulinemia/sangre , Anemia de Diamond-Blackfan/sangre , Dominio Catalítico/genética , Niño , Preescolar , Estudios de Cohortes , Dimerización , Exones , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Trasplante de Células Madre Hematopoyéticas , Homocigoto , Humanos , Péptidos y Proteínas de Señalización Intercelular/sangre , Péptidos y Proteínas de Señalización Intercelular/química , Masculino , Persona de Mediana Edad , Mutación , Poliarteritis Nudosa/sangre , Inmunodeficiencia Combinada Grave/sangre , Adulto JovenRESUMEN
Protein arginine methyltransferase 3 (PRMT3) is a cytosolic enzyme that catalyzes the formation of mono- and asymmetric dimethyl arginines, with ribosomal protein (RP) S2 as its main in vivo substrate. The interplay of PRMT3-RPS2 homologs in yeast is important for regulating the ribosomal subunit ratio and assembly. Prmt3-null mice display slower embryonic growth and development, although this phenotype is milder than in mouse RP gene knockouts. Defects in ribosome maturation are the hallmark of Diamond-Blackfan anemia (DBA). Sequencing of the PRMT3 gene in patients from the Czech DBA registry revealed a heterozygous mutation encoding the Tyr87Cys substitution. Although later analysis excluded this mutation as the cause of disease, we anticipated that this substitution might be important for PRMT3 function and decided to study it in detail. Tyr87 resides in a highly conserved substrate binding domain and has been predicted to be phosphorylated. To address the impact of putative Tyr87 phosphorylation on PRMT3 properties, we constructed two additional PRMT3 variants, Tyr87Phe and Tyr87Glu PRMT3, mimicking non-phosphorylated and phosphorylated Tyr87, respectively. The Tyr87Cys and Tyr87Glu-PRMT3 variants had markedly decreased affinity to RPS2 and, consequently, reduced enzymatic activity compared to the wild-type enzyme. The activity of the Tyr87Phe-PRMT3 mutant remained unaffected. No evidence of Tyr87 phosphorylation was found using mass spectrometric analysis of purified PRMT3, although phosphorylation of serines 25 and 27 was observed. In conclusion, Tyr87 is important for the interaction between PRMT3 and RPS2 and for its full enzymatic activity.
Asunto(s)
Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Anemia de Diamond-Blackfan/enzimología , Anemia de Diamond-Blackfan/genética , Animales , Células HeLa , Humanos , Cinética , Metilación , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Missense , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribosómicas/metabolismo , Tirosina/química , Tirosina/genéticaRESUMEN
We describe a girl with Diamond-Blackfan anemia with accompanying red cell enolase deficiency. At the age of 9 yr old, the patient received allogeneic bone marrow transplantation from her HLA-identical sister who had normal red cell enolase activity. While the post transplant DNA analysis with short tandem repeat has continuously demonstrated a stable mixed chimerism on follow-up, the patient remains transfusion independent and continues to show a steady increase in red cell enolase activity for over two and a half years following bone marrow transplantation.