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 (ÐlogFCÐ > 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/metabolismoRESUMO
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/genéticaRESUMO
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.
Assuntos
Anemia de Diamond-Blackfan , Proteínas Ribossômicas , Anemia de Diamond-Blackfan/genética , Eritrócitos/metabolismo , Feminino , Humanos , Mutação de Sentido Incorreto , Biossíntese de Proteínas , Proteínas Ribossômicas/genéticaRESUMO
To better understand the molecular basis of resistance to azacitidine (AZA) therapy in myelodysplastic syndromes (MDS) and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC), we performed RNA sequencing on pre-treatment CD34+ hematopoietic stem/progenitor cells (HSPCs) isolated from 25 MDS/AML-MRC patients of the discovery cohort (10 AZA responders (RD), six stable disease, nine progressive disease (PD) during AZA therapy) and from eight controls. Eleven MDS/AML-MRC samples were also available for analysis of selected metabolites, along with 17 additional samples from an independent validation cohort. Except for two patients, the others did not carry isocitrate dehydrogenase (IDH)1/2 mutations. Transcriptional landscapes of the patients' HSPCs were comparable to those published previously, including decreased signatures of active cell cycling and DNA damage response in PD compared to RD and controls. In addition, PD-derived HSPCs revealed repressed markers of the tricarboxylic acid cycle, with IDH2 among the top 50 downregulated genes in PD compared to RD. Decreased citrate plasma levels, downregulated expression of the (ATP)-citrate lyase and other transcriptional/metabolic networks indicate metabolism-driven histone modifications in PD HSPCs. Observed histone deacetylation is consistent with transcription-nonpermissive chromatin configuration and quiescence of PD HSPCs. This study highlights the complexity of the molecular network underlying response/resistance to hypomethylating agents.
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 JovemRESUMO
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/farmacologiaRESUMO
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-AtividadeRESUMO
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 JovemRESUMO
In mature erythrocytes, glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) yield NADPH, a crucial cofactor of the enzyme glutathione reductase (GR) converting glutathione disulfide (GSSG) into its reduced state (GSH). GSH is essential for detoxification processes in and survival of erythrocytes. We explored whether the anti-inflammatory compounds Bay 11-7082, parthenolide and dimethyl fumarate (DMF) were able to completely deplete a common target (GSH), and to impair the function of upstream enzymes of GSH recycling and replenishment. Treatment of erythrocytes with Bay 11-7082, parthenolide or DMF led to concentration-dependent eryptosis resulting from complete depletion of GSH. GSH depletion was due to strong inhibition of G6PDH activity. Bay 11-7082 and DMF, but not parthenolide, were able to inhibit the GR activity. This approach "Inhibitors, Detection of their common target that is completely depleted or inactivated when pharmacologically relevant concentrations of each single inhibitor are applied, Subsequent functional analysis of upstream enzymes for this target" (IDS), can be applied to a broad range of inhibitors and cell types according to the selected target. The specific G6PDH inhibitory effect of these compounds may be exploited for the treatment of human diseases with high NADPH and GSH consumption rates, including malaria, trypanosomiasis, cancer or obesity.
Assuntos
Fumarato de Dimetilo/farmacologia , Eriptose/efeitos dos fármacos , Eritrócitos/enzimologia , Glucosefosfato Desidrogenase , Nitrilas/farmacologia , Sesquiterpenos/farmacologia , Sulfonas/farmacologia , Glucosefosfato Desidrogenase/antagonistas & inibidores , Glucosefosfato Desidrogenase/metabolismo , HumanosRESUMO
We report two brothers with mild intellectual deficiency, exercise intolerance, rhabdomyolysis, seizures and no hemolysis. Phosphoglycerate kinase (PGK) activity was strongly decreased in their red blood cells. Subsequent molecular analysis of PGK1 revealed hemizygosity for a novel mutation c.756 + 3A > G, in intron 7. Analysis of the effect of this mutation on pre-mRNA processing demonstrated markedly decreased levels of normal PGK1 mRNA. In addition, the c.756 + 3A > G change resulted in abnormally spliced transcripts. If translated, these transcripts mostly encode for C-terminally truncated proteins. The consequences of the c.756 + 3A > G mutation is discussed, as well as the genotype-to-phenotype correlation with regard to previously described mutations (PGK Fukuroi and PGK Antwerp), which also result in C-terminal truncated proteins.
Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Deficiência Intelectual/complicações , Erros Inatos do Metabolismo/diagnóstico , Erros Inatos do Metabolismo/genética , Mioglobinúria/complicações , Fosfoglicerato Quinase/deficiência , Convulsões/complicações , Adolescente , Doenças Genéticas Ligadas ao Cromossomo X/complicações , Genótipo , Hemólise , Humanos , Masculino , Erros Inatos do Metabolismo/complicações , Músculo Esquelético/patologia , Mutação , Fenótipo , Fosfoglicerato Quinase/genética , Fosfoglicerato Quinase/ultraestrutura , IrmãosRESUMO
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 JovemRESUMO
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/sangueRESUMO
Phosphofructokinase deficiency is a very rare autosomal recessive disorder, which belongs to group of rare inborn errors of metabolism called glycogen storage disease. Here we report on a new mutation in the phosphofructokinase (PFK) gene PFKM identified in a 65-years-old woman who suffered from lifelong intermittent muscle weakness and painful spasms of random occurrence, episodic dark urines, and slight haemolytic anemia. After ruling out the most common causes of chronic haemolytic anemia, the study of a panel of 24 enzyme activities showed a markedly decreased PFK activity in red blood cells (RBCs) from the patient. DNA sequence analysis of the PFKM gene subsequently revealed a novel homozygous mutation: c.926A>G; p.Asp309Gly. This mutation is predicted to severely affect enzyme catalysis thereby accounting for the observed enzyme deficiency. This case represents a prime example of classical PFK deficiency and is the first reported case of this very rare red blood cell disorder in Spain.